Researcher Profile and Settings

Master

Affiliation (Master)

• Faculty of Science　Chemistry　Physical Chemistry

Affiliation (Master)

• Faculty of Science　Chemistry　Physical Chemistry

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Profile and Settings

Degree

• Ph. D.（Kyoto University）

Profile and Settings

• Name (Japanese)

  Ishimori

• Name (Kana)

  Koichiro

• Name

  200901006051594815

Alternate Names

http://barato.sci.hokudai.ac.jp/~stchem/

Achievement

Research Interests

• 構造化学   蛋白質工学   分子分光学   生物無機化学   生物物理学   Molecular Spectroscopy   Protein Engineering   Bioinorganic Chemistry   Biophysics   Structural Chemistry   

Research Areas

• Life sciences / Biophysics
• Nanotechnology/Materials / Basic physical chemistry
• Nanotechnology/Materials / Biochemistry

Research Experience

• 2006 - Today Hokkaido University, Faculty of Science Professor
• 2011/04 - 2021/03 Osaka University Institute for Protein Research
• 2017 Kyoto University Faculty of Science
• 2012 Yamaguchi University Faculty of Agriculture
• 2009 - 2011 Osaka University, Institute of Protein Research Visiting Fellow
• 2011 奈良先端科学技術大学 非常勤講師
• 2010 Tottori University Faculty of Engineering
• 2008 - 2009 Nara Women's University Faculty of Human Life and Environment
• 2005 - 2007 Kyoto University Graduate School of Engineering
• 2005 - 2006 University of Hyogo of Life Science, Graduate School
• 2005 - 2006 Institute of Molecular Science Visiting Professor
• 2005 - 2006 Hokkaido University, Graduate School of Science Professor
• 2005 - 2006 Visiting Professor,Institute of Molecular Sceince
• 2005 - 2006 Professor,Graduate School of Science, Hokkaido University
• 2004 - 2005 Institute of Molecular Science Visiting Associate Professor
• 1995 - 2005 Kyoto University, Graduate School of Engineering Associate Professor
• 1989 - 1995 Kyoto University, Faculty of Engineering Research Associate
• 1993 - 1994 University of Wisconsin-Madison Visiting Researcher
• 1987 - 1989 JSPS Research Fellow

Education

• 1986/03 - 1989/04  Kyoto University  Graduate School of Engineering  Division of Molecular Engineering
• 1984/04 - 1986/03  Kyoto University  Graduate School, Division of Engineering  Division of Molecular Engineering
• 1980/04 - 1984/03  Kyoto University  Faculty of Engineering  Department of Hydrocarbon Chemistry

Published Papers

• A dye-decolorizing peroxidase from Vibrio cholerae can demetallate heme

  Takeshi Uchida, Sayaka Umetsu, Miho Sasaki, Haruka Yoshimura, Issei Omura, Koichiro Ishimori

  Journal of Inorganic Biochemistry 112764 - 112764 0162-0134 2024/10
• Conserved loop of a phase modifier endows protein condensates with fluidity

  Honoka Kawamukai, Motonori Matsusaki, Takanari Tanimoto, Mai Watabe, Ken Morishima, Shunsuke Tomita, Yoichi Shinkai, Tatsuya Niwa, Taro Mannen, Hiroyuki Kumeta, Hitoki Nanaura, Kotona Kato, Takuya Mabuchi, Yuichiro Aiba, Takeru Uehara, Noriyoshi Isozumi, Yoshika Hara, Shingo Kanemura, Hiroyoshi Matsumura, Kazuma Sugie, Koichiro Ishimori, Takahiro Muraoka, Masaaki Sugiyama, Masaki Okumura, Eiichiro Mori, Takuya Yoshizawa, Tomohide Saio

  2024/07/04 

  Abstract Dipeptide repeats (DPRs) that are gene products from abnormal hexanucleotide repeat expansion inC9orf72trigger amyotrophic lateral sclerosis (ALS) through unknown mechanism. This study highlights, importin Karyopherinβ2 (Kapβ2), which is responsible for nuclear transport and phase modification of RNA-binding proteins (RBPs), as a major DPR target. We demonstrate DPR accumulation in the nucleus via Kapβ2-mediated transport, which results in dose-dependent toxicity observed in nematode and yeast models. In vitro interaction studies exploiting chemical probe arrays and biophysical measurements reveal multivalent DPR binding to Kapβ2, including at the conserved acidic loop. Refractive index and fluorescence imaging coupled with biochemical assays unveiled that binding of excess DPRs to the acidic loop turns a phase modifier Kapβ2 into phase disrupter, resulting more condensed and viscous RBP condensates. Our findings provides molecular insight intoC9orf72-ALS related to age and repeat expansion.
• Conformational Distribution of a Multidomain Protein Measured by Single-Pair Small-Angle X-ray Scattering

  Honoka Kawamukai, Shumpei Takishita, Kazumi Shimizu, Daisuke Kohda, Koichiro Ishimori, Tomohide Saio

  The Journal of Physical Chemistry Letters 15 (3) 744 - 750 1948-7185 2024/01/15
• Heat-Induced Conformational Transition Mechanism of Heat Shock Factor 1 Investigated by Tryptophan Probe

  Soichiro Kawagoe, Munehiro Kumashiro, Takuya Mabuchi, Hiroyuki Kumeta, Koichiro Ishimori, Tomohide Saio

  Biochemistry 0006-2960 2022/12/09
• Metal Sensing by a Glycine–Histidine Repeat Sequence Regulates the Heme Degradation Activity of PM0042 from Pasteurella multocida

  Takeshi Uchida, Kazuki Ota, Akinobu Tatsumi, Syota Takeuchi, Koichiro Ishimori

  Inorganic Chemistry 0020-1669 2022/08/12 [Refereed]
  
• Heme binding to cold shock protein D, CspD, from Vibrio cholerae

  Dayeon Nam, Wataru Motegi, Koichiro Ishimori, Takeshi Uchida

  Biochemical and Biophysical Research Communications 0006-291X 2022/07 [Refereed]
  
• Structural and Kinetic Views of Molecular Chaperones in Multidomain Protein Folding.

  Soichiro Kawagoe, Koichiro Ishimori, Tomohide Saio

  International journal of molecular sciences 23 (5) 2022/02/24 [Refereed]
   

  Despite recent developments in protein structure prediction, the process of the structure formation, folding, remains poorly understood. Notably, folding of multidomain proteins, which involves multiple steps of segmental folding, is one of the biggest questions in protein science. Multidomain protein folding often requires the assistance of molecular chaperones. Molecular chaperones promote or delay the folding of the client protein, but the detailed mechanisms are still unclear. This review summarizes the findings of biophysical and structural studies on the mechanism of multidomain protein folding mediated by molecular chaperones and explains how molecular chaperones recognize the client proteins and alter their folding properties. Furthermore, we introduce several recent studies that describe the concept of kinetics-activity relationships to explain the mechanism of functional diversity of molecular chaperones.
• Converting cytochrome c into a DyP-like metalloenzyme

  Issei Omura, Koichiro Ishimori, Takeshi Uchida

  Dalton Transactions 51 (33) 12641 - 12649 1477-9226 2022 [Refereed]
   

  Dye-decolorizing peroxidase (DyP), which can degrade anthraquinone dyes using H₂O₂, is an attractive prospect for potential biotechnological applications for environmental purification. We previously designed an artificial DyP with an optimal...
• Regulation of the expression of the nickel uptake system in Vibrio cholerae by iron and heme via Fur

  Kazuyoshi Muranishi, Koichiro Ishimori, Takeshi Uchida

  Journal of Inorganic Biochemistry 111713 - 111713 0162-0134 2022/01 [Refereed]
  
• Oxidative phase transition of heat shock factor-1

  Soichiro Kawagoe, Motonori Matsusaki, Koichiro Ishimori, Tomohide Saio

  2021/12/03 

  ABSTRACT Heat shock factor 1 (Hsf1) was found as a central upregulator of molecular chaperones in stress adaptation, but it has recently been rediscovered as a major component of persistent nuclear stress bodies (nSBs). When the persistently stressed cells undergo apoptosis, the phase transition of nSBs from fluid to gel-like states is proposed to be an important event in switching the cell fate from survival to death. Nonetheless, how the phase separation and transition of nSBs are driven remain unanswered. In this study, we discovered that Hsf1 formed liquid-liquid phase separation dropletsin vitro, causing the assembly of Hsf1 to drive nSBs formation. Under oxidative conditions, disulfide-bonded and oligomerized Hsf1 formed gel-like and more condensed droplets, confirmed through fluorescence recovery, refractive index imaging, and light scattering. Then, on the basis of our results, we proposed that Hsf1 undergoes oxidative phase transition by sensing redox conditions potentially to drive the cell fate decision by nSBs.
• C9orf72-derived arginine-rich poly-dipeptides impede phase modifiers

  Hitoki Nanaura, Honoka Kawamukai, Ayano Fujiwara, Takeru Uehara, Yuichiro Aiba, Mari Nakanishi, Tomo Shiota, Masaki Hibino, Pattama Wiriyasermkul, Sotaro Kikuchi, Riko Nagata, Masaya Matsubayashi, Yoichi Shinkai, Tatsuya Niwa, Taro Mannen, Naritaka Morikawa, Naohiko Iguchi, Takao Kiriyama, Ken Morishima, Rintaro Inoue, Masaaki Sugiyama, Takashi Oda, Noriyuki Kodera, Sachiko Toma-Fukai, Mamoru Sato, Hideki Taguchi, Shushi Nagamori, Osami Shoji, Koichiro Ishimori, Hiroyoshi Matsumura, Kazuma Sugie, Tomohide Saio, Takuya Yoshizawa, Eiichiro Mori

  Nature Communications 12 (1) 2021/12 [Refereed]
   

  Nuclear import receptors (NIRs) not only transport RNA-binding proteins (RBPs) but also modify phase transitions of RBPs by recognizing nuclear localization signals (NLSs). Toxic arginine-rich poly-dipeptides from C9orf72 interact with NIRs and cause nucleocytoplasmic transport deficit. However, the molecular basis for the toxicity of arginine-rich poly-dipeptides toward NIRs function as phase modifiers of RBPs remains unidentified. Here we show that arginine-rich poly-dipeptides impede the ability of NIRs to modify phase transitions of RBPs. Isothermal titration calorimetry and size-exclusion chromatography revealed that proline:arginine (PR) poly-dipeptides tightly bind karyopherin-β2 (Kapβ2) at 1:1 ratio. The nuclear magnetic resonances of Kapβ2 perturbed by PR poly-dipeptides partially overlapped with those perturbed by the designed NLS peptide, suggesting that PR poly-dipeptides target the NLS binding site of Kapβ2. The findings offer mechanistic insights into how phase transitions of RBPs are disabled in C9orf72-related neurodegeneration.
• Functional cooperativity between the trigger factor chaperone and the ClpXP proteolytic complex

  Kamran Rizzolo, Angela Yeou Hsiung Yu, Adedeji Ologbenla, Sa Rang Kim, Haojie Zhu, Koichiro Ishimori, Guillaume Thibault, Elisa Leung, Yi Wen Zhang, Mona Teng, Marta Haniszewski, Noha Miah, Sadhna Phanse, Zoran Minic, Sukyeong Lee, Julio Diaz Caballero, Mohan Babu, Francis T. F. Tsai, Tomohide Saio, Walid A. Houry

  Nature Communications 12 (1) 281 - 281 2021/12 [Refereed]
   

  A functional association is uncovered between the ribosome-associated trigger factor (TF) chaperone and the ClpXP degradation complex. Bioinformatic analyses demonstrate conservation of the close proximity of tig, the gene coding for TF, and genes coding for ClpXP, suggesting a functional interaction. The effect of TF on ClpXP-dependent degradation varies based on the nature of substrate. While degradation of some substrates are slowed down or are unaffected by TF, surprisingly, TF increases the degradation rate of a third class of substrates. These include λ phage replication protein λO, master regulator of stationary phase RpoS, and SsrA-tagged proteins. Globally, TF acts to enhance the degradation of about 2% of newly synthesized proteins. TF is found to interact through multiple sites with ClpX in a highly dynamic fashion to promote protein degradation. This chaperone-protease cooperation constitutes a unique and likely ancestral aspect of cellular protein homeostasis in which TF acts as an adaptor for ClpXP.
• Zinc-Dependent Oligomerization of Thermus thermophilus Trigger Factor Chaperone

  Haojie Zhu, Motonori Matsusaki, Taiga Sugawara, Koichiro Ishimori, Tomohide Saio

  Biology 10 (11) 1106 - 1106 2021/10/26 [Refereed]
   

  Thermus thermophilus trigger factor (TtTF) is a zinc-dependent molecular chaperone whose folding-arrest activity is regulated by Zn2+. However, little is known about the mechanism of zinc-dependent regulation of the TtTF activity. Here we exploit in vitro biophysical experiments to investigate zinc-binding, the oligomeric state, the secondary structure, and the thermal stability of TtTF in the absence and presence of Zn2+. The data show that full-length TtTF binds Zn2+, but the isolated domains and tandem domains of TtTF do not bind to Zn2+. Furthermore, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectra suggested that Zn2+-binding induces the partial structural changes of TtTF, and size exclusion chromatography-multi-angle light scattering (SEC-MALS) showed that Zn2+ promotes TtTF oligomerization. Given the previous work showing that the activity regulation of E. coli trigger factor is accompanied by oligomerization, the data suggest that TtTF exploits zinc ions to induce the structural change coupled with the oligomerization to assemble the client-binding site, thereby effectively preventing proteins from misfolding in the thermal environment.
• Conformational ensemble of a multidomain protein explored by Gd³⁺ electron paramagnetic resonance

  Tomohide Saio, Soya Hiramatsu, Mizue Asada, Hiroshi Nakagawa, Kazumi Shimizu, Hiroyuki Kumeta, Toshikazu Nakamura, Koichiro Ishimori

  Biophysical Journal 120 (15) 2943 - 2951 0006-3495 2021/08/03 [Refereed]
   

  Despite their importance in function, the conformational state of proteins and its changes are often poorly understood, mainly because of the lack of an efficient tool. MurD, a 47-kDa protein enzyme responsible for peptidoglycan biosynthesis, is one of those proteins whose conformational states and changes during their catalytic cycle are not well understood. Although it has been considered that MurD takes a single conformational state in solution as shown by a crystal structure, the solution nuclear magnetic resonance (NMR) study suggested the existence of multiple conformational state of apo MurD in solution. However, the conformational distribution has not been evaluated. In this work, we investigate the conformational states of MurD by the use of electron paramagnetic resonance (EPR), especially intergadolinium distance measurement using double electron-electron resonance (DEER) measurement. The gadolinium ions are fixed on specific positions on MurD via a rigid double-arm paramagnetic lanthanide tag that has been originally developed for paramagnetic NMR. The combined use of NMR and EPR enables accurate interpretation of the DEER distance information to the structural information of MurD. The DEER distance measurement for apo MurD shows a broad distance distribution, whereas the presence of the inhibitor narrows the distance distribution. The results suggest that MurD exists in a wide variety of conformational states in the absence of ligands, whereas binding of the inhibitor eliminates variation in conformational states. The multiple conformational states of MurD were previously implied by NMR experiments, but our DEER data provided structural characterization of the conformational variety of MurD.
• Radical transfer but not heme distal residues is essential for pH dependence of dye-decolorizing activity of peroxidase from Vibrio cholerae

  Takeshi Uchida, Issei Omura, Sayaka Umetsu, Koichiro Ishimori

  Journal of Inorganic Biochemistry 219 0162-0134 2021/06 

  Dye-decolorizing peroxidase (DyP) is a heme-containing enzyme that catalyzes the degradation of anthraquinone dyes. A main feature of DyP is the acidic optimal pH for dye-decolorizing activity. In this study, we constructed several mutant DyP enzymes from Vibrio cholerae (VcDyP), with a view to identifying the decisive factor of the low pH preference of DyP. Initially, distal Asp144, a conserved residue, was replaced with His, which led to significant loss of dye-decolorizing activity. Introduction of His into a position slightly distant from heme resulted in restoration of activity but no shift in optimal pH, indicating that distal residues do not contribute to the pH dependence of catalytic activity. His178, an essential residue for dye decolorization, is located near heme and forms hydrogen bonds with Asp138 and Thr278. While Trp and Tyr mutants of His178 were inactive, the Phe mutant displayed ~35% activity of wild-type VcDyP, indicating that this position is a potential radical transfer route from heme to the active site on the protein surface. The Thr278Val mutant displayed similar enzymatic properties as WT VcDyP, whereas the Asp138Val mutant displayed significantly increased activity at pH 6.5. On the basis of these findings, we propose that neither distal amino acid residues, including Asp144, nor hydrogen bonds between His178 and Thr278 are responsible while the hydrogen bond between His178 and Asp138 plays a key role in the pH dependence of activity.
• Author Correction: Functional cooperativity between the trigger factor chaperone and the ClpXP proteolytic complex.

  Kamran Rizzolo, Angela Yeou Hsiung Yu, Adedeji Ologbenla, Sa Rang Kim, Haojie Zhu, Koichiro Ishimori, Guillaume Thibault, Elisa Leung, Yi Wen Zhang, Mona Teng, Marta Haniszewski, Noha Miah, Sadhna Phanse, Zoran Minic, Sukyeong Lee, Julio Diaz Caballero, Mohan Babu, Francis T F Tsai, Tomohide Saio, Walid A Houry

  Nature communications 12 (1) 2753 - 2753 2021/05/06
• Mechanistic insights into heme-mediated transcriptional regulation via a bacterial manganese-binding iron regulator, iron response regulator (Irr)

  Dayeon Nam, Yuki Matsumoto, Takeshi Uchida, Mark R. O'Brian, Koichiro Ishimori

  The Journal of biological chemistry 295 (32) 11316 - 11325 2020/08/07 [Refereed][Not invited]
   

  © 2020 Nam et al. The transcription factor iron response regulator (Irr) is a key regulator of iron homeostasis in the nitrogen-fixating bacterium Bradyrhizobium japonicum Irr acts by binding to target genes, including the iron control element (ICE), and is degraded in response to heme binding. Here, we examined this binding activity using fluorescence anisotropy with a 6-carboxyfluorescein-labeled ICE-like oligomer (FAM-ICE). In the presence of Mn2+, Irr addition increased the fluorescence anisotropy, corresponding to formation of the Irr-ICE complex. The addition of EDTA to the Irr-ICE complex reduced fluorescence anisotropy, but fluorescence was recovered after Mn2+ addition, indicating that Mn2+ binding is a prerequisite for complex formation. Binding activity toward ICE was lost upon introduction of substitutions in a His-cluster region of Irr, revealing that Mn2+ binds to this region. We observed that the His-cluster region is also the heme binding site; results from fluorescence anisotropy and electrophoretic mobility shift analyses disclosed that the addition of a half-equivalent of heme dissociates Irr from ICE, likely because of Mn2+ release due to heme binding. We hypothesized that heme binding to another heme binding site, Cys-29, would also inhibit the formation of the Irr-ICE complex because it is proximal to the ICE binding site, which was supported by the loss of ICE binding activity in a Cys-29-mutated Irr. These results indicate that Irr requires Mn2+ binding to form the Irr-ICE complex and that the addition of heme dissociates Irr from ICE by replacing Mn2+ with heme or by heme binding to Cys-29.
• Spectroscopic Characterization of Halorhodopsin Reconstituted into Nanodisks Using Native Lipids.

  Ayumi Yamamoto, Takashi Tsukamoto, Kenshiro Suzuki, Eri Hashimoto, Yoshihiro Kobashigawa, Kousuke Shibasaki, Takeshi Uchida, Fuyuhiko Inagaki, Makoto Demura, Koichiro Ishimori

  Biophysical journal 118 (11) 2853 - 2865 2020/04/29 [Refereed][Not invited]
   

  We successfully reconstituted single Natronomonas pharaonis halorhodopsin (NpHR) trimers into a nanodisk (ND) using the native archaeal lipid (NL) and an artificial lipid having a zwitterionic headgroup, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Incorporation of single trimeric NpHR into NDs was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, size-exclusion chromatography, and visible circular dichroism spectroscopy. The Cl- binding affinity of NpHR in NDs using NL (NL-ND NpHR) or POPC (POPC-ND NpHR) was examined by absorption spectroscopy, showing that the Cl--releasing affinities (Kd,N↔O) of these ND-reconstituted NpHRs are more than 10 times higher than that obtained from native NpHR membrane fragments (MFs) harvested from a NpHR-overexpressing archaeal strain (MF NpHR). The photoreaction kinetics of these ND-reconstituted NpHRs revealed that the Cl- uptake was faster than that of MF NpHR. These differences in the Cl--releasing and uptake properties of ND-reconstituted NpHRs and MF NpHR may arise from suppression of protein conformational changes associated with Cl- release from the trimeric NpHR caused by ND reconstitution, conformational perturbation in the trimeric state, and loss of the trimer-trimer interactions. On the other hand, POPC-ND NpHR demonstrated accelerated Cl- uptake compared to NL-ND NpHR, suggesting that the negative charge on the archaeal membrane surface regulates the photocycle of NpHR. Although NL-ND NpHR and MF NpHR are embedded in the same lipid, the lower Cl--binding affinity at the initial state (Kd,initial) and faster recovering from the NpHR' state to the original state of the photoreaction cycle were observed for NL-ND NpHR, probably because of insufficient interactions with a chromophore in the native membrane, bacterioruberin in reconstituted NDs. Our results indicate that specific interactions of NpHR with surrounding lipids and bacterioruberin, structural flexibility of the membrane, and interactions between trimeric NpHRs may be necessary for efficient Cl- pumping.
• Osmotic pressure effects identify dehydration upon cytochrome c-cytochrome c oxidase complex formation contributing to a specific electron pathway formation

  Wataru Sato, Seiji Hitaoka, Takeshi Uchida, Kyoko Shinzawa-Itoh, Kazunari Yoshizawa, Shinya Yoshikawa, Koichiro Ishimori

  Biochemical Journal 477 (8) 1565 - 1578 0264-6021 2020/04 [Refereed][Not invited]
   

  © 2020 The Author(s). In the electron transfer (ET) reaction from cytochrome c (Cyt c) to cytochrome c oxidase (CcO), we determined the number and sites of the hydration water released from the protein surface upon the formation of the ET complex by evaluating the osmotic pressure dependence of kinetics for the ET from Cyt c to CcO. We identified that ∼20 water molecules were dehydrated in complex formation under turnover conditions, and systematic Cyt c mutations in the interaction site for CcO revealed that nearly half of the released hydration water during the complexation were located around Ile81, one of the hydrophobic amino acid residues near the exposed heme periphery of Cyt c. Such a dehydration dominantly compensates for the entropy decrease due to the association of Cyt c with CcO, resulting in the entropy-driven ET reaction. The energetic analysis of the interprotein interactions in the ET complex predicted by the docking simulation suggested the formation of hydrophobic interaction sites surrounding the exposed heme periphery of Cyt c in the Cyt c-CcO interface (a 'molecular breakwater'). Such sites would contribute to the formation of the hydrophobic ET pathway from Cyt c to CcO by blocking water access from the bulk water phase.
• Biophysical research in Hokkaido University, Japan.

  Tomoyasu Aizawa, Makoto Demura, Kazutoshi Gohara, Hisashi Haga, Koichiro Ishimori, Masataka Kinjo, Tamiki Komatsuzaki, Katsumi Maenaka, Min Yao

  Biophysical reviews 12 (2) 233 - 236 2020/04 [Refereed][Not invited]
  
• A single mutation converts Alr5027 from cyanobacteria Nostoc sp. PCC 7120 to a heme-binding protein with heme-degrading ability

  Nobuhiko Dojun, Kazuyoshi Muranishi, Koichiro Ishimori, Takeshi Uchida

  Journal of Inorganic Biochemistry 203 110916  0162-0134 2020/02 [Refereed][Not invited]
   

  © 2019 Elsevier Inc. HutZ from Vibrio cholerae (VcHutZ) is a dimeric protein that catalyzes oxygen-dependent degradation of heme. The reaction mechanism is the same as that of canonical heme oxygenase (HO), but the structure of HutZ is quite different from that of HO. Thus, we postulate that HutZ has evolved via a different pathway from that of HO. The Alr5027 protein from cyanobacteria possessing proteins potentially related to ancestral proteins utilizing O2 in enzymatic reactions is homologous to HutZ family proteins (67% similarity), but the heme axial ligand of HutZ is not conserved in Alr5027. To investigate whether Alr5027 can bind and degrade heme, we expressed Alr5027 in Escherichia coli and purified it. Although Alr5027 did not bind heme, replacement of Lys164, corresponding to the heme axial ligand of HutZ, with histidine conferred heme-binding capability. The K164H mutant produced verdoheme in the reaction with H2O2, indicating acquisition of heme-degradation ability. Among the mutants, the K164H mutant produced verdoheme most efficiently. Although the K164H mutant did not degrade heme through ascorbic acid, biliverdin, the final product of VcHutZ, was formed by treatment of verdoheme with ascorbic acid. An analysis of Trp103 fluorescence indicated elongation of the distance between protomers in this mutant compared with VcHutZ—the probable cause of the inefficiency of ascorbic acid-supported heme-degradation activity. Collectively, our findings indicate that a single lysine-to-histidine mutation converted Alr5027 to a heme-binding protein that can form verdoheme through H2O2, suggesting that HutZ family proteins have acquired the heme-degradation function through molecular evolution from an ancestor protein of Alr5027.
• Accelerating structural life science by paramagnetic lanthanide probe methods

  Tomohide Saio, Koichiro Ishimori

  Biochimica et Biophysica Acta - General Subjects 1864 (2) 0304-4165 2020/02 [Refereed][Not invited]
   

  © 2019 Elsevier B.V. We describe the recent progress of structural analysis methods exploiting paramagnetic lanthanide ions. In NMR spectroscopy, the paramagnetic effects induced by the trivalent lanthanide ions provide long-range (~40 Å) distance and angular information that can be exploited in protein structure determination, ligand screening, structure-based resonance assignment, and in-cell observation. The paramagnetic lanthanide ions can also be utilized in EPR spectroscopy, providing nanometer-scale distance measurement. These applications of the paramagnetic lanthanide probe are becoming more widespread by the use of a variety of lanthanide binding tags. Here, we introduce the basics of paramagnetic effects, several examples of lanthanide tags, and recent applications of paramagnetic lanthanide ions in NMR and EPR spectroscopy. Collectively, we show how the paramagnetic lanthanide probe accelerates research in protein science and drug design, and consequently life science.
• Integrated bio-metal science: New frontiers of bio-metal science opened with cutting-edge techniques

  Hitomi Sawai, Koichiro Ishimori

  Biophysics and Physicobiology 17 (0) 94 - 97 2020
• Volume profile of protein determined by pressure effects

  Koichiro Ishimori

  Review of High Pressure Science and Technology/Koatsuryoku No Kagaku To Gijutsu 30 (1) 4 - 11 0917-639X 2020 [Refereed][Not invited]
   

  © 2020 Global Research Online. All rights reserved. Pressure is one of the fundamental parameters in thermodynamics, which can perturb physical properties of proteins as well as temperature. Here, I focused on molecular volume, partial molar volume, of proteins which de-pends on the hydration structure on the protein surface. Based on the thermodynamic relations, molecular volume can be estimated from the pressure, both of hydrostatic and osmotic pressure, dependence of equilibri-um constants. In the protein folding of cytochrome c, the molecular volume change revealed that dehydration of hydrophobic heme group promotes the protein folding by increasing entropy. The dehydration of hydrophobic sites also facilitates the electron transfer from cytochrome c to cytochrome oxidase in the respiratory chain, which forms the ``molecular breakwater'' for the effective electron transfer pathway. By using pressure, we can get new insights into physical properties of proteins that can never be gained by other methodologies.
• Quantitative description and classification of protein structures by a novel robust amino acid network: interaction selective network (ISN)

  Shohei Konno, Takao Namiki, Koichiro Ishimori

  Scientific Reports 9 (1) 16654  2019/12/01 [Refereed][Not invited]
   

  © 2019, The Author(s). To quantitatively categorize protein structures, we developed a quantitative coarse-grained model of protein structures with a novel amino acid network, the interaction selective network (ISN), characterized by the links based on interactions in both the main and side chains. We found that the ISN is a novel robust network model to show the higher classification probability in the plots of average vertex degree (k) versus average clustering coefficient (C), both of which are typical network parameters for protein structures, and successfully distinguished between “all-α” and “all-β” proteins. On the other hand, one of the typical conventional networks, the α-carbon network (CAN), was found to be less robust than the ISN, and another typical network, atomic distance network (ADN), failed to distinguish between these two protein structures. Considering that the links in the CAN and ADN are defined by the interactions only between the main chain atoms and by the distance of the closest atom pair between the two amino acid residues, respectively, we can conclude that reflecting structural information from both secondary and tertiary structures in the network parameters improves the quantitative evaluation and robustness in network models, resulting in a quantitative and more robust description of three-dimensional protein structures in the ISN.
• Role of conserved arginine in the heme distal site of HutZ from Vibrio cholerae in the heme degradation reaction.

  Takeshi Uchida, Nobuhiko Dojun, Kazuki Ota, Yukari Sekine, Yuina Nakamura, Sayaka Umetsu, Koichiro Ishimori

  Archives of biochemistry and biophysics 677 108165 - 108165 0003-9861 2019/11/30 [Refereed][Not invited]
   

  HutZ from Vibrio cholerae is a dimeric enzyme that catalyzes degradation of heme. The highly conserved Arg92 residue in the HutZ family is proposed to interact with an iron-bound water molecule in the distal heme pocket. To clarify the specific role of Arg92 in the heme degradation reaction, the residue was substituted with alanine, leucine, histidine or lysine to modulate electrostatic interactions with iron-bound ligand. All four Arg92 mutants reacted with hydrogen peroxide to form verdoheme, a prominent intermediate in the heme degradation process. However, when ascorbic acid was used as an electron source, iron was not released even at pH 6.0 despite a decrease in the Soret band, indicating that non-enzymatic heme degradation occurred. Comparison of the rates of heme reduction, ligand binding and verdoheme formation suggested that proton transfer to the reduced oxyferrous heme, a potential rate-limiting step of heme degradation in HutZ, is hampered by mutation. In our previous study, we found that the increase in the distance between heme and Trp109 from 16 to 18 Å upon lowering the pH from 8.0 to 6.0 leads to activation of ascorbic acid-assisted heme degradation by HutZ. The distance in Arg92 mutants was >19 Å at pH 6.0, suggesting that subunit-subunit interactions at this pH are not suitable for heme degradation, similar to Asp132 and His63 mutants. These results suggest that interactions of Arg92 with heme-bound ligand induce alterations in the distance between subunits, which plays a key role in controlling the heme degradation activity of HutZ.
• Structural basis for the heme transfer reaction in heme uptake machinery from Corynebacteria.

  Norifumi Muraki, Chihiro Kitatsuji, Yasunori Okamoto, Takeshi Uchida, Koichiro Ishimori, Shigetoshi Aono

  Chemical communications (Cambridge, England) 55 (92) 13864 - 13867 1359-7345 2019/11/14 [Refereed][Not invited]
   

  The crystal structures of the conserved region domains of HtaA and HtaB, which act as heme binding/transport proteins in the heme uptake machinery in Corynebacterium glutamicum, are determined for the first time. The molecular mechanism of heme transfer among these proteins is proposed based on the spectroscopic and structural analyses.
• Complete Genome Sequence of Staphylococcus arlettae Strain P2, Isolated from a Laboratory Environment.

  Yu H, Taniguchi M, Uesaka K, Wiseschart A, Pootanakit K, Nishitani Y, Murakami Y, Ishimori K, Miyazaki K, Kitahara K

  Microbiology resource announcements 8 (45) 2019/11 [Refereed][Not invited]
   

  Staphylococcus arlettae is one coagulase-negative species in the bacterial genus Staphylococcus. Here, we describe the closed complete genome sequence of S. arlettae strain P2, which was obtained using a hybrid approach combining Oxford Nanopore long-read and Illumina MiSeq short-read sequencing data.
• Toxic PR poly-dipeptides encoded by the C9orf72 repeat expansion target Kapβ2 and dysregulate phase separation of low-complexity domains

  Hitoki Nanaura, Honoka Kawamukai, Ayano Fujiwara, Takeru Uehara, Mari Nakanishi, Tomo Shiota, Masaki Hibino, Yuichiro Aiba, Pattama Wiriyasermkul, Sotaro Kikuchi, Riko Nagata, Masaya Matsubayashi, Shushi Nagamori, Osami Shoji, Koichiro Ishimori, Hiroyoshi Matsumura, Kazuma Sugie, Tomohide Saio, Takuya Yoshizawa, Eiichiro Mori

  2019/10/21 

  Low-complexity (LC) domains of proteins are found in about one fifth of human proteome, and a group of LC-domains form labile cross-β polymers and liquid-like droplets. Polymers and droplets formed from LC-domains are dynamically regulated by posttranslational modifications and molecular chaperones including nuclear transport receptors. Repeat expansion in the first intron of a gene designated C9orf72, which is the most prevalent form of familial amyotrophic lateral sclerosis (ALS), causes nucleocytoplasmic transport deficit, however, the detailed mechanism remains unsolved. Here we show that the proline:arginine (PR) poly-dipeptides encoded by the C9orf72 repeat expansion bound nuclear transport receptor Kapβ2 through its nuclear localization signal (NLS) recognition motif, and inhibited the ability of Kapβ2 to melt fused in sarcoma (FUS) droplets by competing interaction with FUS. The findings in this study offer mechanistic insights as to how the C9orf72 repeat expansion disables nucleocytoplasmic transport and causes neurodegenerative diseases.
• Role of His63 in HutZ from Vibrio cholerae in the heme degradation reaction and heme binding.

  Takeshi Uchida, Nobuhiko Dojun, Yukari Sekine, Koichiro Ishimori

  Dalton transactions (Cambridge, England : 2003) 48 (16) 5408 - 5416 2019/04/16 [Refereed][Not invited]
   

  HutZ from Vibrio cholerae is a dimeric enzyme that catalyzes oxygen-dependent degradation of heme via a similar catalytic mechanism to mammalian heme oxygenase. However, HutZ oxidizes the β- or δ-meso position of heme at a ∼1 : 1 ratio distinct from heme oxygenase, which initiates the degradation of heme solely at the α-meso position. His63 is a residue that potentially forms hydrogen bond with the heme 7-propionate group. To establish the role of His63 in regioselectivity of heme degradation by HutZ and heme binding, we constructed mutants of His63. Interestingly, the H63L mutant retained a comparable level of β- or δ-regioselectivity as wild-type HutZ. Ascorbic acid-assisted heme degradation by HutZ is pH-dependent, showing activity at pH 6.0 but not above pH 8.0. Compared to the wild-type protein, the H63L mutant was inactive, even at pH 6.0, and affinity for heme was significantly decreased in contrast with a comparable heme binding affinity at pH 8.0, as observed for the mutant of Asp132 to Val, which is located within hydrogen bonding distance of the heme axial ligand His170, but in a different protomer. In addition, the distance between heme and Trp109 increased from 16-18 Å for wild-type HutZ to 24-28 Å for the H63L mutant, indicating that protomer orientation is altered by the mutation, since Trp109 is in another subunit of the heme axial ligand. Our results collectively suggest that His63 positioned near heme does not contribute to regioselectivity of heme degradation but plays a key role in maintaining the orientation of subunits for HutZ to function of heme degradation.
• Subunit-subunit interactions play a key role in the heme-degradation reaction of HutZ from Vibrio cholerae.

  Takeshi Uchida, Kazuki Ota, Yukari Sekine, Nobuhiko Dojun, Koichiro Ishimori

  Dalton transactions (Cambridge, England : 2003) 48 (12) 3973 - 3983 2019/03/19 [Refereed][Not invited]
   

  HutZ, a dimeric protein, from Vibrio cholerae is a protein that catalyzes the oxygen-dependent degradation of heme. Interestingly, the ascorbic acid-supported heme-degradation activity of HutZ depends on pH: less than 10% of heme is degraded by HutZ at pH 8.0, but nearly 90% of heme is degraded at pH 6.0. We examined here pH-dependent conformational changes in HutZ using fluorescence spectroscopy. Trp109 is estimated to be located approximately 21 Å from heme and is present in a different subunit containing a heme axial ligand. Thus, we postulated that the distance between heme and Trp109 reflects subunit-subunit orientational changes. On the basis of resonance energy transfer from Trp109 to heme, we estimated the distance between heme and Trp109 to be approximately 17 Å at pH 8.0, while the distance increased by less than 2 Å at pH 6.0. We presumed that such changes led to a decrease in electron donation from the proximal histidine, resulting in enhancement of the heme-degradation activity. To confirm this scenario, we mutated Ala31, located at the dimer interface, to valine to alter the distance through the subunit-subunit interaction. The distance between heme and Trp109 for the A31V mutant was elongated to 24-27 Å. Although resonance Raman spectra and reduction rate of heme suggested that this mutation resulted in diminished electron donation from the heme axial ligand, ascorbic acid-supported heme-degradation activity was not observed. Based on our findings, it can be proposed that the relative positioning of two protomers is important in determining the heme degradation rate by HutZ.
• Specific heme binding to heme regulatory motifs in iron regulatory proteins and its functional significance

  Nishitani Y, Okutani H, Takeda Y, Uchida T, Iwai K, Ishimori K

  Journal of Inorganic Biochemistry 198 110726 - 110726 0162-0134 2019 [Refereed][Not invited]
  
• Uncovering dehydration in cytochrome c refolding from urea- and guanidine hydrochloride-denatured unfolded state by high pressure spectroscopy

  Shohei Konno, Kentaro Doi, Koichiro Ishimori

  Biophysics and Physicobiology Biophysical Society of Japan 16 (0) 18  2189-4779 2019 [Refereed][Not invited]
  
• Structural insight into proline cis/trans isomerization of unfolded proteins catalyzed by the Trigger Factor chaperone.

  Kawagoe S, Nakagawa H, Kumeta H, Ishimori K, Saio T

  The Journal of Biological Chemistry 293 (39) 15095 - 15106 2018/08 [Refereed][Not invited]
   

  Molecular chaperones often possess functional modules that are specialized in assisting the formation of specific structural elements, such as a disulfide bridges and peptidyl-prolyl bonds in cis form, in the client protein. A ribosome-associated molecular chaperone trigger factor (TF), which has a peptidyl-prolyl cis/trans isomerase (PPIase) domain, acts as a highly efficient catalyst in the folding process limited by peptidyl-prolyl isomerization. Herein we report a study on the mechanism through which TF recognizes the proline residue in the unfolded client protein during the cis/trans isomerization process. The solution structure of TF in complex with the client protein showed that TF recognizes the proline-aromatic motif located in the hydrophobic stretch of the unfolded client protein through its conserved hydrophobic cleft, which suggests that TF preferentially accelerates the isomerization of the peptidyl-prolyl bond that is eventually folded into the core of the protein in its native fold. Molecular dynamics simulation revealed that TF exploits the backbone amide group of Ile195 to form an intermolecular hydrogen bond with the carbonyl oxygen of the amino acid residue preceding the proline residue at the transition state, which presumably stabilizes the transition state and thus accelerates the isomerization. The importance of such intermolecular hydrogen-bond formation during the catalysis was further corroborated by the activity assay and NMR relaxation analysis.
• Dehydration of hydrophobic residues near the heme center in cytochrome c promotes electron transfer to cytochrome c oxidase

  Wataru Sato, Takeshi Uchida, Tomohide Saio, Koichiro Ishimori

  Biochimica et Biophysica Acta - General Subjects 1862 (6) 1339 - 1349 2018/06 [Refereed][Not invited]
  
• Polyethylene glycol promotes autoxidation of cytochrome c

  Wataru Sato, Takeshi Uchida, Tomohide Saio, Koichiro Ishimori

  Biochimica et Biophysica Acta - General Subjects 1862 (6) 1339 - 1349 1872-8006 2018/06/01 [Refereed][Not invited]
   

  Cytochrome c (Cyt c) was rapidly oxidized by molecular oxygen in the presence, but not absence of PEG. The redox potential of heme c was determined by the potentiometric titration to be +236 ± 3 mV in the absence of PEG, which was negatively shifted to +200 ± 4 mV in the presence of PEG. The underlying the rapid oxidation was explored by examining the structural changes in Cyt c in the presence of PEG using UV–visible absorption, circular dichroism, resonance Raman, and fluorescence spectroscopies. These spectroscopic analyses suggested that heme oxidation was induced by a modest tertiary structural change accompanied by a slight shift in the heme position (< 1.0 Å) rather than by partial denaturation, as is observed in the presence of cardiolipin. The near-infrared spectra showed that PEG induced dehydration from Cyt c, which triggered heme displacement. The primary dehydration site was estimated to be around surface-exposed hydrophobic residues near the heme center: Ile81 and Val83. These findings and our previous studies, which showed that hydrated water molecules around Ile81 and Val83 are expelled when Cyt c forms a complex with CcO, proposed that dehydration of these residues is functionally significant to electron transfer from Cyt c to CcO.
• Oligomerization of a molecular chaperone modulates its activity

  Tomohide Saio, Soichiro Kawagoe, Koichiro Ishimori, Charalampos G Kalodimos

  eLife {eLife} Sciences Organisation, Ltd. 7 2050-084X 2018/05 [Refereed][Not invited]
   

  Molecular chaperones alter the folding properties of cellular proteins via mechanisms that are not well understood. Here, we show that Trigger Factor (TF), an ATP-independent chaperone, exerts strikingly contrasting effects on the folding of non-native proteins as it transitions between a monomeric and a dimeric state. We used NMR spectroscopy to determine the atomic resolution structure of the 100 kDa dimeric TF. The structural data show that some of the substrate-binding sites are buried in the dimeric interface, explaining the lower affinity for protein substrates of the dimeric compared to the monomeric TF. Surprisingly, the dimeric TF associates faster with proteins and it exhibits stronger anti-aggregation and holdase activity than the monomeric TF. The structural data show that the dimer assembles in a way that substrate-binding sites in the two subunits form a large contiguous surface inside a cavity, thus accounting for the observed accelerated association with unfolded proteins. Our results demonstrate how the activity of a chaperone can be modulated to provide distinct functional outcomes in the cell.
• Redox-dependent axial ligand replacement and its functional significance in heme-bound iron regulatory proteins

  Mariko Ogura, Ryosuke Endo, Haruto Ishikawa, Yukiko Takeda, Takeshi Uchida, Kazuhiro Iwai, Kazuo Kobayashi, Koichiro Ishimori

  Journal of Inorganic Biochemistry 182 238 - 248 1873-3344 2018/05/01 [Refereed][Not invited]
   

  Iron regulatory proteins (IRPs), regulators of iron metabolism in mammalian cells, control the translation of proteins involved in iron uptake, storage and utilization by binding to specific iron-responsive element (IRE) sequences of mRNAs. Two homologs of IRPs (IRP1 and IRP2) have a typical heme regulatory motif (HRM), a consensus sequence found in “heme-regulated proteins”. However, specific heme binding to HRM has been reported only for IRP2, which is essential for oxidative modification and loss of binding to target mRNAs. In this paper, we confirmed that IRP1 also specifically binds two molar equivalents of heme, and found that the absorption and resonance Raman spectra of heme-bound IRP1 were quite similar to those of heme-bound IRP2. This shows that the heme environmental structures in IRP1 are close to those of proteins using heme as a regulatory molecule. Pulse radiolysis experiments, however, clearly revealed an axial ligand exchange from Cys to His immediately after the reduction of the heme iron to form a 5-coordinate His-ligated heme in heme-bound IRP2, whereas the 5-coordinate His-ligated heme was not observed after the reduction of heme-bound IRP1. Considering that the oxidative modification is only observed in heme-bound IRP2, but not IRP1, probably owing to the structural flexibility of IRP2, we propose that the transient 5-coordinate His-ligated heme is a prerequisite for oxidative modification of heme-bound IRP2, which functionally differentiates heme binding of IRP2 from that of IRP1.
• Heme Binding to Porphobilinogen Deaminase from Vibrio cholerae Decelerates the Formation of 1-Hydroxymethylbilane

  Takeshi Uchida, Takumi Funamizu, Minghao Chen, Yoshikazu Tanaka, Koichiro Ishimori

  ACS Chemical Biology 13 (3) 750 - 760 1554-8937 2018/03/16 [Refereed][Not invited]
   

  Porphobilinogen deaminase (PBGD) is an enzyme that catalyzes the formation of hydroxymethylbilane, a tetrapyrrole intermediate, during heme biosynthesis through the stepwise polymerization of four molecules of porphobilinogen. PBGD from Vibrio cholerae was expressed in Escherichia coli and characterized in this study. Unexpectedly, spectroscopic measurements revealed that PBGD bound one equivalent of heme with a dissociation constant of 0.33 ± 0.01 μM. The absorption and resonance Raman spectra suggested that heme is a mixture of the 5-coordinate and 6-coordinate hemes. Mutational studies indicated that the 5-coordinate heme possessed Cys105 as a heme axial ligand, and His227 was coordinated to form the 6-coordinate heme. Upon heme binding, the deamination activity decreased by approximately 15%. The crystal structure of PBGD revealed that His227 was located near Cys105, but the side chain of His227 did not point toward Cys105. The addition of the cyanide ion to heme-PBGD abolished the effect of heme binding on the enzymatic activity. Therefore, coordination of His227 to heme appeared to induce reorientation of the domains containing Cys105, leading to a decrease in the enzymatic activity. This is the first report indicating that the PBGD activity is controlled by heme, the final product of heme biosynthesis. This finding improves our understanding of the mechanism by which heme biosynthesis is regulated.
• Heme Iron Coordination Structure of Heme Transport Protein HutB from Vibrio Cholerae

  Takeshi Uchida, Takumi Funamizu, Mariko Ogura, Koichiro Ishimori

  BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN 90 (8) 924 - 930 0009-2673 2017/08 [Refereed][Not invited]
   

  HutB is a putative heme transport protein located in the periplasmic space in Vibrio cholerae. Here, we purified HutB and characterized its heme binding properties. An analysis of the Soret band showed that there are two types of heme binding geometries depending on the heme concentration: 404-nm species are dominant at lower concentrations of heme, and 394nm species dominate at higher concentrations. Moreover, a mutational study revealed that either Tyr65 or Tyr198 binds heme with the help of histidine, a property shared with another V. cholerae heme transport protein, HutX, despite the absence of sequence similarity, indicating that HutB acts as a heme transport protein in the periplasm.
• HmuS from Yersinia pseudotuberculosis is a non-canonical heme-degrading enzyme to acquire iron from heme.

  Masato Onzuka, Yukari Sekine, Takeshi Uchida, Koichiro Ishimori, Shin-Ichi Ozaki

  Biochimica et biophysica acta. General subjects 1861 (7) 1870 - 1878 2017/07 [Refereed][Not invited]
   

  Some Gram-negative pathogens import host heme into the cytoplasm and utilize it as an iron source for their survival. We report here that HmuS, encoded by the heme utilizing system (hmu) locus, cleaves the protoporphyrin ring to release iron from heme. A liquid chromatography/mass spectrometry analysis revealed that the degradation products of this reaction are two biliverdin isomers that result from transformation of a verdoheme intermediate. This oxidative heme degradation by HmuS required molecular oxygen and electrons supplied by either ascorbate or NADPH. Electrons could not be directly transferred from NADPH to heme; instead, ferredoxin-NADP+ reductase (FNR) functioned as a mediator. Although HmuS does not share amino acid sequence homology with heme oxygenase (HO), a well-known heme-degrading enzyme, absorption and resonance Raman spectral analyses suggest that the heme iron is coordinated with an axial histidine residue and a water molecule in both enzymes. The substitution of axial His196 or distal Arg102 with an alanine residue in HmuS almost completely eliminated heme-degradation activity, suggesting that Fe-His coordination and interaction of a distal residue with water molecules in the heme pocket are important for this activity.
• Reaction intermediates in the heme degradation reaction by HutZ from Vibrio cholerae.

  Takeshi Uchida, Yukari Sekine, Nobuhiko Dojun, Ariel Lewis-Ballester, Izumi Ishigami, Toshitaka Matsui, Syun-Ru Yeh, Koichiro Ishimori

  Dalton transactions (Cambridge, England : 2003) 46 (25) 8104 - 8109 2017/06/27 [Refereed][Not invited]
   

  HutZ is a heme-degrading enzyme in Vibrio cholerae. It converts heme to biliverdin via verdoheme, suggesting that it follows the same reaction mechanism as that of mammalian heme oxygenase. However, none of the key intermediates have been identified. In this study, we applied steady-state and time-resolved UV-vis absorption and resonance Raman spectroscopy to study the reaction of the heme-HutZ complex with H2O2 or ascorbic acid. We characterized three intermediates: oxyferrous heme, meso-hydroxyheme, and verdoheme complexes. Our data support the view that HutZ degrades heme in a manner similar to mammalian heme oxygenase, despite their low sequence and structural homology.
• Heme Proximal Hydrogen Bonding between His170 and Asp132 Plays an Essential Role in the Heme Degradation Reaction of HutZ from Vibrio cholerae.

  Takeshi Uchida, Nobuhiko Dojun, Yukari Sekine, Koichiro Ishimori

  Biochemistry 56 (21) 2723 - 2734 2017/05/30 [Refereed][Not invited]
   

  HutZ from Vibrio cholerae is an enzyme that catalyzes the oxygen-dependent degradation of heme. The crystal structure of the homologous protein from Helicobacter pylori, HugZ, predicts that Asp132 in HutZ is located within hydrogen-bonding distance of the heme axial ligand His170. Hydrogen bonding between His170 and Asp132 appears to be disfavored in heme-degrading enzymes, because it can contribute to the imidazolate character of the axial histidine, as observed in most heme-containing peroxidases. Thus, we investigated the role of this potential hydrogen bond in the heme degradation reaction by mutating Asp132 to Leu, Asn, or Glu and by mutating His170 to Ala. Heme degradation activity was almost completely lost in D132L and D132N mutants, whereas verdoheme formation through reaction with H2O2 was comparable in the D132E mutant and wild-type enzyme. However, even at pH 6.0, when the heme is in a high-spin state, the D132E mutant was inactive toward ascorbic acid because of a significant reduction in its affinity (Kd) for heme (4.1 μM) compared with that at pH 8.0 (0.027 μM). The heme degradation activity of the H170A mutant was also substantially reduced, although this mutant bound heme with a Kd of 0.067 μM, despite the absence of an axial ligand. Thus, this study showed that proximal hydrogen bonding between Asp132 and His170 plays a role in retaining the heme in an appropriate position for oxygen-dependent heme degradation.
• The Iron Chaperone Protein CyaY from Vibrio cholerae Is a Heme-Binding Protein

  Takeshi Uchida, Noriyuki Kobayashi, Souichiro Muneta, Koichiro Ishimori

  BIOCHEMISTRY 56 (18) 2425 - 2434 0006-2960 2017/05 [Refereed][Not invited]
   

  CyaY is an iron transport protein for iron-sulfur (Fe-S) cluster biosynthetic systems. It also transports iron to ferrochelatase that catalyzes insertion of Fe2+ into protoporphyrin IX. Here, we find that CyaY has the ability to bind heme as well as iron, exhibiting an apparent dissociation constant for heme of 21 +/- 6 nM. Absorption and resonance Raman spectra revealed that both ferric and ferrous forms of heme were bound to an anionic ligand (e.g., tyrosine and/or cysteine). Consistent with this, mutagenesis studies showed that Tyr67 and Cys78 are possible heme ligands of CyaY. The binding of heme to CyaY increased the apparent dissociation constant of CyaY for iron from 65.2 to 87.9 mu M. Circular dichroism spectra of CyaY suggested that binding of heme to CyaY induces rearrangement of aromatic residues. Furthermore, size-exclusion column chromatography demonstrated heme-mediated oligomerization of CyaY. These results suggest that heme binding induces conformational changes, including oligomerization of CyaY, that result in a decrease in the affinity of CyaY for iron. Accordingly, the presence of excess heme in cells would lead to modulation of Fe-S cluster or heme biosynthesis. This report provides the first description of heme dependence of iron transport by CyaY.
• Iron chelators inhibit the heme-degradation reaction by HutZ from Vibrio cholerae.

  Nobuhiko Dojun, Yukari Sekine, Koichiro Ishimori, Takeshi Uchida

  Dalton transactions (Cambridge, England : 2003) 46 (16) 5147 - 5150 2017/04/19 [Refereed][Not invited]
   

  HutZ is a heme-degrading enzyme. We found that the heme-degradation reaction by HutZ is inhibited by the iron chelators. Kinetic analysis of each heme-degradation step suggests that water molecules hydrogen bonded to Thr27 are involved in proton transfer to Fe(iii)-OO-, and that this step is inhibited by iron chelators.
• Dual role of the active-center cysteine in human peroxiredoxin 1: Peroxidase activity and heme binding

  Yuta Watanabe, Koichiro Ishimori, Takeshi Uchida

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 483 (3) 930 - 935 0006-291X 2017/02 [Refereed][Not invited]
   

  HBP23, a 23-kDa heme-binding protein identified in rats, is a member of the peroxiredoxin (Prx) family, the primary peroxidases involved in hydrogen peroxide catabolism. Although HBP23 has a characteristic Cys-Pro heme-binding motif, the significance of heme binding to Prx family proteins remains to be elucidated. Here, we examined the effect of heme binding to human peroxiredoxin-1 (PRX1), which has 97% amino acid identity to HBP23. PRX1 was expressed in Escherichia coli and purified to homogeneity. Spectroscopic titration demonstrated that PRX1 binds heme with a 1:1 stoichiometry and a dissociation constant of 0.17 mu M. UV-vis spectra of heme-PRX1 suggested that Cys52 is the axial ligand of ferric heme. PRX1 peroxidase activity was lost upon heme binding, reflecting the fact that Cys52 is not only the heme-binding site but also the active center of peroxidase activity. Interestingly, heme binding to PRX1 caused a decrease in the toxicity and degradation of heme, significantly suppressing H2O2-dependent heme peroxidase activity and degradation of PRX1-bound heme compared with that of free hemin. By virtue of its cytosolic abundance (similar to 20 mu M), PRX1 thus functions as a scavenger of cytosolic hemin (<1 mu M). Collectively, our results indicate that PRX1 has a dual role; Cys-dependent peroxidase activity and cytosolic heme scavenger. (C) 2017 Elsevier Inc. All rights reserved.
• Energetic basis on interactions between ferredoxin and ferredoxin NADP(+) reductase at varying physiological conditions

  Misaki Kinoshita, Ju Yaen Kim, Satoshi Kume, Yuxi Lin, K. Hun Mok, Yosky Kataoka, Koichiro Ishimori, Natalia Markova, Genji Kurisu, Toshiharu Hase, Young-Ho Lee

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 482 (4) 909 - 915 0006-291X 2017/01 [Refereed][Not invited]
   

  In spite of a number of studies to characterize ferredoxin (Fd):ferredoxin NADP(+) reductase (FNR) interactions at limited conditions, detailed energetic investigation on how these proteins interact under near physiological conditions and its linkage to FNR activity are still lacking. We herein performed systematic Fd:FNR binding thermodynamics using isothermal titration calorimetry (ITC) at distinct pH (6.0 and 8.0), NaCl concentrations (0-200 mM), and temperatures (19-28 degrees C) for mimicking physiological conditions in chloroplasts. Energetically unfavorable endothermic enthalpy changes were accompanied by Fd:FNR complexation at all conditions. This energetic cost was compensated by favorable entropy changes, balanced by conformational and hydrational entropy. Increases in the NaCl concentration and pH weakened inter protein affinity due to the less contribution of favorable entropy change regardless of energetic gains from enthalpy changes, suggesting that entropy drove complexation and modulated affinity. Effects of temperature on binding thermodynamics were much smaller than those of pH and NaCl. NaCl concentration and pH-dependeht enthalpy and heat capacity changes provided clues for distinct binding modes. Moreover, decreases in the enthalpy level in the Hammond's postulate-based energy landscape implicated kinetic advantages for FNR activity. All these energetic interplays were comprehensively demonstrated by the driving force plot with the enthalpy-entropy compensation which may serve as an energetic buffer against outer stresses. We propose that high affinity at pH 6.0 may be beneficial for protection from proteolysis of Fd and FNR in rest states, and moderate affinity at pH 8.0 and proper NaCl concentrations with smaller endothermic enthalpy changes may contribute to increase FNR activity. (C) 2016 Elsevier Inc. All rights reserved.
• Redox-Dependent Dynamics in Heme-Bound Bacterial Iron Response Regulator (Irr) Protein

  Kazuo Kobayashi, Megumi Nakagaki, Haruto Ishikawa, Kazuhiro Iwai, Mark R. O'Brian, Koichiro Ishimori

  BIOCHEMISTRY 55 (29) 4047 - 4054 0006-2960 2016/07 [Refereed][Not invited]
   

  The iron response regulator (Irr) protein from Bradyrhizobium japonicum mediates iron-dependent regulation of heme biosynthesis. In degrades in response to heme availability through a process that involves the binding of heme to Cys-29 in the heme regulatory motif (HRM) in the presence of molecular oxygen. In this work, we assessed the dynamics of one-electron reduction of heme-bound Irr by monitoring the formation of transient intermediates by pulse radiolysis. Hydrated electrons generated by pulse radiolysis reduced heme iron-bound Irr, facilitating the binding of molecular oxygen to the heme iron in Irr through an initial intermediate with an absorption maximum at 420 nm. This initial intermediate was converted to a secondary intermediate with an absorption maximum at 425 nm, with a first-order rate constant of 1.0 x 10(4) s(-1). The Cys-29 -> Ala (C29A) mutant of Irr, on the other hand, did not undergo the secondary phase, implying that ligand exchange of Cys-29 for another ligand takes place during the process. Spectral changes during the reduction of the heme-bound Irr revealed that binding of CO to ferrous heme consisted of two phases with k(on) values of 1.3 x 10(5) and 2.5 x 10(4) M-1 s(-1), a finding consistent with the presence of two distinct hemes in Irr. In aerobic solutions, by contrast, oxidation of the ferrous heme to the ferric form was found to be a two-phase process. The C29A mutant was similarly oxidized, but this occurred as a single-phase process. We speculate that a reactive oxygen species essential for degradation of the protein is generated during the oxidation process.
• Energetic Mechanism of Cytochrome c-Cytochrome c Oxidase Electron Transfer Complex Formation under Turnover Conditions Revealed by Mutational Effects and Docking Simulation

  Wataru Sato, Seiji Hitaoka, Kaoru Inoue, Mizue Imai, Tomohide Saio, Takeshi Uchida, Kyoko Shinzawa-Itoh, Shinya Yoshikawa, Kazunari Yoshizawa, Koichiro Ishimori

  JOURNAL OF BIOLOGICAL CHEMISTRY 291 (29) 15320 - 15331 0021-9258 2016/07 [Refereed][Not invited]
   

  Based on the mutational effects on the steady-state kinetics of the electron transfer reaction and our NMR analysis of the interaction site (Sakamoto, K., Kamiya, M., Imai, M., Shinzawa-Itoh, K., Uchida, T., Kawano, K., Yoshikawa, S., and Ishimori, K. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 1227112276), we determined the structure of the electron transfer complex between cytochrome c (Cyt c) and cytochrome c oxidase (CcO) under turnover conditions and energetically characterized the interactions essential for complex formation. The complex structures predicted by the protein docking simulation were computationally selected and validated by the experimental kinetic data for mutant Cyt c in the electron transfer reaction to CcO. The interaction analysis using the selected Cyt c-CcO complex structure revealed the electrostatic and hydrophobic contributions of each amino acid residue to the free energy required for complex formation. Several charged residues showed large unfavorable (desolvation) electrostatic interactions that were almost cancelled out by large favorable (Columbic) electrostatic interactions but resulted in the destabilization of the complex. The residual destabilizing free energy is compensated by the van der Waals interactions mediated by hydrophobic amino acid residues to give the stabilized complex. Thus, hydrophobic interactions are the primary factors that promote complex formation between Cyt c and CcO under turnover conditions, whereas the change in the electrostatic destabilization free energy provides the variance of the binding free energy in the mutants. The distribution of favorable and unfavorable electrostatic interactions in the interaction site determines the orientation of the binding of Cyt c on CcO.
• Structural Characterization of Heme Environmental Mutants of CgHmuT that Shuttles Heme Molecules to Heme Transporters.

  Norifumi Muraki, Chihiro Kitatsuji, Mariko Ogura, Takeshi Uchida, Koichiro Ishimori, Shigetoshi Aono

  International journal of molecular sciences 17 (6) 2016/05/27 [Refereed][Not invited]
   

  Corynebacteria contain a heme uptake system encoded in hmuTUV genes, in which HmuT protein acts as a heme binding protein to transport heme to the cognate transporter HmuUV. The crystal structure of HmuT from Corynebacterium glutamicum (CgHmuT) reveals that heme is accommodated in the central cleft with His141 and Tyr240 as the axial ligands and that Tyr240 forms a hydrogen bond with Arg242. In this work, the crystal structures of H141A, Y240A, and R242A mutants were determined to understand the role of these residues for the heme binding of CgHmuT. Overall and heme environmental structures of these mutants were similar to those of the wild type, suggesting that there is little conformational change in the heme-binding cleft during heme transport reaction with binding and the dissociation of heme. A loss of one axial ligand or the hydrogen bonding interaction with Tyr240 resulted in an increase in the redox potential of the heme for CgHmuT to be reduced by dithionite, though the wild type was not reduced under physiological conditions. These results suggest that the heme environmental structure stabilizes the ferric heme binding in CgHmuT, which will be responsible for efficient heme uptake under aerobic conditions where Corynebacteria grow.
• Structural Characterization of Heme Environmental Mutants of CgHmuT that Shuttles Heme Molecules to Heme Transporters.

  Norifumi Muraki, Chihiro Kitatsuji, Mariko Ogura, Takeshi Uchida, Koichiro Ishimori, Shigetoshi Aono

  International journal of molecular sciences 17 (6) 2016/05/27 [Refereed][Not invited]
   

  Corynebacteria contain a heme uptake system encoded in hmuTUV genes, in which HmuT protein acts as a heme binding protein to transport heme to the cognate transporter HmuUV. The crystal structure of HmuT from Corynebacterium glutamicum (CgHmuT) reveals that heme is accommodated in the central cleft with His141 and Tyr240 as the axial ligands and that Tyr240 forms a hydrogen bond with Arg242. In this work, the crystal structures of H141A, Y240A, and R242A mutants were determined to understand the role of these residues for the heme binding of CgHmuT. Overall and heme environmental structures of these mutants were similar to those of the wild type, suggesting that there is little conformational change in the heme-binding cleft during heme transport reaction with binding and the dissociation of heme. A loss of one axial ligand or the hydrogen bonding interaction with Tyr240 resulted in an increase in the redox potential of the heme for CgHmuT to be reduced by dithionite, though the wild type was not reduced under physiological conditions. These results suggest that the heme environmental structure stabilizes the ferric heme binding in CgHmuT, which will be responsible for efficient heme uptake under aerobic conditions where Corynebacteria grow.
• Haem-dependent dimerization of PGRMC1/Sigma-2 receptor facilitates cancer proliferation and chemoresistance

  Yasuaki Kabe, Takanori Nakane, Ikko Koike, Tatsuya Yamamoto, Yuki Sugiura, Erisa Harada, Kenji Sugase, Tatsuro Shimamura, Mitsuyo Ohmura, Kazumi Muraoka, Ayumi Yamamoto, Takeshi Uchida, So Iwata, Yuki Yamaguchi, Elena Krayukhina, Masanori Noda, Hiroshi Handa, Koichiro Ishimori, Susumu Uchiyama, Takuya Kobayashi, Makoto Suematsu

  NATURE COMMUNICATIONS 7 2041-1723 2016/03 [Refereed][Not invited]
   

  Progesterone-receptor membrane component 1 (PGRMC1/Sigma-2 receptor) is a haem-containing protein that interacts with epidermal growth factor receptor (EGFR) and cytochromes P450 to regulate cancer proliferation and chemoresistance; its structural basis remains unknown. Here crystallographic analyses of the PGRMC1 cytosolic domain at 1.95 angstrom resolution reveal that it forms a stable dimer through stacking interactions of two protruding haem molecules. The haem iron is five-coordinated by Tyr113, and the open surface of the haem mediates dimerization. Carbon monoxide (CO) interferes with PGRMC1 dimerization by binding to the sixth coordination site of the haem. Haem-mediated PGRMC1 dimerization is required for interactions with EGFR and cytochromes P450, cancer proliferation and chemoresistance against anti-cancer drugs; these events are attenuated by either CO or haem deprivation in cancer cells. This study demonstrates protein dimerization via haem-haem stacking, which has not been seen in eukaryotes, and provides insights into its functional significance in cancer.
• Amorphous Aggregation of Cytochrome c with Inherently Low Amyloidogenicity Is Characterized by the Metastability of Supersaturation and the Phase Diagram

  Yuxi Lin, Jozsef Kardos, Mizue Imai, Tatsuya Ikenoue, Misaki Kinoshita, Toshihiko Sugiki, Koichiro Ishimori, Yuji Goto, Young-Ho Lee

  LANGMUIR 32 (8) 2010 - 2022 0743-7463 2016/03 [Refereed][Not invited]
   

  Despite extensive studies on the folding and function of cytochrome c, the mechanisms underlying its aggregation remain largely unknown. We herein examined the aggregation behavior of the physiologically relevant two types of cytochrome c, metal-bound cytochrome c, and its fragment with high amyloidogenicity as predicted in alcohol/water mixtures. Although the aggregation propensity of holo cytochrome c was low due to high solubility, markedly unfolded apo cytochrome c, lacking the heme prosthetic group, strongly promoted the propensity for amorphous aggregation with increases in hydrophobicity. Silver-bound apo cytochrome c increased the capacity of fibrillar aggregation (i.e., protofibrils or immature fibrils) due to subtle structural changes of apo cytochrome c by strong binding of silver. However, mature amyloid fibrils were not detected for any of the cytochrome c variants or its fragment, even with extensive ultrasonication, which is a powerful amyloid inducer. These results revealed the intrinsically low amyloidogenicity of cytochrome c, which is beneficial for its homeostasis and function by facilitating the folding and minimizing irreversible amyloid formation. We propose that intrinsically low amyloidogenicity of cytochrome c is attributed to the low metastability of supersaturation. The phase diagram constructed based on solubility and aggregate type is useful for a comprehensive understanding of protein aggregation. Furthermore, amorphous aggregation, which is also viewed as a generic property of proteins, and amyloid fibrillation can be distinguished from each other by the metastability of supersaturation.
• Cytoplasmic Heme-Binding Protein (HutX) from Vibrio cholerae Is an Intracellular Heme Transport Protein for the Heme-Degrading Enzyme, HutZ.

  Yukari Sekine, Takehito Tanzawa, Yoshikazu Tanaka, Koichiro Ishimori, Takeshi Uchida

  Biochemistry 55 (6) 884 - 93 2016/02/16 [Refereed][Not invited]
   

  HutZ is a cytoplasmic heme-binding protein from Vibrio cholerae. Although we have previously identified HutZ as a heme-degrading enzyme [Uchida, T., et al. (2012) Chem. Commun. 48, 6741-6743], the heme transport protein for HutZ remained unknown. To identify the heme transport protein for HutZ, we focused on the heme utilization operon, hutWXZ. To this end, we constructed an expression system for HutX in Escherichia coli and purified it to homogeneity. An absorption spectral analysis demonstrated that HutX binds heme with a 1:1 stoichiometry and a dissociation constant of 7.4 nM. The crystal structure of HutX displays a fold similar to that of the homologous protein, ChuX, from E. coli O157:H7. A structural comparison of HutX and ChuX, and resonance Raman spectra of heme-HutX, suggest that the axial ligand of the ferric heme is Tyr90. The heme bound to HutX is transferred to HutZ with biphasic dissociation kinetics of 8.3 × 10(-2) and 1.5 × 10(-2) s(-1), values distinctly larger than those for transfer from HutX to apomyoglobin. Surface plasmon resonance experiments confirmed that HutX interacts with HutZ with a dissociation constant of ∼400 μM. These results suggest that heme is transferred from HutX to HutZ via a specific protein-protein interaction. Therefore, we can conclude that HutX is a cytoplasmic heme transport protein for HutZ.
• Conformational Disorder of the Most Immature Cu, Zn-Superoxide Dismutase Leading to Amyotrophic Lateral Sclerosis

  Yoshiaki Furukawa, Itsuki Anzai, Shuji Akiyama, Mizue Imai, Fatima Joy C. Cruz, Tomohide Saio, Kenichi Nagasawa, Takao Nomura, Koichiro Ishimori

  JOURNAL OF BIOLOGICAL CHEMISTRY 291 (8) 4144 - 4155 0021-9258 2016/02 [Refereed][Not invited]
   

  Misfolding of Cu,Zn-superoxide dismutase (SOD1) is a pathological change in the familial form of amyotrophic lateral sclerosis caused by mutations in the SOD1 gene. SOD1 is an enzyme that matures through the binding of copper and zinc ions and the formation of an intramolecular disulfide bond. Pathogenic mutations are proposed to retard the post-translational maturation, decrease the structural stability, and hence trigger the misfolding of SOD1 proteins. Despite this, a misfolded and potentially pathogenic conformation of immature SOD1 remains obscure. Here, we show significant and distinct conformational changes of apoSOD1 that occur only upon reduction of the intramolecular disulfide bond in solution. In particular, loop regions in SOD1 lose their restraint and become significantly disordered upon dissociation of metal ions and reduction of the disulfide bond. Such drastic changes in the solution structure of SOD1 may trigger misfolding and fibrillar aggregation observed as pathological changes in the familial form of amyotrophic lateral sclerosis.
• Thermodynamics of protein-protein interactions examined by isothermal titration calorimetry

  Ju Yaen Kim, Misaki Kinoshita, Takashi Inui, Genji Kurisu, Yuji Goto, Toshiharu Hase, Koichiro Ishimori, Young Ho Lee

  Protein-Protein Interactions (PPIs): Types, Methods for Detection and Analysis 67 - 79 2016/01/01 [Refereed][Not invited]
   

  © 2017 by Nova Science Publishers, Inc. All rights reserved. Although the thermodynamics of interactions among biomolecules have been extensively examined, the electrostatic contribution to binding thermodynamics has not been elucidated in detail. We herein concisely characterized thermodynamics by which attractive intermolecular electrostatic interactions control thermodynamic driving forces to form complexes using isothermal titration calorimetry, one of the most powerful approaches to study binding thermodynamics. We initially focused on electrostatic interactions between negatively-charged ferredoxin and ferredoxin-NADP(+) reductase or sulfite reductase, which uses positively-charged clusters for binding. ITC thermograms of two binding systems under the same condition showed opposite signs of binding heat with similar binding affinities. We then investigated a number of attractive electrostatic binding systems between oppositelycharged proteins, negatively-charged nucleic acids and proteins, and proteins and positively-charged metal ions were investigated based on previous thermodynamic studies. The results obtained have contributed to a comprehensive general view on the binding thermodynamics and interplay between enthalpy and entropy in which attractive electrostatic interactions are remarkable. The importance of conformational entropy in stabilizing complexes and the usefulness of driving force plots are also discussed.
• Protein oxidation mediated by heme-induced active site conversion specific for heme-regulated transcription factor, iron response regulator

  Chihiro Kitatsuji, Kozue Izumi, Shusuke Nambu, Masaki Kurogochi, Takeshi Uchida, Shin-Ichiro Nishimura, Kazuhiro Iwai, Mark R. O'Brian, Masao Ikeda-Saito, Koichiro Ishimori

  SCIENTIFIC REPORTS 6 18703  2045-2322 2016/01 [Refereed][Not invited]
   

  The Bradyrhizobium japonicum transcriptional regulator Irr (iron response regulator) is a key regulator of the iron homeostasis, which is degraded in response to heme binding via a mechanism that involves oxidative modification of the protein. Here, we show that heme-bound Irr activates O-2 to form highly reactive oxygen species (ROS) with the "active site conversion" from heme iron to non-heme iron to degrade itself. In the presence of heme and reductant, the ROS scavenging experiments show that Irr generates H2O2 from O-2 as found for other hemoproteins, but H2O2 is less effective in oxidizing the peptide, and further activation of H2O2 is suggested. Interestingly, we find a time-dependent decrease of the intensity of the Soret band and appearance of the characteristic EPR signal at g = 4.3 during the oxidation, showing the heme degradation and the successive formation of a non-heme iron site. Together with the mutational studies, we here propose a novel "two-step self-oxidative modification" mechanism, during which O-2 is activated to form H2O2 at the heme regulatory motif (HRM) site and the generated H2O2 is further converted into more reactive species such as OH at the non-heme iron site in the His-cluster region formed by the active site conversion.
• Investigation of the redox-dependent modulation of structure and dynamics in human cytochrome c

  Mizue Imai, Tomohide Saio, Hiroyuki Kumeta, Takeshi Uchida, Fuyuhiko Inagaki, Koichiro Ishimori

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 469 (4) 978 - 984 0006-291X 2016/01 [Refereed][Not invited]
   

  Redox-dependent changes in the structure and dynamics of human cytochrome c (Cyt c) were investigated by solution NMR. We found significant structural changes in several regions, including residues 23-28 (loop 3), which were further corroborated by chemical shift differences between the reduced and oxidized states of Cyt c. These differences are essential for discriminating redox states in Cyt c by cytochrome c oxidase (CcO) during electron transfer reactions. Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments identified that the region around His33 undergoes conformational exchanges on the mu s-ms timescale, indicating significant redox-dependent structural changes. Because His33 is not part of the interaction site for CcO, our data suggest that the dynamic properties of the region, which is far from the interaction site for CcO, contribute to conformational changes during electron transfer to CcO. (C) 2015 Elsevier Inc. All rights reserved.
• A Dye-Decolorizing Peroxidase from Vibrio cholerae

  Takeshi Uchida, Miho Sasaki, Yoshikazu Tanaka, Koichiro Ishimorit

  BIOCHEMISTRY 54 (43) 6610 - 6621 0006-2960 2015/11 [Refereed][Not invited]
   

  The dye-decolorizing peroxidase (DyP) protein from Vibrio cholerae (VcDyP) was expressed in Escherichia coli, and its DyP activity was assayed by monitoring degradation of a typical anthraquinone dye, reactive blue 19 (RB19). Its kinetic activity was obtained by fitting the data to the Michaelis Menten equation, giving K-cat and K-m values of 1.3 +/- 0.3 s(-1) and 50 +/- 20 mu M, respectively, which are comparable to those of other DyP enzymes. The enzymatic activity of VcDyP was highest at pH 4. A mutational study showed that two distal residues, Asp 144 and Arg230, which are conserved in a DyP family, are essential for the DyP reaction. The crystal structure and resonance Raman spectra of VcDyP indicate the transfer of a radical from heme to the protein surface, which was supported by the formation of the intermolecular covalent bond in the reaction with H2O2. To identify the radical site, each of nine tyrosine or two tryptophan residues was substituted. It was clarified that Tyr129 and Tyr235 are in the active site of the dye degradation reaction at lower pH, while Tyr109 and Tyr133 are the sites of an intermolecular covalent bond at higher pH. VcDyP degrades RB19 at lower pH, while it loses activity under neutral or alkaline conditions because of a change in the radical transfer pathway. This finding suggests the presence of a pH-dependent switch of the radical transfer pathway, probably including His178. Although the physiological function of the DyP reaction is unclear, our findings suggest that VcDyP enhances the DyP activity to survive only when it is placed under a severe condition such as being in gastric acid.
• Unique Heme Environmental Structures in Heme-regulated Proteins Using Heme as the Signaling Molecule

  Koichiro Ishimori, Yuta Watanabe

  CHEMISTRY LETTERS 43 (11) 1680 - 1689 0366-7022 2014/11 [Refereed][Not invited]
   

  Heme is a typical and common prosthetic group for various types of proteins and is utilized for active centers in many biologically important processes in vivo. However, heme has also been shown to function as a signaling molecule that regulates the functions of proteins, suggesting that novel signaling cascades are mediated by heme. We focused on the spectroscopic characterization of such "heme-regulated" proteins, showing unique heme environmental structures.
• Heme-Binding Properties of HupD Functioning as a Substrate-Binding Protein in a Heme-Uptake ABC-Transporter System in Listeria monocytogenes

  Yasunori Okamoto, Hitomi Sawai, Mariko Ogura, Takeshi Uchida, Koichiro Ishimori, Takashi Hayashi, Shigetoshi Aono

  BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN 87 (10) 1140 - 1146 0009-2673 2014/10 [Refereed][Not invited]
   

  The HupDCG protein complex is a putative ABC-transporter for heme in the pathogenic Gram-positive bacterium Listeria monocytogenes, where HupD functions as a heme-binding protein. UV-vis absorption, EPR, and resonance Raman spectroscopy have revealed that HupD binds a heme with two histidine residues as the axial ligands. His105 and His259 are identified as the axial ligands by site-directed mutagenesis. HupD is the first example of a heme-binding protein having a bis-histidine coordination environment among the heme-binding proteins working in bacterial heme acquisition systems. While mutation of His259 to Ala resulted in a loss in the heme-binding ability of HupD, the H105A variant of HupD retained its heme-binding ability with lower heme-binding affinity compared with the wild type. These results suggest that His259 is an essential ligand for heme acquisition by HupD and that His105 might be responsible for regulation of the heme-binding affinity of HupD during the heme-transport process.
• Molecular Mechanism for Heme-Mediated Inhibition of 5-Aminolevulinic Acid Synthase 1

  Chihiro Kitatsuji, Mariko Ogura, Takeshi Uchida, Koichiro Ishimori, Shigetoshi Aono

  BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN 87 (9) 997 - 1004 0009-2673 2014/09 [Refereed][Not invited]
   

  Mammalian 5-aminolevulinic acid synthase 1 (ALAS1), an isozyme expressed in all cell types, catalyzes the first reaction in the heme biosynthetic pathway in mitochondria. Heme regulates ALAS1 function at multiple levels including the regulation of transcription, translation, mitochondrial import, protein degradation, and enzyme activity to maintain intracellular heme concentrations in an appropriate range. In this study, we elucidated the molecular mechanism of heme-mediated regulation of enzymatic activity for rat ALAS1. ALAS1 has three putative heme regulatory motifs (HRMs), two of which were found to be the iron(III) heme binding sites in ALAS1. Electronic absorption and resonance Raman spectroscopy demonstrated that (110)Cys and (527)Cys were the axial ligand of the iron(III) hemes bound to ALAS1. The heme binding to the HRMs in ALAS1 was found not to be responsible for heme-mediated inhibition of ALAS1 activity. Protoporphyrin IX, a reaction intermediate of heme biogenesis, was found to inhibit ALAS1 activity more efficiently than heme, indicating the presence of multiple pathways for the feedback regulation of ALAS1 activity.
• Spectroscopic studies on HasA from Yersinia pseudotuberculosis.

  Shin-Ichi Ozaki, Takehiro Sato, Yukari Sekine, Catharina T Migita, Takeshi Uchida, Koichiro Ishimori

  Journal of inorganic biochemistry 138 31 - 38 2014/09 [Refereed][Not invited]
   

  Heme acquisition system A (HasA) is known as a hemophore in Gram-negative pathogens. The ferric heme iron is coordinated by Tyr-75 and His-32 in holo-HasA from Pseudomonas aeruginosa (HasApa). In contrast, in holo-HasA from Yersinia pseudotuberculosis (HasAyp), our spectroscopic studies suggest that only Tyr-75 coordinates to the ferric heme iron. The substitution of Gln-32 with alanine in HasAyp does not alter the spectroscopic properties, indicating that Gln-32 is not an axial ligand for the heme iron. Somewhat surprisingly, the Y75A mutant of HasAyp can capture a free hemin molecule but the rate of hemin uptake is slower than that of wild type, suggesting that the hydrophobic interaction in the heme pocket may also play a role in heme acquisition. Unlike in wild type apoprotein, ferric heme transfer from Hb to Y75A apo-HasAyp has not been observed. These results imply that coordination (bonding/interaction) between Tyr-75 and the heme iron is important for heme transfer from Hb. Interestingly, HasAyp differs from HasApa in its ability to bind the ferrous heme iron. Apo-HasAyp can capture ferrous heme and resonance Raman spectra of ferrous-carbon monoxide holo-HasAyp suggest that Tyr-75 is protonated when the heme iron is in the ferrous state. The ability of HasAyp to acquire the ferrous heme iron might be beneficial to Y. pseudotuberculosis, a facultative anaerobe in the Enterobacteriaceae family.
• 2P130 Analysis of interactions in the electron transfer complex between Cytochrome c and Cytochrome c Oxidase using osmotic pressure(07. Water & Hydration & Electrolyte,Poster,The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))

  Sato Wataru, Imai Mizue, Uchida Takeshi, Ito Kyoko, Yoshikawa Shinya, Ishimori Koichiro

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 54 (1) S216  2014
• 3P053 Dehydration in cytochrome c folding revealed by high pressure spectroscopy(01C. Protein: Property,Poster,The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))

  Konno Shohei, Doi Kentaro, Uchida Takeshi, Ishimori Koichiro

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 54 (1) S257  2014
• 2P041 Conformational Gating for Electron Transfer Reaction from Cytochrome c to Cytochrome c Oxidase in Mitochondrial Respiratory Chain(01B. Protein: Structure & Function,Poster,The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))

  Imai Mizue, Sato Wataru, Inoue Kaoru, Sakamoto Koichi, Shinzawa Kyoko, Uchida Takeshi, Yoshikawa Shinya, Ishimori Koichiro

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 54 (1) S201  2014
• Sequence and temperature dependence of the end-to-end collision dynamics of single-stranded DNA

  Takanori Uzawa, Takashi Isoshima, Yoshihiro Ito, Koichiro Ishimori, Dmitrii E. Makarov, Kevin W. Plaxco

  Biophysical Journal 104 (11) 2485 - 2492 0006-3495 2013/06/04 [Refereed][Not invited]
   

  Intramolecular collision dynamics play an essential role in biomolecular folding and function and, increasingly, in the performance of biomimetic technologies. To date, however, the quantitative studies of dynamics of single-stranded nucleic acids have been limited. Thus motivated, here we investigate the sequence composition, chain-length, viscosity, and temperature dependencies of the end-to-end collision dynamics of single-stranded DNAs. We find that both the absolute collision rate and the temperature dependencies of these dynamics are base-composition dependent, suggesting that base stacking interactions are a significant contributor. For example, whereas the end-to-end collision dynamics of poly-thymine exhibit simple, linear Arrhenius behavior, the behavior of longer poly-adenine constructs is more complicated. Specifically, 20- and 25-adenine constructs exhibit biphasic temperature dependencies, with their temperature dependences becoming effectively indistinguishable from that of poly-thymine above 335 K for 20-adenines and 328 K for 25-adenines. The differing Arrhenius behaviors of poly-thymine and poly-adenine and the chain-length dependence of the temperature at which poly-adenine crosses over to behave like poly-thymine can be explained by a barrier friction mechanism in which, at low temperatures, the energy barrier for the local rearrangement of poly-adenine becomes the dominant contributor to its end-to-end collision dynamics. © 2013 Biophysical Society.
• 3P127 Analysis of interactions in the electron transfer complex between Cytochrome c and Cytochrome c Oxidase using osmotic pressure(07. Water & Hydration & Electrolyte,Poster)

  Sato Wataru, Imai Mizue, Uchida Takeshi, Ito Kyoko, Yoshikawa Shinya, Ishimori Koichiro

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 53 (1) S232  2013
• 3P105 Effects of homotrimer formation on chloride pump activity in membrane mimetics, Nanodisc, embedded Halorhodopsin(03. Membrane proteins,Poster)

  Suzuki Kenshiro, Yamamoto Ayumi, Tsukamoto Takashi, Kobashigawa Toshihiro, Uchida Takeshi, Inagaki Fuyuhiko, Demura Makoto, Ishimori Koichiro

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 53 (1) S229  2013
• A heme degradation enzyme, HutZ, from Vibrio cholerae.

  Takeshi Uchida, Yukari Sekine, Toshitaka Matsui, Masao Ikeda-Saito, Koichiro Ishimori

  Chemical communications (Cambridge, England) 48 (53) 6741 - 3 1359-7345 2012/07/07 [Refereed][Invited]
   

  HutZ, one of the crucial proteins of the iron uptake system in Vibrio cholerae, was purified, which binds to heme at a stoichiometry of 1 : 1. In the presence of ascorbic acid, the HutZ-bound heme degrades via the same intermediates observed in heme oxygenase, suggesting that HutZ works as a heme degradation enzyme.
• Effects of the bHLH domain on axial coordination of heme in the PAS-A domain of neuronal PAS domain protein 2 (NPAS2): Conversion from His119/Cys170 coordination to His119/His171 coordination

  Takeshi Uchida, Ikuko Sagami, Toru Shimizu, Koichiro Ishimori, Teizo Kitagawa

  JOURNAL OF INORGANIC BIOCHEMISTRY 108 188 - 195 0162-0134 2012/03 [Refereed][Invited]
   

  Neuronal PAS domain protein 2 (NPAS2), which is a CO-dependent transcription factor, consists of a basic helix-loop-helix domain (bHLH), and two heme-containing PAS domains (PAS-A and PAS-B). In our previous study on the isolated PAS-A domain, we concluded that His119 and Cys170 are the axial ligands of the ferric heme, while Cys170 is replaced by His171 upon reduction of heme (Uchida et al., J. Biol. Chem. 270, (2005) 21358-21368.). Recently, we characterized the PAS-A domain combined with the N-terminal bHLH domain, and found that some spectroscopic features were different from those of the isolated PAS-A domain (Mukaiyama et al., FEBS J. 273, (2006) 2528-2539.). Therefore, we reinvestigated the coordination structure of heme in the bHLH-PAS-A domain and prepared four histidine and one cysteine mutants. Resonance Raman spectrum of the Cys170Ala mutant is the same as that of wild type with a dominant 6-coordinate heme in the ferric form. In contrast, His119Ala and His171Ala mutants significantly increase amounts of the 5-coordinate species, indicating that His119 and His171, not Cys170, are axial ligands of the ferric heme in the bHLH-PAS-A domain. We had confirmed that the coordination structure of the isolated PAS-A domain is in equilibrium between Cys-Fe-His and His-Fe-His coordinated species but newly found that interaction of the PAS-A domain with the bHLH domain shifts the equilibrium toward the latter structure. Such flexibility in the heme coordination structure seems to be in favor of signal transduction in NPAS2. (C). 2011 Elsevier Inc. All rights reserved.
• Heme binding characteristics of mouse per1, a transcriptional regulatory factor associated with circadian rhythms

  Nagata R, Harada M, Kitanishi K, Igarashi J, Uchida T, Ishimori K, Shimizu T

  Circadian Rhythms: Biology, Cognition and Disorders 133 - 160 2012 [Refereed][Not invited]
  
• Identification and Functional and Spectral Characterization of a Globin-coupled Histidine Kinase from Anaeromyxobacter sp Fw109-5

  Kenichi Kitanishi, Kazuo Kobayashi, Takeshi Uchida, Koichiro Ishimori, Jotaro Igarashi, Toru Shimizu

  JOURNAL OF BIOLOGICAL CHEMISTRY 286 (41) 35522 - 35534 0021-9258 2011/10 [Refereed][Not invited]
   

  Two-component signal transduction systems regulate numerous important physiological functions in bacteria. In this study we have identified, cloned, overexpressed, and characterized a dimeric full-length heme-bound (heme: protein, 1: 1 stoichiometry) globin-coupled histidine kinase (AfGcHK) from Anaeromyxobacter sp. strain Fw109-5 for the first time. The Fe(III), Fe(II)-O(2), and Fe(II)-CO complexes of the protein displayed autophosphorylation activity, whereas the Fe(II) complex had no significant activity. A H99A mutant lost heme binding ability, suggesting that this residue is the heme proximal ligand. Moreover, His-183 was proposed as the autophosphorylation site based on the finding that the H183A mutant protein was not phosphorylated. The phosphate group of autophosphorylated AfGcHK was transferred to Asp-52 and Asp-169 of a response regulator, as confirmed from site-directed mutagenesis experiments. Based on the amino acid sequences and crystal structures of other globin-coupled oxygen sensor enzymes, Tyr-45 was assumed to be the O(2) binding site at the heme distal side. The O(2) dissociation rate constant, 0.10 s(-1), was substantially increased up to 8.0 s(-1) upon Y45L mutation. The resonance Raman frequencies representing nu(Fe-O2) (559 cm(-1)) and nu(O-O) (1149 cm(-1)) of the Fe(II)-O(2) complex of Y45F mutant AfGcHK were distinct from those of the wildtype protein (nu(Fe-O2), 557 cm(-1); nu(O-O), 1141 cm(-1)), supporting the proposal that Tyr-45 is located at the distal side and forms hydrogen bonds with the oxygen molecule bound to the Fe(II) complex. Thus, we have successfully identified and characterized a novel heme-based globin-coupled oxygen sensor histidine kinase, AfGcHK, in this study.
• NMR basis for interprotein electron transfer gating between cytochrome c and cytochrome c oxidase

  Koichi Sakamoto, Masakatsu Kamiya, Mizue Imai, Kyoko Shinzawa-Itoh, Takeshi Uchida, Keiichi Kawano, Shinya Yoshikawa, Koichiro Ishimori

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 108 (30) 12271 - 12276 0027-8424 2011/07 [Refereed][Not invited]
   

  The final interprotein electron transfer (ET) in the mammalian respiratory chain, from cytochrome c (Cyt c) to cytochrome c oxidase (CcO) is investigated by (1)H-(15)N heteronuclear single quantum coherence spectral analysis. The chemical shift perturbation in isotope-labeled Cyt c induced by addition of unlabeled CcO indicates that the hydrophobic heme periphery and adjacent hydrophobic amino acid residues of Cyt c dominantly contribute to the complex formation, whereas charged residues near the hydrophobic core refine the orientation of Cyt c to provide well controlled ET. Upon oxidation of Cyt c, the specific line broadening of N-H signals disappeared and high field (1)H chemical shifts of the N-terminal helix were observed, suggesting that the interactions of the N-terminal helix with CcO are reduced by steric constraint in oxidized Cyt c, while the chemical shift perturbations in the C-terminal helix indicate notable interactions of oxidized Cyt c with CcO. These results suggest that the overall affinity of oxidized Cyt c for CcO is significantly, but not very much weaker than that of reduced Cyt c. Thus, electron transfer is gated by dissociation of oxidized Cyt c from CcO, the rate of which is controlled by the affinity of oxidized Cyt c to CcO for providing an appropriate electron transfer rate for the most effective energy coupling. The conformational changes in Lys13 upon CcO binding to oxidized Cyt c, shown by (1)H- and (1)H, (15)N-chemical shifts, are also expected to gate intraprotein ET by a polarity control of heme c environment.
• Probing Phenylalanine Environments in Oligomeric Structures with Pentafluorophenylalanine and Cyclohexylalanine

  Takao Nomura, Rui Kamada, Issaku Ito, Koichi Sakamoto, Yoshiro Chuman, Koichiro Ishimori, Yasuyuki Shimohigashi, Kazuyasu Sakaguchi

  BIOPOLYMERS 95 (6) 410 - 419 0006-3525 2011/06 [Refereed][Not invited]
   

  Stabilization of protein structures and protein protein interactions are critical in the engineering of industrially useful enzymes and in the design of pharmaceutically valuable ligands. Hydrophobic interactions involving phenylalanine residues play crucial roles in protein stability and protein-protein/peptide interactions. To establish an effective method to explore the hydrophobic environments of phenylalanine residues, we present a strategy that uses pentafluorophenylalanine (F(5)Phe) and cyclohexylalanine (Cha). In this study, substitution of F(5)Phe or Cha for three Phe residues at positions 328, 338, and 341 in the tetramerization domain of the tumor suppressor protein p53 was performed. These residues are located at the interfaces of p53 p53 interactions and are important in the stabilization of the tetrameric structure. The stability of the p53 tetrameric structure did not change significantly when F(5)Phe-containing peptides at positions Phe328 or Phe338 were used. In contrast, the substitution of Cha for Phe341 in the hydrophobic core enhanced the stability of the tetrameric structure with a T(m) value of similar to 100 degrees C. Phe328 and Phe338 interact with each other through pi-interactions, whereas Phe341 is buried in the surrounding alkyl side-chains of the hydrophobic core of the p53 tetramerization domain. Furthermore, high pressure-assisted denaturation analysis indicated improvement in the occupancy of the hydrophobic core. Considerable stabilization of the p53 tetramer was achieved by filling the identified cavity in the hydrophobic core of the p.5.3 tetramer. The results indicate the status of the Phe residues, indicating that the "pair substitution" of Cha and F(5)Phe is highly suitable for probing the environments of Phe residues. (C) 2011 Wiley Periodicals, Inc. Biopolymers 95: 410-419, 2011.
• Unusual Heme Binding in the Bacterial Iron Response Regulator Protein: Spectral Characterization of Heme Binding to the Heme Regulatory Motif

  Haruto Ishikawa, Megumi Nakagaki, Ai Bamba, Takeshi Uchida, Hiroshi Hori, Mark R. O'Brian, Kazuhiro Iwai, Koichiro Ishimori

  BIOCHEMISTRY 50 (6) 1016 - 1022 0006-2960 2011/02 [Refereed][Not invited]
   

  We characterized heme binding in the bacterial iron response regulator (Irr) protein, which is a simple heme-regulated protein having a single "heme-regulatory motif", HRM, and plays a key role in the iron homeostasis of a nitrogen-fixing bacterium. The heme titration to wild-type and mutant Irr clearly showed that Irr has two heme binding sites: one of the heme binding sites is in the HRM, where (29)Cys is the axial ligand, and the other one, the secondary heme binding site, is located outside of the HRM. The Raman line for the Fe-S stretching mode observed at 333 cm(-1) unambiguously confirmed heme binding to Cys. The lower frequency of the Fe-S stretching mode corresponds to the weaker Fe-S bond, and the broad Raman line of the Fe-S bond suggests multiple configurations of heme binding. These structural characteristics are definitely different from those of typical hemoproteins. The unusual heme binding in Irr was also evident in the EPR spectra. The characteristic g-values of the 5-coordinate Cys-ligated heme and 6-coordinate His/His-ligated heme were observed, while the multiple configurations of heme binding were also confirmed. Such multiple heme configurations are not encountered for typical hemoproteins where the heme functions as the active center. Therefore, we conclude that heme binding to HRM in the heme-regulated protein, Irr, is quite different from that in conventional hemoproteins but characteristic of heme-regulated proteins using heme as the signaling molecule.
• 2SJ-02 Interactions in Electron Transfer Complex between Cytochrome c and Cytochrome c Oxidase(2SJ New developments in protein complex research: From molecules to supramolecules and aggregates,The 49th Annual Meeting of the Biophysical Society of Japan)

  Ishimori Koichiro

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 51 S22  2011
• Redox-controlled backbone dynamics of human cytochrome c revealed by N-15 NMR relaxation measurements

  Koichi Sakamoto, Masakatsu Kamiya, Takeshi Uchida, Keiichi Kawano, Koichiro Ishimori

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 398 (2) 231 - 236 0006-291X 2010/07 [Refereed][Not invited]
   

  Redox-controlled backbone dynamics in cytochrome c (Cyt c) were revealed by 2D N-15 NMR relaxation experiments. N-15 T-1 and T-2 values and H-1-N-15 NOEs of uniformly N-15-labeled reduced and oxidized Cyt c were measured, and the generalized order parameters (S-2), the effective correlation time for internal motion (tau(e)), the N-15 exchange broadening contributions (R-ex) for each residue, and the overall correlation time (tau(m)) were estimated by model-free dynamics formalism. These dynamic parameters clearly showed that the backbone dynamics of Cyt c are highly restricted due to the covalently bound heme that functions as the stable hydrophobic core. Upon oxidation of the heme iron in Cyt c, the average S-2 value was increased from 0.88 +/- 0.01 to 0.92 +/- 0.01, demonstrating that the mobility of the backbone is further restricted in the oxidized form. Such increases in the S-2 values were more prominent in the loop regions, including amino acid residues near the thioether bonds to the heme moiety and positively charged region around Lys87. Both of the regions are supposed to form the interaction site for cytochrome c oxidase (CcO) and the electron pathway from Cyt c to CcO. The redox-dependent mobility of the backbone in the interaction site for the electron transfer to CcO suggests an electron transfer mechanism regulated by the backbone dynamics in the Cyt c-CcO system. (C) 2010 Elsevier Inc. All rights reserved.
• Molecular oxygen regulates the enzymatic activity of a heme-containing diguanylate cyclase (HemDGC) for the synthesis of cyclic di-GMP

  Hitomi Sawai, Shiro Yoshioka, Takeshi Uchida, Mamoru Hyodo, Yoshihiro Hayakawa, Koichiro Ishimori, Shigetoshi Aono

  BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 1804 (1) 166 - 172 1570-9639 2010/01 [Refereed][Not invited]
   

  We have studied the structural and enzymatic properties of a diguanylate cyclase from an obligatory anaerobic bacterium Desulfotalea psychrophila, which consists of the N-terminal sensor domain and the C-terminal diguanylate cyclase domain. The sensor domain shows an amino acid sequence homology and spectroscopic properties similar to those of the sensor domains of the globin-coupled sensor proteins containing a protoheme. This heme-containing diguanylate cyclase catalyzes the formation of cyclic di-GMP from GTP only when the heme in the sensor domain binds molecular oxygen. When the heme is in the ferric, deoxy, CO-bound, or NO-bound forms, no enzymatic activity is observed. Resonance Raman spectroscopy reveals that Tyr55 forms a hydrogen bond with the heme-bound O(2), but not with CO. Instead, Gln81 interacts with the heme-bound CO. These differences of a hydrogen bonding network will play a crucial role for the selective O(2) sensing responsible for the regulation of the enzymatic activity. (C) 2009 Elsevier B.V. All rights reserved.
• Optical manipulation of proteins in aqueous solution

  Yasuyuki Tsuboi, Tatsuya Shoji, Masayuki Nishino, Seiji Masuda, Koichiro Ishimori, Noboru Kitamura

  APPLIED SURFACE SCIENCE 255 (24) 9906 - 9908 0169-4332 2009/09 [Refereed][Not invited]
   

  Optical trapping of lysozyme, cytochrome c, or myoglobin based on photon pressure generated by focusing 1064 nm laser beam in an aqueous solution was explored. For all the proteins, microparticle formation was observed at the focal point under an optical microscope. Furthermore, the microparticles were identified to the molecular assemblies of the corresponding protein by means of confocal Raman microspectroscopy. For lysozyme, molecular clusters in solution were optically trapped to form the microparticle and it took more than 1 h to produce the microparticle. By contrast, molecular assembling proceeded within 1 min for cytochrome c and myoglobin. Since heme in cytochrome c or myoglobin would have a high polarizability, that would contribute to rapid assembling of the protein. Thus we demonstrated that a focused laser beam was a powerful tool to manipulate protein molecules in solution. (C) 2009 Elsevier B.V. All rights reserved.
• 3P-080 Identification of amino acid residues responsible for the electron transfer reaction with cytochrome c oxidase in cytochrome c.(Heme proteins,The 47th Annual Meeting of the Biophysical Society of Japan)

  Inoue Kaoru, Sakamoto Koichi, Nomoto Naoko, Uchida Takeshi, Shinzawa-Ito Kyoko, Yoshikawa Shinya, Ishimori Koichiro

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 49 S164  2009
• Hierarchical folding mechanism of apomyoglobin revealed by ultra-fast H/D exchange coupled with 2D NMR

  Takanori Uzawa, Chiaki Nishimura, Shuji Akiyama, Koichiro Ishimori, Satoshi Takahashi, H. Jane Dyson, Peter E. Wright

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 105 (37) 13859 - 13864 0027-8424 2008/09 [Refereed][Not invited]
   

  The earliest steps in the folding of proteins are complete on an extremely rapid time scale that is difficult to access experimentally. We have used rapid-mixing quench-flow methods to extend the time resolution of folding studies on apomyoglobin and elucidate the structural and dynamic features of members of the ensemble of intermediate states that are populated on a submillisecond time scale during this process. The picture that emerges is of a continuum of rapidly interconverting states. Even after only 0.4 ms of refolding time a compact state is formed that contains major parts of the A, G, and H helices, which are sufficiently well folded to protect amides from exchange. The B, C, and E helix regions fold more slowly and fluctuate rapidly between open and closed states as they search docking sites on this core; the secondary structure in these regions becomes stabilized as the refolding time is increased from 0.4 to 6 ms. No further stabilization occurs in the A, G, H core at 6 ms of folding time. These studies begin to time-resolve a progression of compact states between the fully unfolded and native folded states and confirm the presence an ensemble of intermediates that interconvert in a hierarchical sequence as the protein searches conformational space on its folding trajectory.
• Dehydration of main-chain amides in the final folding step of single-chain monellin revealed by time-resolved infrared spectroscopy

  Tetsunari Kimura, Akio Maeda, Shingo Nishiguchi, Koichiro Ishimori, Isao Morishima, Takashi Konno, Yuji Goto, Satoshi Takahashi

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 105 (36) 13391 - 13396 0027-8424 2008/09 [Refereed][Not invited]
   

  Kinetic IR spectroscopy was used to reveal beta-sheet formation and water expulsion in the folding of single-chain monellin (SMN) composed of a five-stranded beta-sheet and an alpha-helix. The time-resolved IR spectra between 100 mu s and 10 s were analyzed based on two consecutive intermediates, I(1) and I(2), appearing within 100 mu s and with a time constant of approximate to 100 ms, respectively. The initial unfolded state showed broad amide I' corresponded to a fluctuating conformation. In contrast, I(1) possessed a feature at 1,636 cm(-1) for solvated helix and weak features assignable to turns, demonstrating the rapid formation of helix and turns. I(2) possessed a line for solvated helix at 1,637 cm(-1) and major and minor lines for beta-sheet at 1,625 and 1,680 cm(-1), respectively. The splitting of the major and minor lines is smaller than that of the native state, implying an incomplete formation of the beta-sheet. Furthermore, both major and minor lines demonstrated a low-frequency shift compared to those of the native state, which was interpreted to be caused by hydration of the C=O group in the beta-sheet. Together with the identification of solvated helix, the core domain of I(2) was interpreted as being hydrated. Finally, slow conversion of the water-penetrated core of I(2) to the dehydrated core of the native state was observed. We propose that both the expulsion of water, hydrogen-bonded to main-chain amides, and the completion of the secondary structure formation contribute to the energetic barrier of the rate-limiting step in SMN folding.
• Heme-binding characteristics of the isolated PAS-A domain of mouse Per2, a transcriptional regulatory factor associated with circadian rhythms

  Kenichi Kitanishi, Jotaro Igarashi, Koya Hayasaka, Naoki Hikage, Islam Saiful, Seigo Yamauchi, Takeshi Uchida, Koichiro Ishimori, Toru Shimizu

  BIOCHEMISTRY 47 (23) 6157 - 6168 0006-2960 2008/06 [Refereed][Not invited]
   

  Neuronal PAS protein 2 (NPAS2), a heme-binding transcriptional regulatory factor, is involved in circadian rhythms. Period homologue (Per) is another important transcriptional regulatory factor that binds to cryptochrome (Cry). The resultant Per/Cry heterodimer interacts with the NPAS2/BMAL1 heterodimer to inhibit the transcription of Per and Cry. Previous cell biology experiments indicate that mouse Per2 (mPef2) is also a heme-binding protein, and heme shuttling between mPer2 and NPAS2 may regulate transcription. In the present study, we show that the isolated PAS-A domain of mPer2 (PAS-A-mPer2) binds the Fe(III) protoporphyrin IX complex (hemin) with a heme:protein stoichiometry of 1:1. Optical absorption and EPR spectroscopic findings suggest that the Fe(III)-bound PAS-A-mPer2 is a six-coordinated low-spin complex with Cys and an unknown axial ligand. A Hg2+ binding study supports the theory that Cys is one of the axial ligands for Fe(III)-bound PAS-A-mPer2. The dissociation rate constant of the Fe(III) complex from PAS-A-mPer2 (6.3 x 10(-4) s(-1)) was comparable to that of the heme-regulated inhibitor (HRI), a heme-sensor enzyme (1.5 x 10(-3) s(-1)), but markedly higher than that of metmyoglobin (8.4 x 10(-7) s(-1)). As confirmed by a Soret absorption spectral shift, heme transferred from the holo basic helix-loop-helix PAS-A of NPAS2 to apoPAS-A-mPer2. The Soret CD spectrum of the C215A mutant PAS-A-mPer2 protein was markedly different from that of the wild-type protein. On the basis of the data, we propose that PAS-A-mPer2 is a heme-sensor protein in which Cys215 is the heme axial ligand.
• Early Research in Biophysics Award : Report on the Third Award Selection Process

  MITAKU Shigeki, ISHIMORI Koichiro

  Seibutsu Butsuri 一般社団法人日本生物物理学会 48 (1) 52 - 55 0582-4052 2008/01/25
• The heme-mediated oxidation mechanism of Irr

  Kitatsuji Chihiro, Nakamura Atsushi, Kurogochi Masaki, Nishimura Shin-Ichiro, O'Brian Mark R, Ishimori Koichiro

  YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 128 43  0031-6903 2008 [Refereed][Not invited]
  
• Unique peroxidase reaction mechanism in prostaglandin endoperoxide H synthase-2 - Compound I in prostaglandin endoperoxide H synthase-2 can be formed without assistance by distal glutamine residue

  Shizuo Ichimura, Takeshi Uchida, Shuhei Taniguchi, Shusuke Hira, Takehiko Tosha, Isao Morishima, Teizo Kitagawa, Koichiro Ishimori

  JOURNAL OF BIOLOGICAL CHEMISTRY 282 (22) 16681 - 16690 0021-9258 2007/06 [Refereed][Not invited]
   

  Prostaglandin-endoperoxide H synthase-2 (PGHS-2) shows peroxidase activity to promote the cyclooxygenase reaction for prostaglandin H-2, but one of the highly conserved amino acid residues in peroxidases, distal Arg, stabilizing the developing negative charge on the peroxide through a hydrogen-bonding interaction, is replaced with a neutral amino acid residue, Gln. To characterize the peroxidase reaction in PGHS-2, we prepared three distal glutamine ( Gln-189) mutants, Arg ( Gln -> Arg), Asn ( Gln3Asn), and Val ( Gln -> Val) mutants, and examined their peroxidase activity together with their structural characterization by absorption and resonance Raman spectra. Although a previous study (Landino, L. M., Crews, B. C., Gierse, J. K., Hauser, S. D., and Marnett, L. ( 1997) J. Biol. Chem. 272, 21565-21574) suggested that the Gln residue might serve as a functionally equivalent residue to Arg, our current results clearly showed that the peroxidase activity of the Val and Asn mutants was comparable with that of the wild-type enzyme. In addition, the Fe-C and C-O stretching modes in the CO adduct were almost unperturbed by the mutation, implying that Gln-189 might not directly interact with the heme-ligated peroxide. Rather, the peroxidase activity of the Arg mutant was depressed, concomitant with the heme environmental change from a six-coordinate to a five-coordinate structure. Introduction of the bulky amino acid residue, Arg, would interfere with the ligation of a water molecule to the heme iron, suggesting that the side chain volume, and not the amide group, at position 189 is essential for the peroxidase activity of PGHS-2. Thus, we can conclude that the O-O bond cleavage in PGHS-2 is promoted without interactions with charged side chains at the peroxide binding site, which is significantly different from that in typical plant peroxidases.
• Molecular basis of guanine nucleotide dissociation inhibitor activity of human neuroglobin by chemical cross-linking and mass spectrometry

  Chihiro Kitatsuji, Masaki Kurogochi, Shin-Ichiro Nishimura, Koichiro Ishimori, Keisuke Wakasugi

  JOURNAL OF MOLECULAR BIOLOGY 368 (1) 150 - 160 0022-2836 2007/04 [Refereed][Not invited]
   

  Oxidized human neuroglobin (Ngb), a heme protein expressed in the brain, has been proposed to act as a guanine nucleotide dissociation inhibitor (GDI) for the GDP-bound form of the heterotrimeric G protein alpha-subunit (G(xi). Here, to elucidate the molecular mechanism underlying the GD1 activity of Ngb, we used an glutathione-S-transferase pun-down assay to confirm that Ngb competes with G-protein beta gamma-subunits (G beta gamma) for binding to G alpha(i), and identified the G alpha(i)-binding site in Ngb by chemical cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and sulfo-N-hydroxysuccinimide, coupled with mass spectrometry (MS). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis for tryptic peptides derived from the cross-linked Ngb-G alpha(i) complex revealed several binding regions in Ngb. Furthermore, MALDI-TOF/TOF MS analysis of the cross-linked Ngb and Gai peptides, together with the MS/MS scoring method, predicted cross-linking between Glu60 (Ngb) and Ser206 (G alpha(i)), and between Glu53 (Ngb) and Ser44 (G alpha(i)). Because Ser206 of G alpha(i) is located in the region that contacts G beta gamma, binding of Ngb could facilitate the release of G beta gamma from G alpha(i). Binding of Ngb to Gai would also inhibit the exchange of GDP for GTP, because Ser44 (G alpha(i)) is adjacent to the GDP-binding site and Glu53 (Ngb), which is cross-linked to Ser44 (G alpha(i)), could be located close to GDP. Thus, we have identified, for the first time, the sites of interaction between Ngb and Gai, enabling us to discuss the functional significance of this binding on the GDI activity of Ngb. (c) 2007 Elsevier Ltd. All rights reserved.
• S14I4 Structural and Functional Characterization of Sensor Proteins Regulated by Heme Binding(Protein-Ligand Interactions)

  Ishimori Koichiro

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 47 S20  2007
• 3P110 Heme Transfer Mechanism from Heme Chaperon to Apocytochrome c(Hemeproteins,Poster Presentations)

  Uchida Takeshi, Kondo Yuka, Ishimori Koichiro

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 47 S230  2007
• Volume profile analysis for protein folding

  Koichiro Ishimori

  Review of High Pressure Science and Technology/Koatsuryoku No Kagaku To Gijutsu 17 (1) 13 - 22 1348-1940 2007 [Refereed][Not invited]
   

  By using spectroscopies under high pressure, we determined the volume changes associated with protein folding of reduced cytochrome c from the unfolded state to the native state. The pressure dependence of the equilibrium constant for the denaturation and the folding rate revealed that the volume change for the protein folding and the activation volume for the native state are negative. Such negative volumes can be accounted for by a decrease in volume resulting from the dehydration of hydrophobic groups, primarily the heme group, and the dehydration is mainly induced in the formation of the transition for the native state. We, therefore, propose that dehydration can compensate for the decreased entropy in the formation of protein structures, entropically promoting the protein folding reactions.
• Time-resolved small-angle X-ray scattering investigation of the folding dynamics of heme oxygenase: Implication of the scaling relationship for the submillisecond intermediates of protein folding

  T Uzawa, T Kimura, K Ishimori, Morishima, I, T Matsui, M Ikeda-Saito, S Takahashi, S Akiyama, T Fujisawa

  JOURNAL OF MOLECULAR BIOLOGY 357 (3) 997 - 1008 0022-2836 2006/03 [Refereed][Not invited]
   

  Polypeptide collapse is generally observed as the initial folding dynamics of proteins with more than 100 residues, and is suggested to be caused by the coil-globule transition explained by Flory's theory of polymers. To support the suggestion by establishing a scaling behavior between radius of gyration (R-g) and chain length for the initial folding intermediates, the folding dynamics of heme oxygenase (HO) was characterized by time-resolved, small-angle X-ray scattering. HO is a highly helical protein without disulfide bridges, and is the largest protein (263 residues) characterized by the method. The folding process of HO was found to contain a transient oligomerization; however, the conformation within 10 ms was demonstrated to be monomeric and to possess R-g of 26.1(+/- 1.1) angstrom. Together with the corresponding data for proteins with different chain lengths, the seven R-g values demonstrated the scaling relationship to chain length with a scaling exponent of 0.35 +/- 0.11, which is close to the theoretical value of 1/3 predicted for globules in solutions where monomer-monomer interactions are favored over monomer-solvent interactions (poor solvent). The finding indicated that the initial folding dynamics of proteins bears the signature of the coil-globule transition, and offers a clue to explain the folding mechanisms of proteins with different chain lengths. (c) 2006 Elsevier Ltd. All rights reserved.
• Japanese Only

  ISHIMORI Koichiro

  Seibutsu Butsuri 一般社団法人日本生物物理学会 46 (1) 1 - 1 0582-4052 2006/01/25
• Dehydration in the folding of reduced cytochrome c revealed by the electron-transfer-triggered folding under high pressure

  T Kimura, K Sakamoto, Morishima, I, K Ishimori

  JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 128 (3) 670 - 671 0002-7863 2006/01 [Refereed][Not invited]
  
• S3f1-4 Generality of Initial Collapse Demonstrated by Scaling Relationship for Submillisecond Intermediates of Protein Folding(S3-f1: "Hydration Effects on Structure and Thermodynamics of Proteins,Symposia,Abstract,Meeting Program of EABS & BSJ 2006)

  Uzawa Takanori, Kimura Tetsunari, Ishimori Koichiro, Morishima Isao, Matsui Toshitaka, Ikeda-Saito Masao, Takahashi Satoshi, Akiyama Shuji, Fujisawa Tetsuro

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 46 (2) S139  2006
• Absence of a detectable intermediate in the compound I formation of horseradish peroxidase at ambient temperature

  M Shintaku, K Matsuura, S Yoshioka, S Takahashi, K Ishimori, Morishima, I

  JOURNAL OF BIOLOGICAL CHEMISTRY 280 (49) 40934 - 40938 0021-9258 2005/12 [Refereed][Not invited]
   

  A microsecond-resolved absorption spectrometer was developed to investigate the elementary steps in hydrogen peroxide (H2O2) activation reaction of horseradish peroxidase (HRP) at ambient temperature. The kinetic absorption spectra of HRP upon the mixing with various concentrations of H2O2 (0.5-3 mM) were monitored in the time range from 50 to 300 mu s. The time-resolved spectra in the Soret region possessed isosbestic points that were close to those between the resting state and compound I. The kinetic changes in the Soret absorbance could be well fitted by a single exponential function. Accordingly, no distinct spectrum of the putative intermediate between the resting state and compound I was identified. These results were consistent with the proposal that the O-O bond activation in heme peroxidases is promoted by the imidazolium form of the distal histidine that exists only transiently. It was estimated that the rate constant for the breakage of the O-O bond in H2O2 by HRP is significantly faster than 1 x 10(4) s(-1).
• Involvement of heme regulatory motif in heme-mediated ubiquitination and degradation of IRP2

  H Ishikawa, M Kato, H Hori, K Ishimori, T Kirisako, F Tokunaga, K Iwai

  MOLECULAR CELL 19 (2) 171 - 181 1097-2765 2005/07 [Refereed][Not invited]
   

  yIron regulatory protein 2 (IRP2), a regulator of iron metabolism, is modulated by ubiquitination and degradation. We have shown that IRP2 degradation is triggered by heme-mediated oxidation. We report here that not only Cys201, an invariant residue in the heme regulatory motif (HRM), but also His204 is critical for IRP2 degradation. Spectroscopic studies revealed that Cys201 binds ferric heme, whereas His204 is a ferrous heme binding site, indicating the involvement of these residues in sensing the redox state of the heme iron and in generating the oxidative modification. Moreover, the HRM in IRP2 has been suggested to play a critical role in its recognition by the HOIL-1 ubiquitin ligase. Although HRMs are known to sense heme concentration by simply binding to heme, the HRM in IRP2 specifically contributes to its oxidative modification, its recognition by the ligase, and its sensing of iron concentration after iron is integrated into heme.
• Specifically collapsed intermediate in the early stage of the folding of ribonuclease A

  T Kimura, S Akiyama, T Uzawa, K Ishimori, Morishima, I, T Fujisawa, S Takahashi

  JOURNAL OF MOLECULAR BIOLOGY 350 (2) 349 - 362 0022-2836 2005/07 [Refereed][Not invited]
   

  Nature of the burst-phase signals of protein folding has been the subject of much debate as to whether the signals represent the formation of early intermediates or the non-specific collapse of unfolded polypeptides. To distinguish the two possibilities, the submillisecond folding dynamics of ribonuclease A (RNase A) was examined, and compared with those of the disulfide bond-ruptured analog of RNase A (r-RNase A). The circular dichroism measurements on RNase A showed the burst-phase signal within 320 mu s after the initiation of the folding reaction, which was identical to that observed for r-RNase A. In contrast, the burst phase increase in the extrinsic fluorescence from 1-anilino-8-naphthalene sulfonate (ANS) was observed for RNase A but not for r-RNase A. The kinetic titration experiment of the ANS fluorescence intensity showed the presence of a specific binding site for ANS in the fast-refolding component of RNase A. The small-angle X-ray scattering measurements at similar to 22 ms after initiating the folding reaction demonstrated that the burst phase conformations of the medium and slow-refolding components of RNase A were distinctly smaller than that of r-RNase A. These results indicated the difference in the burst phase conformations of RNase A and r-RNase A. Since r-RNase A is denatured in the physiological solution condition, the burst-phase signal of RNase A was interpreted as the formation of the folding intermediate with specific conformations. (c) 2005 Elsevier Ltd. All rights reserved.
• Two heme binding sites are involved in the regulated degradation of the bacterial iron response regulator (Irr) protein

  JH Yang, K Ishimori, MR O'Brian

  JOURNAL OF BIOLOGICAL CHEMISTRY 280 (9) 7671 - 7676 0021-9258 2005/03 [Refereed][Not invited]
   

  The iron response regulator (Irr) protein from Bradyrhizobium japonicum is a conditionally stable protein that degrades in response to cellular iron availability. This turnover is heme-dependent, and rapid degradation involves heme binding to a heme regulatory motif (HRM) of Irr. Here, we show that Irr confers iron-dependent instability on glutathione S-transferase (GST) when fused to it. Analysis of Irr-GST derivatives with C-terminal truncations of Irr implicated a second region necessary for degradation, other than the HRM, and showed that the HRM was not sufficient to confer instability on GST. The HRM-defective mutant IrrC29A degraded in the presence of iron but much more slowly than the wild-type protein. This slow turnover was heme-dependent, as discerned by the stability of Irr in a heme-defective mutant strain. Whereas the HRM of purified recombinant Irr binds ferric ( oxidized) heme, a second site that binds ferrous ( reduced) heme was identified based on spectral analysis of truncation and substitution mutants. A mutant in which histidines 117 - 119 were changed to alanines severely diminished ferrous, but not ferric, heme binding. Introduction of these substitutions in an Irr-GST fusion stabilized the protein in vivo in the presence of iron. We conclude that normal iron-dependent Irr degradation involves two heme binding sites and that both redox states of heme are required for rapid turnover.
• Structural diversities of active site in clinical azole-bound forms between sterol 14 alpha-demethylases ( CYP51s) from human and Mycobacterium tuberculosis

  K Matsuura, S Yoshioka, T Tosha, H Hori, K Ishimori, T Kitagawa, Morishima, I, N Kagawa, MR Waterman

  JOURNAL OF BIOLOGICAL CHEMISTRY 280 (10) 9088 - 9096 0021-9258 2005/03 [Refereed][Not invited]
   

  To gain insights into the molecular basis of the design for the selective azole anti-fungals, we compared the binding properties of azole-based inhibitors for cytochrome P450 sterol 14alpha-demethylase (CYP51) from human (HuCYP51) and Mycobacterium tuberculosis (MtCYP51). Spectroscopic titration of azoles to the CYP51s revealed that HuCYP51 has higher affinity for ketoconazole (KET), an azole derivative that has long lipophilic groups, than MtCYP51, but the affinity for fluconazole (FLU), which is a member of the anti-fungal armamentarium, was lower in HuCYP51. The affinity for 4-phenylimidazole (4-PhIm) to MtCYP51 was quite low compared with that to HuCYP51. In the resonance Raman spectra for HuCYP51, the FLU binding induced only minor spectral changes, whereas the prominent high frequency shift of the bending mode of the heme vinyl group was detected in the KET- or 4-PhIm-bound forms. On the other hand, the bending mode of the heme propionate group for the FLU-bound form of MtCYP51 was shifted to high frequency as found for the KET- bound form, but that for 4-PhIm was shifted to low frequency. The EPR spectra for 4-PhIm-bound MtCYP51 and FLU-bound HuCYP51 gave multiple g values, showing heterogeneous binding of the azoles, whereas the single g(x) and g(z) values were observed for other azole-bound forms. Together with the alignment of the amino acid sequence, these spectroscopic differences suggest that the region between the B' and C helices, particularly the hydrophobicity of the C helix, in CYP51s plays primary roles in determining strength of interactions with azoles; this differentiates the binding specificity of azoles to CYP51s.
• Electron transfer reaction in a single protein molecule observed by total internal reflection fluorescence microscopy

  Y Furukawa, T Ban, D Hamada, K Ishimori, Y Goto, Morishima, I

  JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 127 (7) 2098 - 2103 0002-7863 2005/02 [Refereed][Not invited]
   

  To observe an electron transfer (ET) process in a single protein molecule, we constructed a model system, Alexa-HCytb(5), in which cytochrome b(5) (Cytb(5)) is modified with a fluorescent probe, Alexa. Fluor 647 dye. In this model system, intramolecular transfer of an electron from the Alexa dye to heme in Cytb(5) is supposed to oxidize the probe and quench its fluorescence, and the ET reaction at the single-molecule level can be monitored as the intermittent change in the fluorescence intensity. Alexa-HCytb(5) was fixed on the glass surface, and illumination of laser light by the total internal reflection resulted in blinking of the fluorescence from the single Alexa-HCytb(5) molecule in the time scale of several hundred milliseconds. Each Alexa-HCytb(5) molecule is characterized by its own rate constant of the blinking, corresponding to the ET rate constant at the single-molecule level, and its variation ranges between 1 and 10 s(-1). The current system thus enables us to visualize the ET reaction in the single protein molecule, and the protein ET reaction was found to be explained by the distribution of the rate constants. On the basis of the Marcus theory, we suggest that the origin of this rate distribution is the distance change associated with the structural fluctuation in the protein molecule.
• Specific collapse followed by slow hydrogen-bond formation of beta-sheet in the folding of single-chain monellin

  T Kimura, T Uzawa, K Ishimori, Morishima, I, S Takahashi, T Konno, S Akiyama, T Fujisawa

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 102 (8) 2748 - 2753 0027-8424 2005/02 [Refereed][Not invited]
   

  Characterization of the conformational landscapes for proteins with different secondary structures is important in elucidating the mechanism of protein folding. The folding trajectory of single-chain monellin composed of a five-stranded beta-sheet and a helix was investigated by using a pH-jump from the alkaline unfolded to native state. The kinetic changes in the secondary structures and in the overall size and shape were measured by circular dichroism spectroscopy and small-angle x-ray scattering, respectively. The formation of the tertiary structure was monitored by intrinsic and extrinsic fluorescence. A significant collapse was observed within 300 mus after the pH-jump, leading to the intermediate with a small amount of secondary and tertiary structures but with an overall oblate shape. Subsequently, the stepwise formation of secondary and tertiary structures was detected. The current observation was consistent with the theoretical prediction that a more significant collapse precedes the formation of secondary structures in the folding of beta-sheet proteins than that of helical proteins [Shea, J. E., Onuchic, J. N. & Brooks, C. L., III (2002) Proc. Nati. Acad. Sci. USA 99, 16064-16068]. Furthermore, it was implied that the initial collapse was promoted by the formation of some specific structural elements, such as tight turns, to form the oblate shape.
• 3P105 Roles of Glutamine189 at the Heme Distal Site in the Peroxidase Reaction of Prostaglandin H Synthase-2

  Ichimura S., Taniguchi S., Morishima I., Ishimori K.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 45 S230  2005
• 3P104 Spectroscopic Characterization of Heme Coordination Structure in Iron Regulatory Protein 2 (IRP2)

  Endo R., Ishikawa H., Uchida T., Kobayashi K., Kitagawa T., Iwai K., Ishimori K.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 45 S229  2005
• 3P081 Heme Binding in Iron Response Regulator (Irr) and its Regulation Mechanism

  Nakagaki M., Uchida T., Kobayashi K., Ishikawa H., Kitagawa H., Iwai K., O'Brian Mark, Ishimori K.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 45 S224  2005
• Regulation Mechanism of Cytochrome P450cam-Catalyzed Oxygenation Reaction upon Putidaredoxin Binding

  Takehiko TOSHA, Koichiro ISHIMORI, Isao MORISHIMA

  Seibutsu Butsuri Biophysical Society of Japan 45 (2) 78 - 83 0582-4052 2005 [Refereed][Not invited]
   

  Structural changes in cytochrome P450 camphor monooxygenase (P450cam) upon the binding of the electron donor, putidaredoxin (Pdx), have been believed to be crucial for the P450cam catalysis. However, the regulation mechanism for the P450cam-catalyzed reaction by Pdx binding, so-called "effector function" of Pdx, was unclear due to the lack of the structural information on the Pdx-induced structural changes in P450cam. Here we summarize the recent progress in characterizing the Pdx-induced structural changes in P450cam by using NMR spectroscopy and site-directed mutagenesis. The current information would help us to understand the effector function of Pdx in the P450cam catalysis.
• Steric effects of isoleucine 107 on heme reorientation reaction in human myoglobin

  H Ishikawa, S Takahashi, K Ishimori, Morishima, I

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 324 (3) 1095 - 1100 0006-291X 2004/11 [Refereed][Not invited]
   

  Structural factors to regulate the heme reorientation reaction in myoglobin were examined and we found that the side chain at position 107 (Ile107), which is located between the 2-vinyl and 3-methyl groups of heme, forms a kinetic barrier for the heme rotation about the alpha-gamma axis. The phenylalanine-substituted mutant showed an extremely slow heme reorientation rate, compared to that of the wild-type protein, while replacement by the decreased side chain, valine, at position 107 accelerated the reorientation reaction. Considering that the spectroscopic data show only minor structural changes in the heme environments of the Ile107 mutants, the side chain at position 107 sterically interacts with the heme peripheral groups in the activation state for the heme reorientation, which supports the intramolecular mechanism that the heme rotates about the alpha-gamma axis without leaving the "protein cage." (C) 2004 Elsevier Inc. All rights reserved.
• Structural and functional characterization of "laboratory evolved" cytochrome P450cam mutants showing enhanced naphthalene oxygenation activity

  K Matsuura, T Tosha, S Yoshioka, S Takahashi, K Ishimori, Morishima, I

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 323 (4) 1209 - 1215 0006-291X 2004/10 [Refereed][Not invited]
   

  To elucidate molecular mechanisms for the enhanced oxygenation activity in the three mutants of cytochrome P450cam screened by 'laboratory evolution' [Nature 399 (1999) 670], we purified the mutants and characterized their functional and structural properties. The electronic absorption and resonance Raman spectra revealed that the structures of heme binding site of all purified mutants were quite similar to that of the wild-type enzyme, although the fraction of the inactivated form, called "P420," was increased. In the reaction with H2O2, only trace amounts of the naphthalene hydroxylation product were detected by gas chromatography. We, therefore, conclude that the three mutants do not exhibit significant changes in the structural and functional properties from those of wild-type P450cam except for the stability of the axial ligand in the reduced form. The enhanced fluorescence in the whole-cell assay would reflect enhancement in the oxygenation activity below the detectable limit of the gas chromatography and/or contributions of other reactions catalyzed by the heme iron. (C) 2004 Elsevier Inc. All rights reserved.
• L358P mutation on cytochrome P450cam simulates structural changes upon putidaredoxin binding - The structural changes trigger electron transfer to oxy-P450cam from electron donors

  T Tosha, S Yoshioka, K Ishimori, Morishima, I

  JOURNAL OF BIOLOGICAL CHEMISTRY 279 (41) 42836 - 42843 0021-9258 2004/10 [Refereed][Not invited]
   

  To investigate the functional and structural characterization of a crucial cytochrome P450cam ( P450cam)putidaredoxin (Pdx) complex, we utilized a mutant whose spectroscopic property corresponds to the properties of the wild type P450cam in the presence of Pdx. The H-1 NMR spectrum of the carbonmonoxy adduct of the mutant, the Leu- 358 --> Pro mutant (L358P), in the absence of Pdx showed that the ring current-shifted signals arising from D-camphor were upfield-shifted and observed as resolved signals, which are typical for the wild type enzyme in the presence of Pdx. Signals from the beta-proton of the axial cysteine and the gamma-methyl group of Thr-252 were also shifted upfield and downfield, respectively, in the L358P mutant as observed for Pdx-bound wild type P450cam. The close similarity in the NMR spectra suggests that the heme environment of the L358P mutant mimics that of the Pdx-bound enzyme. The functional analysis of the L358P mutant has revealed that the oxygen adduct of the L358P mutant can promote the oxygenation reaction for D-camphor with nonphysiological electron donors such as dithionite and ascorbic acid, showing that oxygenated L358P is "activated" to receive electron from the donor. Based on the structural and functional characterization of the L358P mutant, we conclude that the Pdx-induced structural changes in P450cam would facilitate the electron transfer from the electron donor, and the Pdx binding to P450cam would be a trigger for the electron transfer to oxygenated P450cam.
• Crystal structure of the cytochrome P450cam mutant that exhibits the same spectral perturbations induced by putidaredoxin binding

  S Nagano, T Tosha, K Ishimori, Morishima, I, TL Poulos

  JOURNAL OF BIOLOGICAL CHEMISTRY 279 (41) 42844 - 42849 0021-9258 2004/10 [Refereed][Not invited]
   

  The cytochrome P450cam active site is known to be perturbed by binding to its redox partner, putidaredoxin (Pdx). Pdx binding also enhances the camphor monooxygenation reaction (Nagano, S., Shimada, H., Tarumi, A., Hishiki, T., Kimata- Ariga, Y., Egawa, T., Suematsu, M., Park, S.- Y., Adachi, S., Shiro, Y., and Ishimura, Y. ( 2003) Biochemistry 42, 14507 - 14514). These effects are unique to Pdx because nonphysiological electron donors are unable to support camphor monooxygenation. The accompanying H-1 NMR paper ( Tosha, T., Yoshioka, S., Ishimori, K., and Morishima, I. ( 2004) J. Biol. Chem. 279, 42836 - 42843) shows that the conformation of active site residues, Thr-252 and Cys-357, and the substrate in the ferrous ( Fe(II)) CO complex of the L358P mutant mimics that of the wild-type enzyme complexed to Pdx. To explore how these changes are transmitted from the Pdx-binding site to the active site, we have solved the crystal structures of the ferrous and ferrous-CO complex of wild-type and the L358P mutant. Comparison of these structures shows that the L358P mutation results in the movement of Arg-112, a residue known to be important for putidaredoxin binding, toward the heme. This change could optimize the Pdx-binding site leading to a higher affinity for Pdx. The mutation also pushes the heme toward the substrate and ligand binding pocket, which relocates the substrate to a position favorable for regio-selective hydroxylation. The camphor is held more firmly in place as indicated by a lower average temperature factor. Residues involved in the catalytically important proton shuttle system in the I helix are also altered by the mutation. Such conformational alterations and the enhanced reactivity of the mutant oxy complex with nonphysiological electron donors suggest that Pdx binding optimizes the distal pocket for monooxygenation of camphor.
• Activation mechanisms of transcriptional regulator CooA revealed by small-angle X-ray scattering

  S Akiyama, T Fujisawa, K Ishimori, Morishima, I, S Aon

  JOURNAL OF MOLECULAR BIOLOGY 341 (3) 651 - 668 0022-2836 2004/08 [Refereed][Not invited]
   

  CooA, a heme-containing transcriptional activator, binds CO to the heme moiety and then undergoes a structural change that promotes the specific binding to the target DNA. To elucidate the activation mechanism coupled to CO binding, we investigated the CO-dependent structural transition of CooA with small-angle X-ray scattering (SAXS). In the absence of CO, the radius of gyration (Rg) and the second virial coefficient (A(2)) were 25.3(+/-0.5) A and - 0.39(+/-0.25) x 10(-4) ml mol g(-2), respectively. CO binding caused a slight increase in R-g (by 0.5 Angstrom) and a marked decrease in A(2) (by 5.09 x 10(-4) ml mol g(-2)). The observed decrease in A(2) points to higher attractive interactions between CO-bound CooA molecules in solution compared with CO-free CooA. Although the minor alternation of R-g rules out changes in the overall structure, the marked change in the surface properties points to a CO-induced conformational transition. The experimental Rg and SAXS curves of the two states did not agree with the crystal structure of CO-free CooA. We thus simulated the solution structures of CooA based on the experimental data using rigid-body refinements as well as low-resolution model reconstructions. Both results demonstrate that the hinge region connecting the N-terminal heme domain and C-terminal DNA-binding domain is kinked in CO-free CooA, so that the two domains are positioned close to each other. The CO-dependent structural change observed by SAXS corresponds to a slight swing of the DNA-binding domains away from the heme domains coupled with their rotation by about 8degrees around the axis of 2-fold symmetry. (C) 2004 Elsevier Ltd. All rights reserved.
• Dioxygen reduction by bo-type quinol oxidase from Escherichia coli studied by submillisecond-resolved freeze-quench EPR spectroscopy

  K Matsuura, S Yoshioka, S Takahashi, K Ishimori, T Mogi, H Hori, Morishima, I

  BIOCHEMISTRY 43 (8) 2288 - 2296 0006-2960 2004/03 [Refereed][Not invited]
   

  The mechanism of the dioxygen (O-2) reduction conducted by cytochrome bo-type quinol oxidase was investigated using submillisecond-resolved freeze-quench EPR spectroscopy. The fully reduced form of the wild-type enzyme (WT) with the bound ubiquinone-8 at the high-affinity quinone-binding site was mixed with an O-2-saturated solution, and the subsequent reaction was quenched at different time intervals from 0.2 to 50 ms. The EPR signals derived from the binuclear center and heme b were weak in the time domain from 0.2 to 0.5 ms. The signals derived from the ferric heme b and hydroxide-bound ferric heme o increased simultaneously after I ms, indicating that the oxidation of heme b is coupled to the formation of hydroxy heme o. In contrast, the enzyme without the bound ubiquinone-8 (DeltaUbiA) showed the faster oxidation of heme b and the slower formation of hydroxy heme o than WT. It is interpreted that the F-I intermediate possessing ferryl-oxo heme o. cupric Cu-B, and ferric heme b is converted to the F-II intermediate within 0.2 ms by an electron transfer from the bound ubiquinonol-8 to ferric heme b. The conversion of the F-II intermediate to the hydroxy intermediate occurred after 1 ms and was accompanied by the one-electron transfer from heme b to the binuclear center. Finally, it is suggested that the hydroxy intermediate possesses no bridging ligand between heme o and Cu-B and is the final intermediate in the turnover cycle of cytochrome bo under steady-state conditions.
• Identification of crucial histidines for heme binding in the N-terminal domain of the heme-regulated eIF2 alpha kinase

  T Inuzuka, BG Yun, H Ishikawa, S Takahashi, H Hori, RL Matts, K Ishimori, Morishima, I

  JOURNAL OF BIOLOGICAL CHEMISTRY 279 (8) 6778 - 6782 0021-9258 2004/02 [Refereed][Not invited]
   

  The heme-regulated eukaryotic initiation factor-2alpha (eIF2alpha) kinase (HRI) regulates the initiation of protein synthesis in reticulocytes. The binding of NO to the N-terminal heme-binding domain (NTD) of HRI positively modulates its kinase activity. By utilizing UV-visible absorption, resonance Raman, EPR and CD spectroscopies, two histidine residues have been identified that are crucial for the binding of heme to the NTD. The UV-visible absorption and resonance Raman spectra of all the histidine to alanine mutants constructed were similar to those of the unmutated NTD. However, the change in the CD spectra of the NTD construct containing mutation of His(78) to Ala (H78A) indicated loss of the specific binding of heme. The EPR spectrum for the ferric H78A mutant was also substantially perturbed. Thus, His(78) is one of the axial ligands for the NTD of HRL Significant changes in the EPR spectrum of the H123A mutant were also observed, and heme readily dissociated from both the H123A and the H78A NTD mutants, suggesting that His(123) was also an axial heme ligand. However, the CD spectrum for the Soret region of the H123A mutant indicated that this mutant still bound heme specifically. Thus, while both His(78) and His(123) are crucial for stable heme binding, the effects of their mutations on the structure of the NTD differed. His(78) appears to play the primary role in the specific binding of heme to the NTD, acting analogously to the "proximal histidine" ligand of globins, while His(123) appears to act as the "distal" heme ligand.
• Collapse and search dynamics of apomyoglobin folding revealed by submillisecond observations of alpha-helical content and compactness

  T Uzawa, S Akiyama, T Kimura, S Takahashi, K Ishimori, Morishima, I, T Fujisawa

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 101 (5) 1171 - 1176 0027-8424 2004/02 [Refereed][Not invited]
   

  The characterization of protein folding dynamics in terms of secondary and tertiary structures is important in elucidating the features of intraprotein interactions that lead to specific folded structures. Apomyoglobin (apoMb), possessing seven helices termed A-E, G, and H in the native state, has a folding intermediate composed of the A, G, and H helices, whose formation in the submillisecond time domain has not been clearly characterized. In this study, we used a rapid-mixing device combined with circular dichroism and small-angle x-ray scattering to observe the submillisecond folding dynamics of apoMb in terms of helical content (f(H)) and radius of gyration (R-g), respectively. The folding of apoMb from the acid-unfolded state at pH 2.2 was initiated by a pH jump to 6.0. A significant collapse, corresponding to approximate to50% of the overall change in R-g from the unfolded to native conformation, was observed within 300 Its after the pH jump. The collapsed intermediate has a f(H) of 33% and a globular shape that involves >80% of all its atoms. Subsequently, a stepwise helix formation was detected, which was interpreted to be associated with a conformational search for the correct tertiary contacts. The characterized folding dynamics of apoMb indicates the importance of the initial collapse event, which is suggested to facilitate the subsequent conformational search and the helix formation leading to the native structure.
• 2P077 Regulation of Heme Peroxidase Reactions by Controlling the Position of the Radical Center

  Ichimura S., Tosha T., Sugihara Y., Kitagawa T., Ishimori K., Morishima I.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 44 S129  2004
• 2P062 Heme Binding Affinity of Iron Response Regulator (Irr) and its Regulation Mechanism

  Nakagaki M., Bamba A., Ishikawa H., Iwai K., O'Brian Mark, Morishima I., Is K.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 44 S125  2004
• 2P066 Activation Mechanisms of Transcriptional Regulator CooA Revealed by Small-angle X-ray Scattering

  Akiyama S., Fujisawa T., Ishimori K., Morishima I., Aono S.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 44 S126  2004
• 2P063 Spectroscopic Characterization of Heme Binding and Coordination Structure in Iron Regulatory Protein 2(IRP2)

  Endo R., Ishikawa H., Iwai K., Morishima I., Ishimori K.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 44 S125  2004
• 2P076 The Stabilization of Novel Heme Proteins Using Phage Display

  Nogami K., Takata K., Ishimori K., Morishima I.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 44 S128  2004
• 3P017 Contribution of dehydration to reduced cytochrome c folding revealed by high pressure measurements

  Kimura T., Sekiyama N., Sakamoto K., Morishima I., Ishimori K.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 44 S194  2004
• 3P023 Significance of oligomerization and rapid collapse in hemeoxygenase folding

  Uzawa T., Kimura T., Takahashi S., Ishimori K., Akiyama S., Fujisawa T., Matsui T., Saito M.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 44 S195  2004
• NMR study on the structural changes of cytochrome P450cam upon the complex formation with putidaredoxin - Functional significance of the putidaredoxin-induced structural changes

  T Tosha, S Yoshioka, S Takahashi, K Ishimori, H Shimada, Morishima, I

  JOURNAL OF BIOLOGICAL CHEMISTRY 278 (41) 39809 - 39821 0021-9258 2003/10 [Refereed][Not invited]
   

  We investigated putidaredoxin-induced structural changes in carbonmonoxy P450cam by using NMR spectroscopy. The resonance from the beta-proton of the axial cysteine was upfield shifted by 0.12 ppm upon the putidaredoxin binding, indicating that the axial cysteine approaches to the heme-iron by about 0.1 Angstrom. The approach of the axial cysteine to the heme-iron would enhance the electronic donation from the axial thiolate to the heme-iron, resulting in the enhanced heterolysis of the dioxygen bond. In addition to the structural perturbation on the axial ligand, the structural changes in the substrate and ligand binding site were observed. The resonances from the 5-exo- and 9-methyl-protons of d-camphor, which were newly identified in this study, were upfield shifted by 1.28 and 0.20 ppm, respectively, implying that d-camphor moves to the heme-iron by 0.15-0.7 Angstrom. Based on the radical rebound mechanism, the approach of d-camphor to the heme-iron could promote the oxygen transfer reaction. On the other hand, the downfield shift of the resonance from the gamma-methyl group of Thr-252 reflects the movement of the side chain away from the heme-iron by similar to0.25 Angstrom. Because Thr-252 regulates the heterolysis of the dioxygen bond, the positional rearrangement of Thr-252 might assist the scission of the dioxygen bond. We, therefore, conclude that putidaredoxin induces the specific heme environmental changes of P450cam, which would facilitate the oxygen activation and the oxygen transfer reaction.
• Kinetic and spectroscopic characterization of a hydroperoxy compound in the reaction of native myoglobin with hydrogen peroxide

  T Egawa, S Yoshioka, S Takahashi, H Hori, S Nagano, H Shimada, K Ishimori, Morishima, I, M Suematsu, Y Ishimura

  JOURNAL OF BIOLOGICAL CHEMISTRY 278 (43) 41597 - 41606 0021-9258 2003/10 [Refereed][Not invited]
   

  The reaction of metmyoglobin with H2O2 was investigated in a pH range between 8.5 and 6.0 with the aid of stopped flow-rapid scan and rapid freezing-EPR techniques. Singular value decomposition analyses of the stopped flow data at pH 8.5 revealed that a spectral species previously unknown accumulated during the reaction and exhibited a Soret absorption maximum at greater than or equal to423 nm. In the EPR experiments, the new species exhibited a set of g values at 2.32, 2.19, and 1.94, indicating that the species was assignable to a ferric hydroperoxy (Fe(III)[O-O-H](-)) compound. In contrast, the hydroperoxy compound scarcely accumulated in the reaction at pH 6.0, and the dominant intermediate species accumulated was compound I, which was derived from the oxygen-oxygen bond cleavage of the hydroperoxy compound. The accumulated amount of the hydroperoxy compound relative to compound I showed a pH dependence with an apparent pK(a) (pK(a)(app)) from 6.95 to 7.27 depending on the metmyoglobins examined. This variation in pK(a)(app) paralleled that in pK(a) of the acid-alkaline transition (pK(a)(AB)) of metmyoglobins, suggesting that the accumulation of hydroperoxy compound is controlled by the distal histidine. We propose that the H2O2 activation by metmyoglobin is promoted at the acidic condition due to the imidazolium form of the distal histidine, and we further propose that the controlled protonation state of the distal histidine is important for the facile O-O bond cleavage in heme peroxidases.
• The observation of the dioxygen activation of cytochrome bo from Escherichia coli by submillisecond-resolved freeze quench EPR spectroscopy

  K Matsuura, S Yoshioka, S Takahashi, K Ishimori, T Mogi, H Hori, Morishima, I

  JOURNAL OF INORGANIC BIOCHEMISTRY 96 (1) 188 - 188 0162-0134 2003/07 [Refereed][Not invited]
  
• Identification of the ubiquitin-protein ligase that recognizes oxidized IRP2

  K Yamanaka, H Ishikawa, Y Megumi, F Tokunaga, M Kanie, TA Rouault, Morishima, I, N Minato, K Ishimori, K Iwai

  NATURE CELL BIOLOGY 5 (4) 336 - 340 1465-7392 2003/04 [Refereed][Not invited]
   

  The ubiquitin system is involved in several basic cellular functions(1-3). Ubiquitination is carried out by a cascade of three reactions catalysed by the El, E2 and E3 enzymes. Among these, the E3 ubiquitin-protein ligases have a pivotal role in determining the specificity of the system by recognizing the target substrates through defined targeting motifs(1-3). Although RING finger proteins constitute an important family of E3 ligases(4), only a few post-transcriptional modifications, including phosphorylation(1), proline hydroxylation(5,6) and glycosylation(7), are known to function as recognition signals for E3. Iron regulatory protein 2 (IRP2), a modulator of iron metabolism, is regulated by iron-induced ubiquitination and degradation(8). Here we show that the RING finger protein HOIL-1 functions as an E3 ligase for oxidized IRP2, suggesting that oxidation is a specific recognition signal for ubiquitination. The oxidation of IRP2 is generated by haem, which binds to IRP2 in iron-rich cells, and by oxygen, indicating that the iron sensing of IRP2 depends on the synthesis and availability of haem.
• Iron hemiporphycene as a functional prosthetic group for myoglobin

  S Neya, K Imai, H Hori, H Ishikawa, K Ishimori, D Okuno, S Nagatomo, T Hoshino, M Hata, N Funasaki

  INORGANIC CHEMISTRY 42 (5) 1456 - 1461 0020-1669 2003/03 [Refereed][Not invited]
   

  The iron complex of hemiporphycene, a molecular hybrid of porphyrin with porphycene, was incorporated into the apomyoglobin pocket to examine ligand binding ability of the iron atom in the novel porphyrinoid. Apomyoglobin was successfully coupled with a stoichiometric amount of ferric hemiporphycene to afford the reconstituted myoglobin equipped with the iron coordination structure of native protein. Cyanide, imidazole, and fluoride coordinated to the ferric protein with affinities comparable with those for native myoglobin. The ferrous myoglobin was functionally active to bind O-2 and CO reversibly at pH 7.4 and 20 degreesC. The O-2 affinity is 12-fold higher than that of native myoglobin while the CO affinity is slightly lower, suggesting decreased discrimination between O-2 and CO in the heme pocket. The functional anomaly was interpreted to reflect increased sigma-bonding character in the Fe(II)-O-2 bond. In contrast with 6-coordinate native NO protein, the NO myoglobin containing ferrous hemiporphycene is in a mixed 5- and 6-coordinate state. This observation suggests that the in-plane configuration of the iron atom in hemiporphycene is destabilized by NO. Influence of the core deformation was also detected with both the infrared absorption for the ferrous CO derivative and electron paramagnetic resonance for ferric imidazole complex. Anomalies in the ferric and ferrous derivatives were ascribed to the modified iron-N(pyrrole) interactions in the asymmetric metallo core of hemiporphycene.
• Activation of hydrogen peroxide in horseradish peroxidase occurs within similar to 200 mu s observed by a new freeze-quench device

  M Tanaka, K Matsuura, S Yoshioka, S Takahashi, K Ishimori, H Hori, Morishima, I

  BIOPHYSICAL JOURNAL 84 (3) 1998 - 2004 0006-3495 2003/03 [Refereed][Not invited]
   

  To observe the formation process of compound I in horseradish peroxidase (HRP), we developed anew freeze-quench device with similar to200 mus of the mixing-to-freezing time interval and observed the reaction between HRP and hydrogen peroxide (H2O2). The developed device consists of a submillisecond solution mixer and rotating copper or silver plates cooled at 77 K; it freezes the small droplets of mixed solution on the surface of the rotating plates. The ultraviolet-visible spectra of the sample quenched at similar to1 ms after the mixing of HRP and H2O2 suggest the formation of compound 1. The electron paramagnetic resonance spectra of the same reaction quenched at similar to200 mus show a convex peak at g = 2.00, which is identified as compound I due to its microwave power and temperature dependencies. The absence of ferric signals in the electron paramagnetic resonance spectra of the quenched sample indicates that compound I is formed within similar to200 mus after mixing HRP and H2O2. We conclude that the activation of H2O2 in HRP at ambient temperature completes within similar to200 mus. The developed device can be generally applied to investigate the electronic structures of short-lived intermediates of metalloenzymes.
• Activation of hydrogen peroxide in horseradish peroxidase occurs within approximately 200 micro s observed by a new freeze-quench device.

  Motomasa Tanaka, Koji Matsuura, Shiro Yoshioka, Satoshi Takahashi, Koichiro Ishimori, Hiroshi Hori, Isao Morishima

  Biophysical journal 84 (3) 1998 - 2004 0006-3495 2003/03 [Refereed][Not invited]
   

  To observe the formation process of compound I in horseradish peroxidase (HRP), we developed a new freeze-quench device with approximately 200 micro s of the mixing-to-freezing time interval and observed the reaction between HRP and hydrogen peroxide (H(2)O(2)). The developed device consists of a submillisecond solution mixer and rotating copper or silver plates cooled at 77 K; it freezes the small droplets of mixed solution on the surface of the rotating plates. The ultraviolet-visible spectra of the sample quenched at approximately 1 ms after the mixing of HRP and H(2)O(2) suggest the formation of compound I. The electron paramagnetic resonance spectra of the same reaction quenched at approximately 200 micro s show a convex peak at g = 2.00, which is identified as compound I due to its microwave power and temperature dependencies. The absence of ferric signals in the electron paramagnetic resonance spectra of the quenched sample indicates that compound I is formed within approximately 200 micro s after mixing HRP and H(2)O(2). We conclude that the activation of H(2)O(2) in HRP at ambient temperature completes within approximately 200 micro s. The developed device can be generally applied to investigate the electronic structures of short-lived intermediates of metalloenzymes.
• Regulation Mechanism of CooA Studied by Small-Angle X-Ray Scattering

  Akiyama S., Fujisawa T., Maeda Y., Ishimori K., Morishima I., Aono S.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 43 S91  2003
• Characterization of hydrophobic cluster in early step of cytochrome c folding

  Yane A., takahashi S., Ishimori K., Morishima I.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 43 S59  2003
• Main-chain collapse and subsequent tertiary structure formation during apomyoglobin folding

  Uzawa T., Kimura T., Takahashi S., Ishimori K., Morishima I., Akiyama S., Fujisawa T.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 43 S60  2003
• Roles of the N-terminal fluctuating domain in folding of coiled coil GCN4-pl'

  Kinoshita M., Kimura T., Takahashi S., Ishimori K., Morishima I.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 43 S61  2003
• Folding Mechanism of Single-chain Monellin Based on the Secondary Structure Formation and the Packing of the Hydrophobic Residues

  Kimura T., Takahashi S., Konno T., Ishimori K., Morishima I.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 43 S59  2003
• Roles of the proximal hydrogen bonding network in cytochrome P450(cam)-catalyzed oxygenation

  S Yoshioka, T Tosha, S Takahashi, K Ishimori, H Hori, Morishima, I

  JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 124 (49) 14571 - 14579 0002-7863 2002/12 [Refereed][Not invited]
   

  Structural and functional roles of the hydrogen bonding network that surrounds the heme-thiolate coordination of P450(cam) from Pseudomonas putida were investigated. A hydrogen bond between the side chain amide of Gln360 and the carbonyl oxygen of the axial Cys357 was removed in Q360L. The side chain hydrogen bond and the electrostatic interaction between the polypeptide amide proton of Gln360 and the sulfur atom of Cys357 were simultaneously removed in Q360P. The increased electron donation of the axial thiolate in Q360L and Q360P was evidenced by negative shifts of their reduction potentials by 45 and 70 mV, respectively. Together with the results on L358P in which the amide proton at position 358 was removed (Yoshioka, S., Takahashi, S., Ishimori, K., Morishima, I.J. Inorg. Biochem. 2000, 81, 141-151), we propose that the side chain hydrogen bond and the electrostatic interaction of the amide proton with the thiolate ligand cause similar to45 and similar to35 mV of positive shifts, respectively, of the redox potential of the heme in P450(cam). The resonance Raman spectra of the ferrous-CO form of the Q360 mutants showed a downshifted Fe-CO stretching mode at 482similar to483 cm(-1) compared with that of wild-type P450(cam) at 484 cm(-1). The Q360 mutants also showed the upshift by 4similar to5 cm-1 of the Fe-NO stretching mode in the ferrous-NO form. These Raman results indicate the increase in the sigma-electron donation of the thiolate ligand in the reduced state of the 0360 mutants and were in contrast to the increased pi-back-do nation of the thiolate in L358P having an upshifted Fe-CO stretching mode at 489 cm(-1). The catalytic activities of the Q360 mutants for the unnatural substrates were similar to those of the wild-type enzyme, indicating that the increased sigma-electron donation does not promote the O-O bond heterolysis in the Q360 mutants, although the increased pi-electron donation in L358P promoted the heterolysis of the O-O bond. We conclude that the functions of the proximal hydrogen bonding network in P450(cam) are to stabilize the heme-thiolate coordination, and to regulate the redox potential of the heme iron. Furthermore, we propose that the pi-electron donation, not the sigma-electron donation, of the thiolate ligand promotes the heterolysis of the O-O bond of dioxygen.
• NO-induced activation mechanism of the heme-regulated eIF2 alpha kinase

  H Ishikawa, BG Yun, S Takahashi, H Hori, RL Matts, K Ishimori, Morishima, I

  JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 124 (46) 13696 - 13697 0002-7863 2002/11 [Refereed][Not invited]
  
• Molecular mechanism of the electron transfer reaction in cytochrome P450(cam)-putidaredoxin: Roles of glutamine 360 at the heme proximal site

  T Tosha, S Yoshioka, H Hori, S Takahashi, K Ishimori, Morishima, I

  BIOCHEMISTRY 41 (47) 13883 - 13893 0006-2960 2002/11 [Refereed][Not invited]
   

  We characterized electron transfer (ET) from putidaredoxin (Pdx) to the mutants of cytochrome P450(cam) (P450(cam)), in which one of the residues located on the putative binding site to Pdx, Gln360, was replaced with Glu, Lys, and Len. The kinetic analysis of the ET reactions from reduced Pdx to ferric P450(cam) (the first ET) and to ferrous oxygenated P450(cam) (the second ET) showed the dissociation constants (K) that were moderately perturbed for the Lys and Len mutants and the distinctly increased for the Glu mutant. Although the alterations in K-m indicate that Gln360 is located at the Pdx binding site, the effects of the Gln360 mutations (0.66-20-fold of that of wild type) are smaller than those of the Arg112 mutants (25-2500-fold of that of wild type) [Unno, M., et al. (1996) J. Biol. Chem. 271, 17869-17874], allowing us to conclude that Gln360 much less contributes to the complexation with Pdx than Arg112. The first ET rate (35 s(-1) for wild-type P450(cam)) was substantially reduced in the Ght mutant (5.4 s(-1)), while less perturbation was observed for the Lys (53 s(-1)) and Leu (23 s(-1)) mutants. In the second ET reaction, the retarded ET rate was detected only in the Ght mutant but not in the Lys and Len mutants. These results showed the smaller mutational effects of Gln360 on the ET reactions than those of the Arg112 mutants. In contrast to the moderate perturbations in the kinetic parameters, the mutations at Gln360 significantly affected both the standard enthalpy and entropy of the redox reaction of P450(cam), which cause the negative shift of the redox potentials for the Fe3+/Fe2+ couple by 20-70 mV. Since the arnide group of Gln360 is located near the carbonyl oxygen of the amide group of the axial cysteine, it is plausible that the mutation at Gln360 perturbs the electronic interaction of the axial ligand with heme iron, resulting in the reduction of the redox potentials. We, therefore, conclude that Gln360 primarily regulates the ET reaction of P450cam by modulating the redox potential of the heme iron and not by the specific interaction with Pdx or the formation of the ET pathway that are proposed as the regulation mechanism of Arg 112.
• Direct observation of the multistep helix formation of poly-L-glutamic acids

  T Kimura, S Takahashi, S Akiyama, T Uzawa, K Ishimori, Morishima, I

  JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 124 (39) 11596 - 11597 0002-7863 2002/10 [Refereed][Not invited]
  
• Oxidation-state-dependent protein docking between cytochrome c and cytochrome b(5): High-pressure laser flash photolysis study

  Y Furukawa, K Ishimori, Morishima, I

  BIOCHEMISTRY 41 (31) 9824 - 9832 0006-2960 2002/08 [Refereed][Not invited]
   

  To characterize the protein-protein interaction during electron transfer, we used Zn-substituted cytochrome c (ZnCytc) as a model of ferrous Cytc and determined the volume change, DeltaV(d)(Zn), for the dissociation of its complex with ferric cytochrome b(5) (Cytb(5)) by the pressure dependence of its photoinduced electron-transfer kinetics. Under ambient pressure, the dissociation constant, K-d(Zn), of the ZnCytc/Cytb(5) complex was dependent on the buffer concentration, 1.5 and 12 muM in 2 and 10 mM Tris-HCl, pH 7.4, respectively, which was consistent with formation of salt bridges in its complexation. The dissociation of one salt bridge is usually associated with large volume changes of -10 to -30 cm(3) mol(-1), while pressure dependence of K-d(Zn) resulted in smaller value of DeltaV(d)(Zn), -8.5 cm(3) mol(-1). Therefore, the interaction between ZnCytc and Cytb5 cannot be explained only by salt bridge interaction, and the partial cancellation by the positive volume change due to the additional hydrophobic interaction is a plausible explanation for the observed DeltaV(d)(Zn). In addition, DeltaV(d)(Zn) of -8.5 cm(3) mol(-1) was considerably smaller than the previously reported volume change, DeltaV(d)(Fe), of -122 cm(3) mol(-1) in the ferric Cytc/Cytb(5) complex dissociation [Rodgers and Sligar (1991) J. Mol. Biol. 221, 1453-1460]. ZnCytc used here has been assumed to be a reliable model of ferrous Cytc, and thus the discrepancy between our present DeltaV(d)(Zn) and the previous DeltaV(d)(Fe) is discussed on the basis of the protein docking dependent on the oxidation states of heme iron in Cytc.
• Investigation of the electron-transfer mechanism by cross-linking between Zn-substituted myoglobin and cytochrome b(5)

  Y Furukawa, F Matsuda, K Ishimori, Morishima, I

  JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 124 (15) 4008 - 4019 0002-7863 2002/04 [Refereed][Not invited]
   

  We have investigated the photoinduced electron transfer (ET) in the 1: 1 cross-linked complex (CL-ZnMb/b(5)) formed by a cross-linking reagent, EDC, between Zn-substituted myoglobin (ZnMb) and cytochrome b(5) (Cytb(5)) to reveal the mechanism of the inter-protein ET reactions under the condition of multiple encounter complexes. A variety of the ZnMb-Cytb(5) orientations was suggested because of failure to identify the single and specific cross-linking site on Cytb(5) by the peptide-mapping analysis using mass spectrometry. In CL-ZnMb/b(5), a laser pulse generates the triplet excited state of the ZnMb domain ((3)ZnMb(*)), which can transfer one electron to the Cytb(5) domain. The decay kinetics of (3)ZnMb(*) in CL-ZnMb/b(5) consists of a facile power-law ET phase to Cytb(5) domain (similar to30%) and a slower single-exponential phase (similar to70%). The application of the Marcus equation to this power-law phase indicates that CL-ZnMb/b(5) has a variety of ZnMb-Cytb(5) orientations for the facile ET in which the distance between the redox centers (D-A distance) is distributed over 13-20 Angstrom. The single-exponential phase in the (3)ZnMb(*) decay kinetics of CL-ZnMb/b(5) is similar to the intrinsic decay of (3)ZnMb(*) in its rate constant, 65 s(-1), This implies that the ET is impeded in about 70% of the total ZnMb-Cytb(5) orientations due to the D-A distance larger than 20 Angstrom. Combined with the results of the Brownian dynamics simulations for the encounter complexes, the overall bimolecular ET rate, k(app), can be reproduced by the sum of the ET rates for the minor encounter complexes of which D-A distance is less than 20 Angstrom. On the other hand, the encounter complexes with longer D-A distance, which are the majority of the encounter complexes between ZnMb and Cytb(5), have little contribution to the overall bimolecular ET rate. These observations experimentally demonstrate that ZnMb forms a variety of encounter complexes with Cytb(5), among which a minor set of the complexes with the shorter D-A distance < &SIM;20 &ANGS;) regulates the overall bimolecular ET between the proteins.
• Conformational landscape of cytochrome c folding studied by microsecond-resolved small-angle x-ray scattering

  S Akiyama, S Takahashi, T Kimura, K Ishimori, Morishima, I, Y Nishikawa, T Fujisawa

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 99 (3) 1329 - 1334 0027-8424 2002/02 [Refereed][Not invited]
   

  To investigate protein folding dynamics in terms of compactness, we developed a continuous-flow mixing device to make small-angle x-ray scattering measurements with the time resolution of 160 mus and characterized the radius of gyration (R-g) of two folding intermediates of cytochrome c (cyt c). The early intermediate possesses approximate to20 Angstrom of R-g, which is smaller by approximate to4 Angstrom than that of the acid-unfolded state. The R-g of the later intermediate is approximate to18 Angstrom, which is close to that of the molten globule state. Considering the alpha-helix content (f(H)) of the intermediates, we clarified the folding pathway of cyt c on the conformational landscape defined by R-g and f(H). Cyt c folding proceeds with a collapse around a specific region of the protein followed by a cooperative acquisition of secondary structures and compactness.
• Myoglobin

  2002/02 [Refereed][Not invited]
  
• 3M1030 Observation on the folding process of single-chain monellin studied by microsecond-resolved FTIR spectroscopy

  Kimura T., Takahashi S., Konno T., Ishimori K., Morishima I.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 42 (2) S190  2002
• 1K0945 Electronic states of the redox centers in dioxygen reduction of cytochrome bo from Escherichia coli by rapid-freezing EPR Method

  Matsuura K., Yoshioka S., Takahashi S., Ishimori K., Morishima I., Mogi T., Hori H.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 42 (2) S60  2002
• 1L1700 Design of Horseradish Peroxidases with the Enhanced Catalase Activity and their Mechanism of Enhancing the Catalase Activity

  Hirose S., Tosha T., Shimada H., Takahashi S., Ishimori K., Morishima I.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 42 (2) S72  2002
• Pressure Effects on the Intramolecular Electron Transfer Reactions in Hemoproteins

  Yoshiaki Furukawa, Yoichi Sugiyama, Satoshi Takahashi, Koichiro Ishimori, Isao Morishima

  Springer Nature 187  2002 [Refereed][Not invited]
  
• Binding of CO at the Pro(2) side is crucial for the activation of CO-sensing transcriptional activator CooA - H-1 NMR spectroscopic studies

  K Yamamoto, H Ishikawa, S Takahashi, K Ishimori, Morishima, I, H Nakajima, S Aono

  JOURNAL OF BIOLOGICAL CHEMISTRY 276 (15) 11473 - 11476 0021-9258 2001/04 [Refereed][Not invited]
   

  CooA is a heme-containing transcriptional activator that anaerobically binds to DNA at CO atmosphere. To obtain information on the conformational transition of CooA induced by CO binding to the heme, we assigned ring current-shifted H-1 NMR signals of CooA using two mutants whose axial Ligands of the heme were replaced. In the absence of CO, the NMR spectral pattern of H77T CooA in which the axial histidine (His(77)) was replaced with tyrosine, was similar to that of wild-type CooA. In contrast, the spectra of CooA Delta N5, in which the NH2 termini including the other axial ligand (Pro(2)) were deleted, were drastically modulated. We assigned three signals of wild-type CooA at -4.5, -3.6, and -2.8 ppm to delta (1)-, alpha-, and delta (2)-protons of Pro(2), respectively. The Pro(2) signals were undetectable in the upfield region of the spectrum of the CO-bound state, which confirms that CO displaces Pro(2), Interestingly, the Pro(2) signals were observed for CO-bound H77Y CooA implying that CO binds to the trans position of Pro(2) in H77Y CooA. The abolished CO-dependent transcriptional activity of H77Y CooA is therefore the consequence of Pro(2) ligation. These observations are consistent with the view that the movement of the NH2 terminus triggers the conformational transition to the DNA binding form.
• Ligand migration in human myoglobin: Steric effects of isoleucine 107(G8) on O-2 and CO binding

  H Ishikawa, T Uchida, S Takahashi, K Ishimori, Morishima, I

  BIOPHYSICAL JOURNAL 80 (3) 1507 - 1517 0006-3495 2001/03 [Refereed][Not invited]
   

  To investigate the ligand pathway in myoglobin, some mutant myoglobins, in which one of the amino acid residues constituting a putative ligand-docking site, Ile107, is replaced by Ala, Val, Leu, or Phe, were prepared and their structural and ligand binding properties were characterized. The kinetic barrier for the ligand entry to protein inside was lowered by decreasing the side-chain volume at position 107, indicating that the bulky side chain interferes with the formation of the activation state for the ligand migration and the free space near position 107 would be filled with the ligand in the activation state. Another prominent effect of the reduced side-chain volume at position 107 is to stabilize the ligand-binding intermediate state. Because the stabilization can be ascribed to decrease of the positive enthalpy, the enlarged free space near position 107 would relieve unfavorable steric interactions between the ligand and nearby amino acid residues. The side-chain volume at position 107, therefore, is crucial for the kinetic barrier for the ligand migration and free energy of the ligand-binding intermediate state, which allows us to propose that some photodissociated O-2 moves toward position 107 to be trapped and then expelled to the solvent.
• Ligand migration in human myoglobin: Steric effects of isoleucine 107(G8) on O-2 and CO binding

  H Ishikawa, T Uchida, S Takahashi, K Ishimori, Morishima, I

  BIOPHYSICAL JOURNAL 80 (3) 1507 - 1517 0006-3495 2001/03 [Refereed][Not invited]
   

  To investigate the ligand pathway in myoglobin, some mutant myoglobins, in which one of the amino acid residues constituting a putative ligand-docking site, Ile107, is replaced by Ala, Val, Leu, or Phe, were prepared and their structural and ligand binding properties were characterized. The kinetic barrier for the ligand entry to protein inside was lowered by decreasing the side-chain volume at position 107, indicating that the bulky side chain interferes with the formation of the activation state for the ligand migration and the free space near position 107 would be filled with the ligand in the activation state. Another prominent effect of the reduced side-chain volume at position 107 is to stabilize the ligand-binding intermediate state. Because the stabilization can be ascribed to decrease of the positive enthalpy, the enlarged free space near position 107 would relieve unfavorable steric interactions between the ligand and nearby amino acid residues. The side-chain volume at position 107, therefore, is crucial for the kinetic barrier for the ligand migration and free energy of the ligand-binding intermediate state, which allows us to propose that some photodissociated O-2 moves toward position 107 to be trapped and then expelled to the solvent.
• Time-resolved hole-burning study on myoglobin: fluctuation of restricted water within distal pocket

  Shibata Y, Ishikawa H, Ishimori K, Morishima I

  Biophysical Journal 80 1013 - 1023 2001/02 [Refereed][Not invited]
  
• Functional difference between IRE-recongnation and IRE-binding sites in IRPs

  Watanabe K., Ishikawa H., Iwai K., Ishimori K., Morishima I.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 41 S180  2001
• Apomyoglobin folding studied by submillisecond small-angle X-ray scattering

  Uzawa T, Akiyama S, Kimura T, Takahashi S, Ishimori K, Morishima I, Nishikawa Y, Fujisawa T

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 41 S166  2001
• Structural Studies on P450cam-Putidaredoxin Complex Using a P450cam Mutant

  Tosha T., Yoshioka S., Harada H., Ishimori K., Yano Jason, Poulos Thomas, Morishima I.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 41 S117  2001
• The recognition mechanism in the protein-protein electron transfer

  Furukawa T., Matsuda F., Ishimori K., Morishima I.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 41 S99  2001
• Proximal cysteine residue is essential for the enzymatic activities of cytochrome P450(cam)

  S Yoshioka, S Takahashi, H Hori, K Ishimori, Morishima, I

  EUROPEAN JOURNAL OF BIOCHEMISTRY 268 (2) 252 - 259 0014-2956 2001/01 [Refereed][Not invited]
   

  To investigate the functional and structural roles of the proximal thiolate ligand in cytochrome P450(cam), we prepared the C357H mutant of the enzyme in which the axial cysteine residue (Cys357) was replaced with a histidine residue. We obtained the unstable C357H mutant by developing a new preparation procedure involving in vitro folding of P450(cam) from the inclusion bodies. The C357H mutant in the ferrous-CO form exhibited the Soret peak at 420 nm and the Fe-CO stretching line at 498 cm(-1), indicating a neutral histidine residue as the axial ligand. However, another internal ligand is coordinated to the heme iron as the sixth ligand in the ferric and ferrous forms of the C357H mutant, suggesting the collapse of the substrate-binding site. The C357H mutant showed no catalytic activity for camphor hydroxylation and the reduced heterolytic/homolytic ratio of the O-O bond scission in the reaction with cumene hydroperoxide. The present observations indicate that the thiolate coordination in P450(cam) is important for the construction of the heme pocket and the heterolysis of the O-O bond.
• Proximal cysteine residue is essential for the enzymatic activities of cytochrome P450(cam)

  S Yoshioka, S Takahashi, H Hori, K Ishimori, Morishima, I

  EUROPEAN JOURNAL OF BIOCHEMISTRY 268 (2) 252 - 259 0014-2956 2001/01 [Refereed][Not invited]
   

  To investigate the functional and structural roles of the proximal thiolate ligand in cytochrome P450(cam), we prepared the C357H mutant of the enzyme in which the axial cysteine residue (Cys357) was replaced with a histidine residue. We obtained the unstable C357H mutant by developing a new preparation procedure involving in vitro folding of P450(cam) from the inclusion bodies. The C357H mutant in the ferrous-CO form exhibited the Soret peak at 420 nm and the Fe-CO stretching line at 498 cm(-1), indicating a neutral histidine residue as the axial ligand. However, another internal ligand is coordinated to the heme iron as the sixth ligand in the ferric and ferrous forms of the C357H mutant, suggesting the collapse of the substrate-binding site. The C357H mutant showed no catalytic activity for camphor hydroxylation and the reduced heterolytic/homolytic ratio of the O-O bond scission in the reaction with cumene hydroperoxide. The present observations indicate that the thiolate coordination in P450(cam) is important for the construction of the heme pocket and the heterolysis of the O-O bond.
• Conversion of an electron-transfer protein into an oxygen binding protein: The axial cytochrome b(5) mutant with an unusually high O-2 affinity

  M Ihara, M Shintaku, S Takahashi, K Ishimori, Morishima, I

  JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 122 (46) 11535 - 11536 0002-7863 2000/11 [Refereed][Not invited]
  
• Identification of histidine 77 as the axial heme ligand of carbonmonoxy CooA by picosecond time-resolved resonance Raman spectroscopy

  T Uchida, H Ishikawa, K Ishimori, Morishima, I, H Nakajima, S Aono, Y Mizutani, T Kitagawa

  BIOCHEMISTRY 39 (42) 12747 - 12752 0006-2960 2000/10 [Refereed][Not invited]
   

  The heme proximal ligand of carbonmonoxy CooA, a CO-sensing transcriptional activator, in the CO-bound form was identified to be His77 by using picosecond time-resolved resonance Raman spectroscopy. On the basis of the inverse correlation between Fe-CO and C-O stretching frequencies, we proposed previously that His77 is the axial ligand trans to CO [Uchida et al. (1998) J. Biol. Chem. 273, 19988-19992], whereas later a possibility of displacement of His77 by CO with retention of another unidentified axial ligand was reported [Vogel et al. (1999) Biochemistry 38, 2629-2682]. Although our previous resonance Raman study failed to detect the Fe-His stretching [v(Fe-His)] mode of CO-photodissociated CooA of the carbonmonoxy adduct due to the rapid recombination, application of the picosecond time-resolved resonance Raman technique enabled us to observe a new intense line assignable to v(Fe-His) at 211 cm(-1) immediately after photolysis, while it became nondiscernible after 100-ps delay. The low v(Fe-His) frequency of photodissociated CooA indicates the presence of some strain in the Fe-His bond in CO-bound CooA. This and the rapid recombination of CO characterize the heme pocket of CooA. The 211 cm(-1) band was completely absent in the spectrum of the CO-photodissociated form of the His77-substituted mutant but the Fe-Im stretching band was observed in the presence of exogenous imidazole (Im). Thus, we conclude that His77 is the axial ligand of CO-bound CooA and CO displaces the axial ligand trans to His77 with retention of ligated His77 to activate CooA as the transcriptional activator.
• Electron transfer reactions in Zn-substituted cytochrome P450cam

  Y Furukawa, K Ishimori, Morishima, I

  BIOCHEMISTRY 39 (36) 10996 - 11004 0006-2960 2000/09 [Refereed][Not invited]
   

  We have investigated photoinduced electron transfer (ET) reactions between zinc-substituted cytochrome P450cam (ZnP450) and several inorganic reagents by using the laser flash photolysis method, to reveal roles of the electrostatic interactions in the regulation of the ET reactions. The laser pulse irradiation to ZnP450 yielded a strong reductant, the triplet excited state of ZnP450, (3)ZnP450*, which was able to transfer one electron to anionic redox partners, OsCl62- and Fe(CN)(6)(3-), with formation of the porphyrin pi-cation radical, ZnP450(+). In contrast, the ET reactions from (3)ZnP450* to cationic redox partners, such as Ru(NH3)(6)(3+) and Co(phen)(3)(3+), were not observed even in the presence of 100-fold excess of the oxidant. One of the possible interpretations for the preferential ET to the anionic redox partner is that the cationic patch on the P450cam surface, a putative interaction site for the anionic reagents, is located near the heme (less than 10 Angstrom from the heme edge), while the anionic surface is far from the heme moiety (more than 16 Angstrom from the heme edge), which would yield 8000-fold faster ET rates through the cationic patch. The ET rate through the anionic patch to the cationic partner would be substantially slower than that of the phosphorescence process in (3)ZnP450*, resulting in no ET reactions to the cationic reagents. These results demonstrate that the asymmetrical charge distribution on the protein surface is critical for the ET reaction in P450cam.
• Unusual pressure effects on ligand rebinding to the human myoglobin leucine 29 mutants

  T Uchida, K Ishimori, Morishima, I

  JOURNAL OF BIOLOGICAL CHEMISTRY 275 (39) 30309 - 30316 0021-9258 2000/09 [Refereed][Not invited]
   

  Using high pressure flash photolysis, we revealed that the side chain of Leu(29) controls the reaction volume of the ligand migration process in myoglobin, which is the primary factor for the unusual activation volume of ligand binding in some Leu(29) mutants. As we previously reported (Adachi, S., Sunohara, N., Ishimori, K., and Morishima, I. (1992) J. Biol. Chem. 267, 12614-12621), CO bimolecular rebinding in the L29A mutant was unexpectedly decelerated by pressurization, suggesting that the rate-determining step is switched to ligand migration. However, very slow CO bimolecular rebinding of the mutants implies that bond formation is still the rate-determining step. To gain further insights into effects of the side chain on ligand binding, we prepared some new Leu(29) mutants to measure the CO and O-2 rebinding reaction rates under high hydrostatic pressure. CO bimolecular rebinding in the mutants bearing Gly or Ser at position 29 was also decelerated upon pressurization, resulting in apparent positive activation volumes (Delta V-double dagger), as observed for O-2 binding. Based on the three-state model, we concluded that the increased space available to ligands in these mutants enhances the volume difference between the geminate and deoxy states (Delta V-32), which shifts the apparent activation volume to the positive side, and that the apparent positive activation volume is not due to contribution of the ligand migration process to the rate-determining step.
• Roles of the axial push effect in cytochrome P450cam studied with the site-directed mutagenesis at the heme proximal site

  S Yoshioka, S Takahashi, K Ishimori, Morishima, I

  JOURNAL OF INORGANIC BIOCHEMISTRY 81 (3) 141 - 151 0162-0134 2000/08 [Refereed][Not invited]
   

  To examine the roles of the axial thiolate in cytochrome P450-catalyzed reactions, a mutant of cytochrome P450cam, L358P, was prepared to remove one of the conserved amide protons that are proposed to neutralize the negative charge of the thiolate sulfur. The increased push effect of the thiolate in L358P was evidenced by the reduced reduction potential of the heme. The N-15-NMR and resonance Raman spectra of the mutant in the ferric-CN- and in the ferrous-CO forms, respectively, also supported the increased push effect. The maintenance of stereo- and regioselectivities for d-camphor hydroxylation by the mutant suggests the minimum structural change at the distal site. The heterolysis/homolysis ratios of cumene hydroperoxide were the same for wild-type and L358P. However, we observed the enhanced monooxygenations of the unnatural substrates using dioxygen and electrons supplied from the reconstituted system, which indicate the significant role of the push effect in dioxygen activation. We interpret that the enhanced push effect inhibits the protonation of the inner oxygen atom and/or promotes the protonation of the outer oxygen atom in the putative iron-hydroperoxo intermediate (Fe3+-O-OH) of P450cam. This work is the first experimental indication of the significance of the axial cysteine for the P450 reactivity. (C) 2000 Elsevier Science S.A. All rights reserved.
• Stepwise formation of alpha-helices during cytochrome c folding

  S Akiyama, S Takahashi, K Ishimori, Morishima, I

  NATURE STRUCTURAL BIOLOGY 7 (6) 514 - 520 1072-8368 2000/06 [Refereed][Not invited]
   

  Two models have been proposed to describe the folding pathways of proteins. The framework model assumes the initial formation of the secondary structures whereas the hydrophobic collapse model supposes their formation after the collapse of backbone structures. To differentiate between these models for real proteins, we have developed a novel CD spectrometer that enables us to observe the submillisecond time frame of protein folding and have characterized the timing of secondary structure formation in the folding process of cytochrome c (cyt c). We found that similar to 20% of the native helical content was organized in the first phase of folding, which is completed within milliseconds. Furthermore, we suggest the presence of a second intermediate, which has alpha-helical content resembling that of the molten globule state. Our results indicate that many of the alpha-helices are organized after collapse in the folding mechanism of cyt c.
• Functions of fluctuation in the heme-binding loops of cytochrome b(5) revealed in the process of heme incorporation

  M Ihara, S Takahashi, K Ishimori, Morishima, I

  BIOCHEMISTRY 39 (20) 5961 - 5970 0006-2960 2000/05 [Refereed][Not invited]
   

  Cytochrome b(5) (cyt b(5)) holds heme using two axial histidines, His63 and His39, that are located in the centers of the two heme-binding loops. The previous NMR study on the apo form of cyt b(5) (apocyt b(5)) revealed that the loop including His63 exhibits a larger fluctuation compared to the other loop including His39 [Falzone, C. J., Mayer, M. R., Whiteman, E. L., Moore, C. D., and Lecomte, J. T. (1996) Biochemistry 35, 6519-6526]. To understand the significance of the fluctuation, the heme association and dissociation rates of the two loops were compared using two mutants of cyt b(5) in which one of the axial histidines was replaced with leucine. It was demonstrated that the fluctuating loop possesses a significantly slower heme dissociation rate and a faster heme association rate than the other loop. To further verify the importance of the fluctuating loop, the heme association process of wild-type apocyt b(5) was investigated using optical absorption and CD spectroscopies. It was indicated that the process proceeds through the two pathways, and that the dominant pathway involves the initial coordination of His63 located in the fluctuating loop. The urea concentration dependency of the rate constants revealed that the folding of the fluctuating loop is associated with the coordination of His63. It was suggested that the fluctuation enables the loop to have a larger heme-loop contact in the heme-bound conformation. The fluctuating heme-binding loops might be useful for the artificial design of heme-binding proteins.
• Substitution of the heme binding module in hemoglobin alpha- and beta-subunits - Implication for different regulation mechanisms of the heme proximal structure between hemoglobin and myoglobin

  K Inaba, K Ishimori, K Imai, Morishima, I

  JOURNAL OF BIOLOGICAL CHEMISTRY 275 (17) 12438 - 12445 0021-9258 2000/04 [Refereed][Not invited]
   

  In our previous work, we demonstrated that the replacement of the "heme binding module," a segment from F1 to G5 site, in myoglobin with that of hemoglobin alpha-subunit converted the heme proximal structure of myoglobin into the alpha-subunit type (Inaba, K., Ishimori, K. and Morishima, I. (1998) J. Mel. Biol. 283, 311-327). To further examine the structural regulation by the heme binding module in hemoglobin, we synthesized the beta alpha(HBM)-subunit, in which the heme binding module (HBM) of hemoglobin beta-subunit was replaced by that of hemoglobin a-subunit. Based on the gel chromatography, the beta alpha(HBM)-subunit was preferentially associated with the alpha-subunit to form a heterotetramer, alpha(2)[beta alpha(HBM)(2)], just as is native beta-subunit. Deoxy-alpha(2)[beta alpha(HBM)(2)] tetramer exhibited the hyperfine-shifted NMR resonance from the proximal histidyl NdeltaH proton and the resonance Raman band from the Fe-His vibrational mode at the same positions as native hemoglobin. Also, NMR spectra of carbonmonoxy and cyanomet alpha(2)[beta alpha(HBM)(2)] tetramer were quite similar to those of native hemoglobin. Consequently, the heme environmental structure of the beta alpha(HBM)-subunit in tetrameric alpha(2)[beta alpha(HBM)(2)] was similar to that of the beta-subunit in native tetrameric Hb A, and the structural conversion by the module substitution was not clear in the hemoglobin subunits, The contrastive structural effects of the module substitution on myoglobin and hemoglobin subunits strongly suggest different regulation mechanisms of the heme proximal structure between these two globins, Whereas the heme proximal structure of monomeric myoglobin is simply determined by the amino acid sequence of the heme binding module, that of tetrameric hemoglobin appears to be closely coupled to the subunit interactions.
• Pressure dependence of the intramolecular electron transfer reaction in myoglobin reinvestigated

  Y Furukawa, K Ishimori, Morishima, I

  JOURNAL OF PHYSICAL CHEMISTRY B 104 (8) 1817 - 1825 1089-5647 2000/03 [Refereed][Not invited]
   

  The activation volumes (Delta V-not equal) for intramolecular electron transfer (ET) reactions in Ru-modified Zn-porphyrin (ZnP) substituted myoglobins (Ru-ZnMb) have been determined to investigate the pressure effects on the redox potentials and donor-acceptor distance (D-A distance) for the ET reaction. Three Ru-ZnMbs, in which D-A distances for the ET reactions are 12.7 Angstrom (His48Mb), 15.5 Angstrom (His83Mb), and 19.3 Angstrom (His81Mb), were constructed. The activation volumes for the forward ET reactions (Delta V-f(not equal)) were -1.6 (His83Mb), +3.7 (His81Mb), and +6.5 cm(3) mol(-1) (His48Mb). We also measured the pressure dependence of the back ET reactions (from Ru2+ complex to ZnP+), showing that the back ET reactions exhibited negative activation volumes (Delta V-b(not equal)) for all of the Ru--ZnMbs: -11, -5.3, and -6.2 cm(3) mol(-1) for His83Mb, His81Mb, and His48Mb, respectively. On the basis of these activation volumes, the pressure dependence of the redox potentials, (partial derivative Delta G degrees/partial derivative P)(T) was estimated as about 2.94 x 10(-4) eV MPa-1, regardless of the position of the Ru complex. Since (partial derivative Delta G degrees/partial derivative P)T in the present study is close to that of RU(NH3)(6)(2+/3+) (2.97 x 10(-4) eV MPa-1), the pressure-induced redox changes of the Ru complex were primarily responsible for that of the ET reaction and the contribution of ZnP to the pressure dependence of the redox potential on the ET reactions would be small. In sharp contrast to (partial derivative Delta G degrees/partial derivative P)(T), the pressure dependence of the D-A distance, (partial derivative d/partial derivative P)(T), highly depends on the ET pathway and microenvironments of the redox centers. The linear compressibility, (-1/d(0))(partial derivative d/partial derivative P)(T), was (2.2 +/- 0.1) x 10(-10), (5.1 +/- 0.5) x 10(-11), and (-2.6 +/- 3.2) x 10(-11) m(2) N-1 for His83Mb, His81Mb, and His48Mb, respectively. The different linear compressibility for the three ET reaction systems suggests that the structural fluctuation in proteins is not unique in protein structure and site specific local fluctuations would be one of the factors regulating the protein ET reactions.
• Pressure dependence of the intramolecular electron transfer reaction in myoglobin reinvestigated

  Y Furukawa, K Ishimori, Morishima, I

  JOURNAL OF PHYSICAL CHEMISTRY B 104 (8) 1817 - 1825 1089-5647 2000/03 [Refereed][Not invited]
   

  The activation volumes (Delta V-not equal) for intramolecular electron transfer (ET) reactions in Ru-modified Zn-porphyrin (ZnP) substituted myoglobins (Ru-ZnMb) have been determined to investigate the pressure effects on the redox potentials and donor-acceptor distance (D-A distance) for the ET reaction. Three Ru-ZnMbs, in which D-A distances for the ET reactions are 12.7 Angstrom (His48Mb), 15.5 Angstrom (His83Mb), and 19.3 Angstrom (His81Mb), were constructed. The activation volumes for the forward ET reactions (Delta V-f(not equal)) were -1.6 (His83Mb), +3.7 (His81Mb), and +6.5 cm(3) mol(-1) (His48Mb). We also measured the pressure dependence of the back ET reactions (from Ru2+ complex to ZnP+), showing that the back ET reactions exhibited negative activation volumes (Delta V-b(not equal)) for all of the Ru--ZnMbs: -11, -5.3, and -6.2 cm(3) mol(-1) for His83Mb, His81Mb, and His48Mb, respectively. On the basis of these activation volumes, the pressure dependence of the redox potentials, (partial derivative Delta G degrees/partial derivative P)(T) was estimated as about 2.94 x 10(-4) eV MPa-1, regardless of the position of the Ru complex. Since (partial derivative Delta G degrees/partial derivative P)T in the present study is close to that of RU(NH3)(6)(2+/3+) (2.97 x 10(-4) eV MPa-1), the pressure-induced redox changes of the Ru complex were primarily responsible for that of the ET reaction and the contribution of ZnP to the pressure dependence of the redox potential on the ET reactions would be small. In sharp contrast to (partial derivative Delta G degrees/partial derivative P)(T), the pressure dependence of the D-A distance, (partial derivative d/partial derivative P)(T), highly depends on the ET pathway and microenvironments of the redox centers. The linear compressibility, (-1/d(0))(partial derivative d/partial derivative P)(T), was (2.2 +/- 0.1) x 10(-10), (5.1 +/- 0.5) x 10(-11), and (-2.6 +/- 3.2) x 10(-11) m(2) N-1 for His83Mb, His81Mb, and His48Mb, respectively. The different linear compressibility for the three ET reaction systems suggests that the structural fluctuation in proteins is not unique in protein structure and site specific local fluctuations would be one of the factors regulating the protein ET reactions.
• Pressure effects on the conformational gating mechanism in protein electron transfer

  Furukawa Y., Ishimori K., Morishima I.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 40 S45  0582-4052 2000
• The study on the burst phase of ribonuclease A folding using a rapid mixing device

  Kimura T., Akiyama S., Takahashi S., Harada Y., Ishimori K., Morishima I.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 40 S161  0582-4052 2000
• Study on DNA-binding Activity of Heme-containing Transcriptional Regulator CooA by Using Fluorescence Anisotropy

  Yamamoto K., Ishikawa H., Ishimori K., Morishima I., Nakajima H., Aono S.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 40 S93  0582-4052 2000
• Study on control mechanism of the oxgen affinity in the isolated betachain of hemoglobin by using Leucine 28 mutants

  Konishi K., Ishikawa S., Takahashi S., Ishimori K., Morishima I.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 40 S45  0582-4052 2000
• Luminol activity of horseradish peroxidase mutants mimicking a proposed binding site for luminol in Arthromyces ramosus peroxidase

  M Tanaka, K Ishimori, Morishima, I

  BIOCHEMISTRY 38 (32) 10463 - 10473 0006-2960 1999/08 [Refereed][Not invited]
   

  To enhance the oxidation activity for luminol in horseradish peroxidase (HRP), we have prepared three HRP mutants by mimicking a possible binding site for luminol in Arthromyces ramosus peroxidase (ARP) which shows 500-fold higher oxidation activity for luminol than native HRP. Spectroscopic studies by H-1 NMR revealed that the chemical shifts of 7-propionate and 8-methyl protons of the heme in cyanide-ligated ARP were deviated upon addition of luminol (4 mM), suggesting that the charged residues, Lys49 and Glu190, which are located near the 7-propionate and 8-methyl groups of the heme. are involved in the specific binding to luminol. The positively charged Lys and negatively charged Glu were introduced into the corresponding positions of Ser35 (S35K) and Gln176 (Q176E) in HRP, respectively, to build the putative binding site for luminol. A double mutant, S35K/Q176E, in which both Ser35 and Gln176 were replaced, was also prepared. Addition of luminol to the HRP mutants induced more pronounced effects on the resonances from the heme substituents and heme environmental residues in the H-1 NMR spectra than that to the wild-type enzyme, indicating that the mutations in this study induced interactions with luminol in the vicinity of the heme. The catalytic efficiencies (V-max/K-m) for luminol oxidation of the S35K and S35K/Q176E mutants were 1.5- and 2-fold improved, whereas that of the Q176E mutant was slightly depressed. The increase in luminol activity of the S35K and S35K/Q176E mutants was rather small but significant, suggesting that the electrostatic interactions between the positive charge of Lys35 and the negative charge of luminol can contribute to the effective binding for the luminol oxidation. On the other hand, the negatively charged residue would not be so crucial for the luminol oxidation. The absence of drastic improvement in the luminol activity suggests that introduction of the charged residues into the heme vicinity is not enough to enhance the oxidation activity for luminol as observed for ARP.
• Direct electron transfer catalysed by recombinant forms of horseradish peroxidase: insight into the mechanism

  A Lindgren, M Tanaka, T Ruzgas, L Gorton, Gazaryan, I, K Ishimori, Morishima, I

  ELECTROCHEMISTRY COMMUNICATIONS 1 (5) 171 - 175 1388-2481 1999/05 [Refereed][Not invited]
   

  The paper presents the first results on recombinant horseradish peroxidase (HRP) electrochemistry obtained on graphite with a rotating disk electrode system. Recombinant MP demonstrates a higher percentage of properly oriented molecules than the native enzyme. The first important conclusion based on the recombinant HRP electrochemistry is that glycosylation hinders direct electron transfer (ET). The single-point mutants with limited activity toward phenolic substrates, viz. Asn70Val and Asn70Asp showed no changes in the registered current upon the addition of p-cresol, catechol, p-aminophenol and guaiacol and, thus, in this particular case mediated ET was not more advantageous than direct ET. The rate constants for direct ET were comparable for all mutants tested in this study demonstrating that direct ET does not depend on the enzyme's ability or inability to oxidise phenolic substrates. The results obtained in this study demonstrate the true mediatorless nature of enzyme-catalysed direct ET. (C) 1999 Elsevier Science S.A. All rights reserved.
• Crystal structure of a protein with an artificial exon-shuffling, module M4-substituted chimera hemoglobin beta alpha, at 2.5 angstrom resolution

  T Shirai, M Fujikake, T Yamane, K Inaba, K Ishimori, Morishima, I

  JOURNAL OF MOLECULAR BIOLOGY 287 (2) 369 - 382 0022-2836 1999/03 [Refereed][Not invited]
   

  The crystal structure of the homotetramer of a chimera beta alpha-subunit of human hemoglobin was refined at 2.5 Angstrom resolution. The chimera subunit was constructed by replacing an exon-encoded module M4 of the beta-subunit with that of the alpha-subunit, simulating an exon-shuffling event. The implanted module M4 retained the native alpha-subunit structure, while module M3 was disturbed around the site where a new type of intron was recently found. Some of the residues were found in alternative conformations that avoid steric hindrance at the subunit interface. The modules are modestly rigid in their backbone structures by using side-chains to compensate for interface incompatibility. (C) 1999 Academic Press.
• CD Measurements on the Early Folding Intermediates of Cytochrome c Using Fast Flow Mixer

  Akiyama S., Takahashi S., Ishimori K., Morishima I.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 39 S149  0582-4052 1999
• 2PA087 ヘモグロビンβ鎖の疎水性残基Leu28による配位子結合反応の制御に関する研究

  小西 一誠, 石川 春人, 近藤 詩乃, 高橋 聡, 石森 浩一郎, 森島 績

  生物物理 一般社団法人 日本生物物理学会 39 S122  0582-4052 1999
• 2PA094 転写調節因子CooAの機能発現に関与するヘム軸配位子の研究

  山本 克彦, 石川 春人, 高橋 聡, 石森 浩一郎, 森島 績, 中島 洋, 青野 重利

  生物物理 一般社団法人 日本生物物理学会 39 S124  0582-4052 1999
• Electron Transfer Mechanism in Zn-substituted Cytochrome P450cam

  Furukawa Y., Ishimori K., Morishima I.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 39 S122  0582-4052 1999
• Dynamics of Protein Folding Studied Using the Fast Flow Mixer

  Takahashi Satoshi, Akiyama Shuji, Ishimori Koichiro, Morishima Isao

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 39 S13  0582-4052 1999
• CD measurements on the early folding intermediate of cytochrome c using the fast flow mixer

  S Takahashi, S Akiyama, K Ishimori, Morishima, I

  OLD AND NEW VIEWS OF PROTEIN FOLDING 1194 75 - 84 0531-5131 1999 [Refereed][Not invited]
   

  The early process of cytochrome c folding consists of the first (tau = 30 similar to 70 mu s) and second (tau = 400 similar to 1400 mu s) kinetic phases that correspond to the formation of the folding intermediate and its conversion to the native state, respectively (Shastry and Roder, Nature Struct. Biol. 5, 385, 1998). To obtain structural information on the folding intermediate of cytochrome c, we developed the fast flow mixer that was combined with the circular dichroism (CD) spectrometer. Using the device, the refolding of cytochrome c from the guanidine-unfolded state was investigated in the time range from 750 mu s to 6 ms following the initiation of refolding. The kinetic CD spectrum at the earliest detectable point (750 mu s) shows that the alpha-helix content is ca. 25 % of the native structure. The alpha-helix content increases at the process after 1 ms and reaches ca. 80 % of the native content at 6 ms. Since the first kinetic phase should be already finished at 750 mu s in our experimental condition, the results demonstrate that the intermediate contains only a partial amount of the alpha-helix. We propose that a large part of the alpha-helix formation occurs after the formation of the collapsed intermediate in the folding process of cytochrome c.
• Detection of a tryptophan radical as an intermediate species in the reaction of horseradish peroxidase mutant (Phe-221 → Trp) and hydrogen peroxide

  Morimoto A, Tanaka M, Takahashi S, Ishimori K, Hori H, Morishima I, Pond A.E, Dawson J.H

  Chemtracts 12 (2) 87 - 95 1999 [Refereed][Not invited]
  
• The artificial alpha 1 beta 1-contact mutant hemoglobin, Hb Phe-35 beta, shows only small functional abnormalities

  T Nakatsukasa, N Nomura, G Miyazaki, K Imai, Y Wada, K Ishimori, Morishima, I, H Morimoto

  FEBS LETTERS 441 (1) 93 - 96 0014-5793 1998/12 [Refereed][Not invited]
   

  It was previously reported that Hb Philly with a mutation of Phe for Tyr at 35(C1)beta showed non-cooperative oxygen binding with a very high affinity and instability leading to hemolysis, Further, it lacked the H-1-NMR signal at 13.1 ppm from 2,2-dimethyl-2-silapentane-5-sulfonate in normal hemoglobin (Hb A), so that this signal was assigned to a hydrogen bond formed by Tyr-35(C1)beta. Surprisingly, our artificial mutant hemoglobin with the same mutation as hb Philly showed slightly lowered oxygen affinity, almost normal cooperativity, the H-1-NMR signal at 13.1 ppm and no sign of instability. Our results indicate that the mutation reported for Hb Philly and the assignment of the 13.1 ppm signal need reexamination. (C) 1998 Federation of European Biochemical Societies.
• Structural and functional roles of heme binding module in globin proteins: Identification of the segment regulating the heme binding structure

  K Inaba, K Ishimori, Morishima, I

  JOURNAL OF MOLECULAR BIOLOGY 283 (1) 311 - 327 0022-2836 1998/10 [Refereed][Not invited]
   

  To investigate structural and functional significance of a newly proposed structural unit in globins, the "heme binding module", we synthesized a "heme binding module"-substituted chimeric globin and characterized its function and structure. In our previous study we proposed that the heme binding module, corresponding to the segment from Leu(F1) to Phe(G5) in hemoglobin alpha-subunit, plays a key role in constructing the heme proximal structure in globins. The replacement of the heme binding module in myoglobin with that of hemoglobin alpha-subunit converted the absorption spectra into that of the alpha-subunit, and, in the resonance Raman spectra, the vibration mode characteristic of myoglobin completely disappeared after the module replacement. The hyperfine-shifted NMR resonances for the cyanide-bound form of the module-substituted myoglobin also revealed that the orientation of the axial histidine is close to that of the alpha-subunit rather than that of myoglobin, while the deviations of the resonance positions of the NMR signals from the amino acid residues located in the distal site were subtle, supporting the preferential structural alterations in the heme proximal site. The present finding for the structural alterations in the module-substituted myoglobin confirms that the heme binding module can be a segment regulating the heme proximal structure in globin proteins. (C) 1998 Academic Press.
• Heme environmental structure of CooA is modulated by the target DNA binding

  T Uchida, H Ishikawa, S Takahashi, K Ishimori, Morishima, I, K Ohkubo, H Nakajima, S Aono

  JOURNAL OF BIOLOGICAL CHEMISTRY 273 (32) 19988 - 19992 0021-9258 1998/08 [Refereed][Not invited]
   

  In order to investigate the gene activation mechanism triggered by the CO binding to CooA, a heme-containing transcriptional activator, the heme environmental structure and the dynamics of the CO rebinding and dissociation have been examined in the absence and presence of its target DNA. In the absence of DNA, the Fe-CO and C=O stretching Raman lines of the GO-bound CooA were observed at 487 and 1969 cm(-l), respectively, suggesting that a neutral histidine is an axial ligand trans to CO. The frequency of nu(Fe-CO) implies an open conformation of the distal heme pocket, indicating that the Ligand replaced by CO is located away from the bound CO. When the target DNA was added to GO-bound CooA, an appearance of a new nu(Fe-CO) line at 519 cm(-l) and narrowing of the main line at 486 cm(-1) were observed. Although the rate of the CO dissociation was insensitive to the additions of DNA, the CO rebinding was decelerated in the presence of the target DNA, but not in the presence of nonsense DNA. These observations demonstrate the structural alterations in the heme distal site in response to binding of the target DNA and support the activation mechanism proposed for CooA, which is triggered by the movement of the heme distal ligand to modify the conformation of the DNA binding domain.
• Design, construction, crystallization, and preliminary X-ray studies of a fine-tuning mutant (F133V) of module-substituted chimera hemoglobin

  T Shirai, M Fujikake, T Yamane, K Inaba, K Ishimori, Morishima, I

  PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 32 (3) 263 - 267 0887-3585 1998/08 [Refereed][Not invited]
   

  A chimera beta alpha-subunit of human hemoglobin was crystallized into a carbon-monoxy form, The protein was assembled by substituting the structural portion of a beta-subunit of hemoglobin (M4 module of the subunit) for its counterpart in the alpha-subunit. In order to overcome the inherent instability in the crystallization of the chimera subunit, a site-directed mutagenesis (F133V) technique was employed based on a computer model. The crystal was used for an X-ray diffraction study yielding a data set with a resolution of 2.5 Angstrom. The crystal belongs to the monoclinic space group P2(1), with cell dimensions of a = 62.9, b = 81.3, c = 55.1 Angstrom, and beta = 91.0 degrees. These dimensions are similar to the crystallographic parameters of the native beta-subunit tetramers in three different ligand states, one of which is a cyanide form that was also crystallized in this study. Proteins 32:263-267, 1998. (C) 1998 Wiley-Liss, Inc.
• Design, construction, crystallization, and preliminary X-ray studies of a fine-tuning mutant (F133V) of module-substituted chimera hemoglobin

  T Shirai, M Fujikake, T Yamane, K Inaba, K Ishimori, Morishima, I

  PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 32 (3) 263 - 267 0887-3585 1998/08 [Refereed][Not invited]
   

  A chimera beta alpha-subunit of human hemoglobin was crystallized into a carbon-monoxy form, The protein was assembled by substituting the structural portion of a beta-subunit of hemoglobin (M4 module of the subunit) for its counterpart in the alpha-subunit. In order to overcome the inherent instability in the crystallization of the chimera subunit, a site-directed mutagenesis (F133V) technique was employed based on a computer model. The crystal was used for an X-ray diffraction study yielding a data set with a resolution of 2.5 Angstrom. The crystal belongs to the monoclinic space group P2(1), with cell dimensions of a = 62.9, b = 81.3, c = 55.1 Angstrom, and beta = 91.0 degrees. These dimensions are similar to the crystallographic parameters of the native beta-subunit tetramers in three different ligand states, one of which is a cyanide form that was also crystallized in this study. Proteins 32:263-267, 1998. (C) 1998 Wiley-Liss, Inc.
• Roles of negatively charged surface residues of putidaredoxin in interactions with redox partners in P450cam monooxygenase system

  M Aoki, K Ishimori, Morishima, I

  BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY 1386 (1) 157 - 167 0167-4838 1998/07 [Refereed][Not invited]
   

  To investigate the interaction of putidaredoxin (Pdx) with its redox partners in the cytochrome P450cam system, we focused on the role of negatively charged surface amino acid residues. The amino acid residues we examined in this mutational study are Asp-58, Glu-65, Glu-72, and Glu-77, which are located on the alpha-helical segment to form a negatively charged region on the surface of Pdx and have been supposed to play key roles in the association with the redox partners, NADH-putidaredoxin reductase (PdR) and P450cam. The neutralization of the single negative charge on these amino acid residues did not significantly inhibit the electron-transfer reaction with the redox partners, except for the mutation at Glu-72. Together with the previous results, we can conclude that the negatively charged cluster on the alpha-helical segment is not so crucial for the electron transfer of the Pdx/PdR complex, and, instead of the negative charges, the steric hindrance is essential for the binding of Pdx with PdR. In the electron transfer from Pdx to P450cam, the alpha-helical region would not be included in the binding site with P450cam and some specific hydrogen bonds on the surface loop near the Fe-S center contribute to the electron transfer to P450cam. Such different binding sites and interactions for Pdx will shed light on the electron-transfer mechanism mediated by Pdx, the shuttle mechanism. (C) 1998 Elsevier Science B.V. All rights reserved.
• NMR studies of putidaredoxin: associations of putidaredoxin with NADH-putidaredoxin reductase and cytochrome P450cam

  M Aoki, K Ishimori, Morishima, I

  BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY 1386 (1) 168 - 178 0167-4838 1998/07 [Refereed][Not invited]
   

  To characterize the electron-transfer reaction in the P450cam monooxygenation system, the binding regions of putidaredoxin (Pdx) to NADH-putidaredoxin reductase (PdR) and P450cam were investigated using isotope-filtered NMR experiments in which uniformly N-15-labeled Pdx ([U-N-15]Pdx) is mixed with unlabeled PdR and P450cam. By addition of PdR to Pdx, site specific signal broadening was observed for the N-H correlation peaks from Val-28, Glu-72, Ile-88, and Gln-105. Although previous studies have suggested the contribution from acidic amino acid residues on the G-helix of Pdx to the binding with PdR, no site specific broadening was observed for the resonances from these residues except for Glu-72. The lesser contribution of electrostatic interactions to the Pdx/PdR complex formation was also suggested by our previous study (M. Aoki, K. Ishimori, H. Fukada, K. Takahashi, I. Morishima, Biochim. Biophys. Acta 1384 (1998) 180-188), which is in sharp contrast to the complex formation between adrenodoxin and adrenodoxin reductase. Upon the complex formation between Pdx and P450cam, the site specific NMR line broadening was observed for several amino acid residues distributed near the iron-sulfur cluster, corresponding to the large binding site in the complex formation with P450cam. Since some of the amino acid residues included in the binding site are not conserved for the electron-transfer iron-sulfur proteins such as ferredoxin and adrenodoxin, the interactions formed by these amino acid residues would be highly specific to the binding with P450cam, consistent with very low cross-reactivity to other iron-sulfur proteins in the P450cam monooxygenation system. (C) 1998 Elsevier Science B.V. All rights reserved.
• Roles of negatively charged surface residues of putidaredoxin in interactions with redox partners in P450cam monooxygenase system

  M Aoki, K Ishimori, Morishima, I

  BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY 1386 (1) 157 - 167 0167-4838 1998/07 [Refereed][Not invited]
   

  To investigate the interaction of putidaredoxin (Pdx) with its redox partners in the cytochrome P450cam system, we focused on the role of negatively charged surface amino acid residues. The amino acid residues we examined in this mutational study are Asp-58, Glu-65, Glu-72, and Glu-77, which are located on the alpha-helical segment to form a negatively charged region on the surface of Pdx and have been supposed to play key roles in the association with the redox partners, NADH-putidaredoxin reductase (PdR) and P450cam. The neutralization of the single negative charge on these amino acid residues did not significantly inhibit the electron-transfer reaction with the redox partners, except for the mutation at Glu-72. Together with the previous results, we can conclude that the negatively charged cluster on the alpha-helical segment is not so crucial for the electron transfer of the Pdx/PdR complex, and, instead of the negative charges, the steric hindrance is essential for the binding of Pdx with PdR. In the electron transfer from Pdx to P450cam, the alpha-helical region would not be included in the binding site with P450cam and some specific hydrogen bonds on the surface loop near the Fe-S center contribute to the electron transfer to P450cam. Such different binding sites and interactions for Pdx will shed light on the electron-transfer mechanism mediated by Pdx, the shuttle mechanism. (C) 1998 Elsevier Science B.V. All rights reserved.
• NMR studies of putidaredoxin: associations of putidaredoxin with NADH-putidaredoxin reductase and cytochrome P450cam

  M Aoki, K Ishimori, Morishima, I

  BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY 1386 (1) 168 - 178 0167-4838 1998/07 [Refereed][Not invited]
   

  To characterize the electron-transfer reaction in the P450cam monooxygenation system, the binding regions of putidaredoxin (Pdx) to NADH-putidaredoxin reductase (PdR) and P450cam were investigated using isotope-filtered NMR experiments in which uniformly N-15-labeled Pdx ([U-N-15]Pdx) is mixed with unlabeled PdR and P450cam. By addition of PdR to Pdx, site specific signal broadening was observed for the N-H correlation peaks from Val-28, Glu-72, Ile-88, and Gln-105. Although previous studies have suggested the contribution from acidic amino acid residues on the G-helix of Pdx to the binding with PdR, no site specific broadening was observed for the resonances from these residues except for Glu-72. The lesser contribution of electrostatic interactions to the Pdx/PdR complex formation was also suggested by our previous study (M. Aoki, K. Ishimori, H. Fukada, K. Takahashi, I. Morishima, Biochim. Biophys. Acta 1384 (1998) 180-188), which is in sharp contrast to the complex formation between adrenodoxin and adrenodoxin reductase. Upon the complex formation between Pdx and P450cam, the site specific NMR line broadening was observed for several amino acid residues distributed near the iron-sulfur cluster, corresponding to the large binding site in the complex formation with P450cam. Since some of the amino acid residues included in the binding site are not conserved for the electron-transfer iron-sulfur proteins such as ferredoxin and adrenodoxin, the interactions formed by these amino acid residues would be highly specific to the binding with P450cam, consistent with very low cross-reactivity to other iron-sulfur proteins in the P450cam monooxygenation system. (C) 1998 Elsevier Science B.V. All rights reserved.
• Detection of a tryptophan radical as an intermediate species in the reaction of horseradish peroxidase mutant (Phe-221 -> Trp) and hydrogen peroxide

  A Morimoto, M Tanaka, S Takahashi, K Ishimori, H Hori, Morishima, I

  JOURNAL OF BIOLOGICAL CHEMISTRY 273 (24) 14753 - 14760 0021-9258 1998/06 [Refereed][Not invited]
   

  The crucial reaction intermediate in the reaction of peroxidase with hydrogen peroxide (H2O2), compound I, contains a porphyrin pi-cation radical in horseradish peroxidase (HRP), which catalyzes oxidation of small organic and inorganic compounds, whereas cytochrome c peroxidase (CcP) has a radical center on the tryptophan residue (Trp-191) and oxidizes the redox partner, cytochrome c. To investigate the roles of the amino acid residue near the heme active center in discriminating the function of the peroxidases in these two enzymes, we prepared a CcP-like HRP mutant, F221W (Phe-221 --> Trp). Although the rapid spectral scanning and stopped-flow experiments confirmed that the F221W mutant reacts with H2O2 to form the porphyrin pi-cation radical at the same rate as for the wild-type enzyme, the characteristic spectral features of the porphyrin pi-cation radical disappeared rapidly, and were converted to the compound II-type spectrum. The EPR spectrum of the resultant species produced by reduction of the porphyrin pi-cation radical, however, was quite different from that of compound II in HRP, showing typical signals from a Trp radical as found for CcP. The sequential radical formation from the porphyrin ring to the Trp residue implies that the proximal Trp is a key residue in the process of the radical transfer from the porphyrin ring, which differentiates the function of peroxidases.
• Roles of valine-98 and glutamic acid-72 of putidaredoxin in the electron-transfer complexes with NADH-putidaredoxin reductase and P450cam

  M Aoki, K Ishimori, Morishima, I, Y Wada

  INORGANICA CHIMICA ACTA 272 (1-2) 80 - 88 0020-1693 1998/05 [Refereed][Not invited]
   

  In the P450cam reaction cycle, the interactions between putidaredoxin (Pdx) and P450cam or NADH-putidaredoxin reductase (PdR) have been considered to be essential in the electron-transfer process. In this study, three mutant putidaredoxins were prepared to examine the hydrophobic and electrostatic interactions in the reaction complexes. Val-98, which is exposed to solvent and has been previously suggested to play a key role in hydrophobic interactions, was substituted by alanine (V98A) or threonine (V98T) to perturb the hydrophobicity at the 98 position. Another mutation site was Glu-72 which is located in the middle of the negative charge-rich region. This glutamate was altered to glutamine (E72Q) to neutralize one of the negative charges on the surface of Pdx. The electronic absorption and H-1 NMR spectra of oxidized and reduced forms of these mutants, and their redox potentials were similar to those of wild type Pdx, indicating that the environment of the Fe-2-S-2 cluster was not very seriously affected by these mutations. In cytochrome c reduction activity, however, the ionic strength dependence of E72Q mutant differs slightly from that of the wild type protein. The mutation at glutamine at the 72 position weakened the association to PAR, indicating that Glu-72 is one of the amino acid residues contributing to form the reaction complex for the electron-transfer between Pdx and PdR. Based on the NADH consumption activity of these mutants, the hydrophobic interactions at Val-98 are involved in binding to P450cam, while electrostatic interactions at Glu-72 are rather small to affect the reaction between Pdx and P450cam. These different effects of the mutations suggest that the interaction sites to P450cam on Pdx are not completely superimposed on that to PUR. (C) 1998 Elsevier Science S.A. All rights reserved.
• Roles of valine-98 and glutamic acid-72 of putidaredoxin in the electron-transfer complexes with NADH-putidaredoxin reductase and P450cam

  M Aoki, K Ishimori, Morishima, I, Y Wada

  INORGANICA CHIMICA ACTA 272 (1-2) 80 - 88 0020-1693 1998/05 [Refereed][Not invited]
   

  In the P450cam reaction cycle, the interactions between putidaredoxin (Pdx) and P450cam or NADH-putidaredoxin reductase (PdR) have been considered to be essential in the electron-transfer process. In this study, three mutant putidaredoxins were prepared to examine the hydrophobic and electrostatic interactions in the reaction complexes. Val-98, which is exposed to solvent and has been previously suggested to play a key role in hydrophobic interactions, was substituted by alanine (V98A) or threonine (V98T) to perturb the hydrophobicity at the 98 position. Another mutation site was Glu-72 which is located in the middle of the negative charge-rich region. This glutamate was altered to glutamine (E72Q) to neutralize one of the negative charges on the surface of Pdx. The electronic absorption and H-1 NMR spectra of oxidized and reduced forms of these mutants, and their redox potentials were similar to those of wild type Pdx, indicating that the environment of the Fe-2-S-2 cluster was not very seriously affected by these mutations. In cytochrome c reduction activity, however, the ionic strength dependence of E72Q mutant differs slightly from that of the wild type protein. The mutation at glutamine at the 72 position weakened the association to PAR, indicating that Glu-72 is one of the amino acid residues contributing to form the reaction complex for the electron-transfer between Pdx and PdR. Based on the NADH consumption activity of these mutants, the hydrophobic interactions at Val-98 are involved in binding to P450cam, while electrostatic interactions at Glu-72 are rather small to affect the reaction between Pdx and P450cam. These different effects of the mutations suggest that the interaction sites to P450cam on Pdx are not completely superimposed on that to PUR. (C) 1998 Elsevier Science S.A. All rights reserved.
• Structure-activity relation of horseradish peroxidases as studied with mutations at heme distal and proximal sites

  M Tanaka, A Morimoto, K Ishimori, Morishima, I

  PURE AND APPLIED CHEMISTRY 70 (4) 911 - 916 0033-4545 1998/04 [Refereed][Not invited]
   

  The distal His-Asn-Glu hydrogen bond network in the heme distal site is highly conserved among various fungal and plant peroxidases. To gain an insight into the functional roles of this hydrogen network in peroxidase activity, we have mutated the Asn70 to Val (N70V) or Asp (N70D), the Glu64 to Pro (E64P), or GIy (E64G) or Ser (E64S) in horseradish peroxidase. All the mutants disrupted the distal His-Asn hydrogen bond with maintaining the heme electronic structures as revealed by H-1 NMR spectra and exhibited substantial depression of the peroxidase activity. The depression of the activity was ascribed to the decreased basicity and dislocation of the distal His induced by these mutations. The replacement of the distal His by Glu (H42E) to mimic the heme distal site of chloroperoxidase impaired the peroxidase activity. We have also studied F221W HRP mutant in which the proximal Phe is replaced with Trp to mimic the heme proximal structure of cytochrome c peroxidase and shown that the HRP-type compound I was formed, followed by transformation to the CcP-type Trp radical.
• Structural and functional effects of pseudo-module substitution in hemoglobin subunits - New structural and functional units in globin structure

  K Inaba, K Ishimori, K Imai, Morishima, I

  JOURNAL OF BIOLOGICAL CHEMISTRY 273 (14) 8080 - 8087 0021-9258 1998/04 [Refereed][Not invited]
   

  Functional and structural significance of the "module" in proteins has been investigated for globin proteins. Our previous studies have revealed that some modules in globins are responsible for regulating the subunit association and heme environmental structures, whereas the module substitution often induces fatal structural destabilization, resulting in failure of functional regulation. In this paper, to gain further insight into functional and structural significance of the modular structure in globins, we focused upon the "pseudo-module" in globin structure where boundaries are located at the center of modules. Although the pseudo-module has been supposed not to retain a compactness, the beta alpha(PM3)-subunit, in which one of the pseudo-modules, the F1-H6 regoin, of the alpha-subunit is implanted into the beta-subunit, conserved stable globin structure, and its association property was converted into that of the alpha-subunit, as the case for the module substituted globin, the beta alpha(M4)-subunit. These results suggest that modules are not unique structural and functional units for globins. Interestingly, however, the recent reconsideration of the module boundary indicates that the modules in globins can be further divided into two small modules, and one of the boundaries for the new small modules coincides with that of the pseudo-module we substituted in this study. Although it would be premature to conclude the significance of the modular structure in globins, it can be safely said that we have found new structural units in globin structure, probably new modules.
• Isothermal titration calorimetric studies on the associations of putidaredoxin to NADH-putidaredoxin reductase and P450cam

  M Aoki, K Ishimori, H Fukada, K Takahashi, Morishima, I

  BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY 1384 (1) 180 - 188 0167-4838 1998/04 [Refereed][Not invited]
   

  Putidaredoxin (Pdx), an iron-sulfur protein containing a 2Fe-2S cluster, serves as a physiological electron mediator from NADH-putidaredoxin reductase (PdR) to P450cam in the P450cam monooxygenation reaction cycle, Previous studies have revealed that the associations of Pdx with P450cam and PdR are not strongly dominated by electrostatic interactions, although such interactions stabilize most electron-transfer complexes [A.R. De Pascalis, I. Jelesarov, F. Ackermann, W.H. Koppenol, M. Hiroasawa, D.B. Knaff, H.R. Bosshard, Protein Sci. 2 (1993) 1126-1135]. In the present study, to elucidate the interactions dominating the specific associations in the electron-transfer reaction mediated by Pdx, the thermodynamic properties-entropy (Delta S), enthalpy (Delta H), and heat capacity changes (Delta Cp)-for PdR/Pdx and P450cam/Pdx association reactions have been examined by isothermal titration calorimetry (ITC). Although the binding enthalpy change, Delta H-bind, for the PdR/Pdx association is positive at 10 degrees C, it declines linearly with temperature in the range 10-22 degrees C and becomes negative above 11 degrees C. On the other hand, the binding entropy changer Delta S-bind, is positive at all temperatures examined in this study, indicating that the association of Pdx to PdR is entropically driven. On the basis of the temperature dependence of Delta H-bind, Delta Cp-bind for the association of Pdx to PdR was estimated as -1.24 kJ mol(-1) K-1. This value is larger than those reported for other electron-transfer protein systems (e.g., -0.68 kJ mol(-1) K-1 for ferredoxin/ferredoxin:NADP(+) reductase), suggesting that the PdR/Pdx association may be dominated by hydrophobic rather than electrostatic components. For the P450cam/Pdx association. the negative Delta S-bind and highly favorable Delta H-bind were observed, behavior that stands in sharp contrast to the association reactions in other electron-transfer proteins. The energetics of the P450cam/Pdx association are similar to those of binding reaction of antibody to antigen in which van der Waals and hydrogen bonding interactions are dominant, resulting in high specificity in the association of Pdx with P450cam. (C) 1998 Elsevier Science B.V.
• Structural roles of the highly conserved Glu residue in the heme distal site of peroxidases

  M Tanaka, K Ishimori, Morishima, I

  BIOCHEMISTRY 37 (8) 2629 - 2638 0006-2960 1998/02 [Refereed][Not invited]
   

  One of the highly conserved amino acid residues in the heme distal site of various fungal and plant peroxidases, glutamic acid 64 (Glu64) in horseradish peroxidase (HRP), interacts with a distal calcium ion through a hydrogen bond with a water molecule and its peptide carbonyl oxygen on the main-chain forms the hydrogen bond network to the distal His via the adjacent Asn residue, suggesting that the Glu residue is related to the stabilization of the calcium ion and catalytic activity of peroxidase [Nagano, S., Tanaka, M., Ishimori, K,, Watanabe, Y., and Morishima, I, (1996) Biochemistry 35, 14251-14258]. To perturb the hydrogen bond with the adjacent Asn, we replaced the Glu with Pro (E64G) or Gly (E64G), which would alter the configuration of the main chain at position 64. Both of the mutants exhibited substantially depressed oxidation activities for hydroquinone and elementary reaction rates in the catalytic cycle. However, the E64S (Glu64 --> Ser) mutant, in which the configuration of the main chain and the hydrogen bond with Asn70 would not be affected but the interactions with the calcium ion are seriously perturbed by removal of the carboxylate, also showed quite low catalytic activity as observed for the E64P and E64G mutants. Spectral features for the E64S mutant are similar to those of the other mutants: the reorientation of the distal His, disruption of the hydrogen bond between the distal His and Asn70, and loss of the calcium ion. Thus, we can conclude that, in addition to forming the hydrogen bond network in the distal site, the Glu residue is a key residue for stable binding of the calcium ion, which maintains the structural integrity of the distal cavity, resulting in high peroxidase activity.
• Catalytic roles of the distal site hydrogen bond network of peroxidases

  S Nagano, M Tanaka, K Ishimori, Morishima, I, Y Watanabe, M Mukai, T Ogura, T Kitagawa

  OXYGEN HOMEOSTASIS AND ITS DYNAMICS 1 354 - 358 1998 [Refereed][Not invited]
   

  There are highly conserved hydrogen bonds between the distal histidine and adjacent asparagine in many peroxidases. To investigate the functional roles of the hydrogen bond between the distal histidine and Asn-70, Asn-70 in horseradish peroxidase (HRP) was replaced with Val or Asp. The disruption of the Asn-70-His-52 couple decreases the rate of compound I formation to less than 10% of that of native enzyme. Based on resonance Raman spectroscopy, the midpoint pH value of the Fe(II)-His stretching frequency in the acid-base transition was decreased by the mutation of Asn-70, suggesting that the distal histidine became less basic. With a less basic distal histidine, proton abstraction from hydrogen peroxide is harder for the mutants, resulting in remarkable deceleration of compound I formation.
• Structureactivity relation of horseradish peroxidases as studied with mutations at heme distal and proximal sites

  M. Tanaka, A. Morimoto, Koichiro Ishimori, I. Morishima

  Pure and Applied Chemistry Walter de Gruyter {GmbH} 70 (4) 1998/01 [Refereed][Not invited]
  
• The effects of heme pocket hydrophobicity on the ligand binding dynamics in myoglobin as studied with leucine 29 mutants

  T Uchida, K Ishimori, Morishima, I

  JOURNAL OF BIOLOGICAL CHEMISTRY 272 (48) 30108 - 30114 0021-9258 1997/11 [Refereed][Not invited]
   

  To examine the effects of heme pocket hydrophobicity on the ligand binding in myoglobin, some artificial mutants of human myoglobin have been prepared, in which less hydrophobic amino acid residue (Ala, Gly, Ser) is located at the Leu(29) (10th residue of the B helix) position, CO rebinding rates for the mutants were markedly decelerated, while the H-1, and N-15 NMR spectra of the mutants show that the structural changes around the heme iron for these mutants are rather small. The kinetic and structural properties of the mutants indicate that the ligand binding rate depends on the hydrophobicity inside the heme cavity for these mutants in addition to the volume of the side chain at the 29-position, On the basis of the IR stretching frequency of liganded CO, invasion of water molecules into the heme pocket in the mutants is suggested, which would be induced by the decrease in the hydrophobicity due to the amino acid substitution. A slight red shift of the position of the Soret peak for the serine mutant L29S also supports the reduced hydrophobicity inside the heme cavity, We can concluded that, together with the kinetic properties of the mutants, the hydrophobicity of the heme pocket is one of the key factors in regulating the ligand binding to the heme iron.
• Structural and functional roles of modules in hemoglobin - substitution of module M4 in hemoglobin subunits

  K Inaba, K Wakasugi, K Ishimori, T Konno, M Kataoka, Morishima, I

  JOURNAL OF BIOLOGICAL CHEMISTRY 272 (48) 30054 - 30060 0021-9258 1997/11 [Refereed][Not invited]
   

  The alpha- and beta-subunits of human hemoglobin consist of the modules M1, M2 + M3, and M4, which correspond to the exons 1, 2, and 3, respectively (Go, M. (1981) Nature 291, 90-92). To gain further insight into functional and structural significance of the modules, we designed two kinds of chimeric hemoglobin subunits (chimeric alpha alpha beta- and beta beta alpha-subunits), in which the module M4 was replaced by the partner subunits. CD spectra in the far-UV region showed that the secondary structure of the chimeric alpha alpha beta-subunit drastically collapsed, while the chimeric beta beta alpha-subunit conserved the native globin structure (Wakasugi, K., Ishimori, It., Imai, IT., Wada, Y., and Morishima, I. (1994) J. Biol. Chem. 269, 18750-18756). SAXS data also suggested a partially disordered structure of the chimeric alpha alpha beta-subunit. Based on tryptophan-fluorescence spectra and computer modeling from x-ray structures of native globins, steric constraint between Trp(14) and Tyr(125) would be induced in the chimeric alpha alpha beta-subunit, which would perturb the packing of the A- and H-helices and destabilize the globule structure, On the other hand, such a steric constraint was not found for the counterpart chimeric subunit, the beta beta alpha-subunit The different stabilities of these module-substituted globins imply that nodules would not always be stable ''structural'' units, and interactions between modules are crucial to construct stable globin subunits.
• Pressure effects on electron transfer rates in zinc/ruthenium modified myoglobins

  Y Sugiyama, S Takahashi, K Ishimori, Morishima, I

  JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 119 (40) 9582 - 9583 0002-7863 1997/10 [Refereed][Not invited]
  
• 'Module'-substituted globins: Artificial exon shuffling among myoglobin, hemoglobin alpha- and beta-subunits

  K Wakasugi, K Ishimori, Morishima, I

  BIOPHYSICAL CHEMISTRY 68 (1-3) 265 - 273 0301-4622 1997/10 [Refereed][Not invited]
   

  Based on the detailed structural analysis of proteins, Go [M. Go, Nature 291 (1981) 90-92] found that protein structures can be divided into some structural units, 'modules,' which correspond to peptides coded by exons. In the present study, to investigate functional and structural roles of modular structures in proteins, we have engineered eight chimera globins, in which the exons are shuffled among human myoglobin, human hemoglobin alpha- and beta-subunits, in addition to the chimera beta beta alpha-globin described previously [K. Wakasugi, K. Ishimori, K. Imai, Y. Wada, I. Morishima, J. Biol. Chem. 269 (1994) 18750-18756]. Although all of the chimera globins stoichiometrically bound the heme and their alpha-helical contents increased by heme incorporation as found for native globins, the alpha-helical contents of the chimera globins were significantly lower than those of native globins, suggesting that 'module' substitutions seriously affect the protein folding and stability in globins. The comparisons among several chimera globins demonstrated that such structural alterations are mainly attributed to loss of some key intermodular interactions for protein folding. By simultaneous substitution of the modules M1 and M4 from the same globin, the protein structure was stabilized, which indicates that the module packing between modules M1 and M4 would be one of the crucial interaction to stabilize the globin fold. Present results allow us to conclude that module substitutions would be available for designing and producing novel functional proteins if we can reproduce the stable modular packing in the 'module'-substituted proteins. (C) 1997 Elsevier Science B.V.
• 'Module'-substituted globins: Artificial exon shuffling among myoglobin, hemoglobin alpha- and beta-subunits

  K Wakasugi, K Ishimori, Morishima, I

  BIOPHYSICAL CHEMISTRY Elsevier {BV} 68 (1-3) 265 - 273 0301-4622 1997/10 [Refereed][Not invited]
   

  Based on the detailed structural analysis of proteins, Go [M. Go, Nature 291 (1981) 90-92] found that protein structures can be divided into some structural units, 'modules,' which correspond to peptides coded by exons. In the present study, to investigate functional and structural roles of modular structures in proteins, we have engineered eight chimera globins, in which the exons are shuffled among human myoglobin, human hemoglobin alpha- and beta-subunits, in addition to the chimera beta beta alpha-globin described previously [K. Wakasugi, K. Ishimori, K. Imai, Y. Wada, I. Morishima, J. Biol. Chem. 269 (1994) 18750-18756]. Although all of the chimera globins stoichiometrically bound the heme and their alpha-helical contents increased by heme incorporation as found for native globins, the alpha-helical contents of the chimera globins were significantly lower than those of native globins, suggesting that 'module' substitutions seriously affect the protein folding and stability in globins. The comparisons among several chimera globins demonstrated that such structural alterations are mainly attributed to loss of some key intermodular interactions for protein folding. By simultaneous substitution of the modules M1 and M4 from the same globin, the protein structure was stabilized, which indicates that the module packing between modules M1 and M4 would be one of the crucial interaction to stabilize the globin fold. Present results allow us to conclude that module substitutions would be available for designing and producing novel functional proteins if we can reproduce the stable modular packing in the 'module'-substituted proteins. (C) 1997 Elsevier Science B.V.
• Hydrogen bond network in the distal site of peroxidases: Spectroscopic properties of Asn70->Asp horseradish peroxidase mutant

  M Tanaka, S Nagano, K Ishimori, Morishima, I

  BIOCHEMISTRY 36 (32) 9791 - 9798 0006-2960 1997/08 [Refereed][Not invited]
   

  The distal His in peroxidases forms a hydrogen bond with the adjacent Asn, which is highly conserved among many plant and fungal peroxidases. Our previous work [Nagano, S., Tanaka, M., Ishimori, K., Watanabe, Y., & Morishima, I. (1996) Biochemistry 35, 14251-14258] has revealed that the replacement of Asn70 in horseradish peroxidase C (HRP) by Val (N70V) and Asp (N70D) discourages the oxidation activity for guaiacol, and the elementary reaction rate constants for the mutants was decreased by 10-15-fold. In order to delineate the structure-function relationship of the His-Asn couple in peroxidase activity, heme environmental structures of the HRP mutant, N70D, were investigated by CD, H-1 NMR, and IR spectroscopies as well as Fe2+/Fe3+ redox potential measurements. While N70D mutant exhibited quite similar CD spectra and redox potential to those of native enzyme, the paramagnetic NMR spectrum clearly showed that the hydrogen bond between the distal His and Asp70 is not formed in the mutant. The disappearance of the splitting in the H-1 NMR signal. of heme peripheral g-methyl group observed in 50% H2O/50% D2O solution of N70D-CN suggests that the hydrogen bond between the distal His and heme-bound cyanide is also disrupted by the mutation, which was supported by the low C-N vibration frequency and large dissociation constant of the heme-bound cyanide in the mutant. Together with the results from various spectroscopies and redox potentials, we can conclude that the improper positioning of the distal His induced the cleavages of the hydrogen bonds around the distal His, resulting in the substantial decrease of the catalytic activity without large structural alterations of the enzyme. The His-Asn hydrogen bond in the distal site of peroxidases, therefore, is essential for the catalytic activity by controlling the precise location of the distal His.
• Catalytic activities and structural properties of horseradish peroxidase distal His42->Glu or Gln mutant

  M Tanaka, K Ishimori, M Mukai, T Kitagawa, Morishima, I

  BIOCHEMISTRY 36 (32) 9889 - 9898 0006-2960 1997/08 [Refereed][Not invited]
   

  The distal histidine (His) is highly conserved in peroxidases and has been considered to play a major role as a general acid-base catalyst for peroxidase reaction cycle. Recently, however, the X-ray structure of chloroperoxidase from the marine fungus Caldariomyces fumago has revealed that a glutamic acid is located at the position where most of the peroxidase has a histidine residue, suggesting that the carboxyl group in the glutamic acid (Glu) can also assist cleavage of an O-O bond in peroxides [Sundaramoorthy, M., Terner, J., & Poulos, T. L. (1995) Structure 3, 1367-1377]; In order to investigate catalytic roles of the glutamic acid at the distal cavity, two horseradish peroxidase mutants were prepared, in which the distal His42 has been replaced by Glu (H42E) or Gln (H42Q). The formation rate of compound I in the H42E mutant was significantly greater than that for the H42Q mutant, indicating that the distal Glu can play a role as a general acid-base catalyst. However, the peroxidase activity of the H42E mutant was still lower, compared to that for native enzyme. On the basis of the CD, resonance Raman, and EPR spectra, it was suggested that the basicity of the distal Glu is lower than that of the distal His and the position of the distal Glu is not fixed at the optimal position as a catalytic amino acid residue, although no prominent structural changes around heme environment were detected. The less basicity and improper positioning of the distal Glu would destabilize the heme-H2O2-distal Glu ternary intermediate for the peroxidase reaction. Another characteristic feature in the mutants was the enhancement of the peroxygenase activity. Since the peroxygenase activity was remarkably enhanced in the H42E mutant, the distal Glu is also crucial to facilitate the peroxygenase activity as well as the enlarged distal cavity caused by the amino acid substitution. These observations indicate that the distal amino acid residue is essential for function of peroxidases and subtle conformational changes around the distal cavity would control the catalytic reactions in peroxidase.
• Mechanism of peroxidase activity as studied with some recombinant horseradish peroxidases

  Motomasa Tanaka, Atsushi Morimoto, Koichiro Ishimori, Isao Morishima

  Journal of Inorganic Biochemistry Elsevier {BV} 67 (1-4) 80  1997/07 [Refereed][Not invited]
  
• Effects of concerted hydrogen bonding of distal histidine on active site structures of horseradish peroxidase. Resonance Raman studies with Asn70 mutants

  M Mukai, S Nagano, M Tanaka, K Ishimori, Morishima, I, T Ogura, Y Watanabe, T Kitagawa

  JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 119 (7) 1758 - 1766 0002-7863 1997/02 [Refereed][Not invited]
   

  Effects of a highly conserved hydrogen bond between the distal histidine (His) and a nearby asparagine residue (Asn) of peroxidases upon the active site structures were investigated using resonance Raman (RR) and EPR spectroscopy. Although there is no crystallographic data for horseradish peroxidase (HRP), Asn70 is deduced to be the hydrogen bond acceptor for HRP. Accordingly, site-directed mutagenesis of Asn70 to Val (N70V) and to Asp (N70D) was carried out with HRP, and their RR spectra were compared with those of native and wild type (WT) enzymes in the resting, reduced, CN-bound ferric and compound II states. In the resting state, the six-coordinate high-spin structure is the main component for N70V and N70D mutants, while the five-coordinate high-spin structure is dominant for the native and WT HRPs. This was confirmed with EPR spectra. The Fe-III-CN stretching (nu(Fe-CN)) and bending RR bands of the linear and bent forms were identified using (CN)-C-12-N-15 and (CN)-C-13-N-14 isotopes. The nu(Fe-CN) frequency of the linear form is lower for the mutants than for native enzyme, and the spectral patterns of the mutants at pH 7.0 resemble that of the basic form of native HRP. The Fe-histidine stretching bands of reduced HRPs exhibit pH dependent frequency shifts, and the midpoint pH values were 7.2, 5.9, and 5.5 for native, N70V, and N70D, respectively. This change is ascribed to the acid-base transition of the distal His. While the Fe-IV=0 stretching (nu(Fe=0)) frequency of compound LI at pD 7.0 is lower than that at pD 10.0 for native enzyme, the nu(Fe=0) band of the mutants show no pH dependent frequency shifts between pD 7.0 and pD 10.0. However, the H2O/D2O frequency change of nu(Fe=0) and the oxygen atom exchange with bulk water suggested the presence of the hydrogen bond between the oxygen ligand of the ferryloxo heme and distal His for these mutants at pD 10.0. On the basis of these observations, it is proposed that the hydrogen bond between the N-delta-proton of distal His and Asn70 regulates the pK(a) of the N-epsilon- proton (and thus the reactivity of compound LI at the distal side) and also affects the Fe-His bond at the proximal side via tertiary structure changes.
• Effects of the intramolecular disulfide bond on ligand binding dynamics in myoglobin

  T Uchida, M Unno, K Ishimori, Morishima, I

  BIOCHEMISTRY 36 (2) 324 - 332 0006-2960 1997/01 [Refereed][Not invited]
   

  In order to investigate the effects of an intramolecular disulfide bond on protein structure and ligand binding dynamics in myoglobin, we prepared a mutant myoglobin having a disulfide bond at the EF corner by introducing two cysteine at the position of Ile 75 and Glu 85. On the basis of the spectral features of the mutant, the formation of the disulfide bond only affected minor structural deviations of the heme environmental structure in the carbonmonoxy form, whereas more substantial structural alterations were induced in the deoxygenated form. Laser photolysis experiments for carbon monoxide rebinding clearly showed that the artificial S-S bond accelerates the bimolecular rebinding rate from 1.0 to 1.8 mu M(-1) s(-1) and increases the geminate yield from 0.072 to 0.092. The ligand migration rate from the solvent to the heme pocket and the bond formation rate from the heme pocket to the heme iron also increased. The free energy diagram for the mutants indicates that the energy barrier for the bond formation was raised as well as that for the ligand migration by introduction of the disulfide bond. However, the effects of the disulfide linkage at the EF corner on the kinetic parameter is much smaller than those of the amino acid substitutions located in the heme cavity. We can conclude that the perturbation of the protein fluctuations by formation of the disulfide bond would be localized at the mutation site or the contributions from other regions and motions might be more important for the Ligand binding dynamics.
• Characterization of a mutant RecA protein that facilitates homologous genetic recombination but not recombinational DNA repair: RecA423

  K Ishimori, S Sommer, A Bailone, M Takahashi, MM Cox, R Devoret

  JOURNAL OF MOLECULAR BIOLOGY 264 (4) 696 - 712 0022-2836 1996/12 [Refereed][Not invited]
   

  A recA mutant (recA423; Arg(169) --> His), with properties that should help clarify the relationship between the biochemical properties of RecA protein and its two major functions, homologous genetic recombination and recombinational DNA repair, has been isolated. The mutant has been characterized in vivo and the purified RecA423 protein has been studied in vitro. The recA423 cells are nearly as proficient in conjugational recombination, transductional recombination, and recombination of lambda red(-) gam(-) phage as wild-type cells. At the same time, the mutant cells are deficient for intra-chromosomal recombination and nearly as sensitive to UV irradiation as a recA deletion strain. The cells are proficient in SOS induction, and results indicate the defect involves the capacity of RecA protein to participate directly in recombinational DNA repair. In vitro, the RecA423 protein binds to single-stranded DNA slowly, with an associated decline in the ATP hydrolytic activity. The RecA423 protein promoted a limited DNA strand exchange reaction when the DNA substrates were homologous, but no bypass of a short heterologous insert in the duplex DNA substrate was observed. These results indicate that poor binding to DNA and low ATP hydrolysis activity can selectively compromise certain functions of RecA protein. The RecA423 protein can promote recombination between homologous DNAs during Hfr crosses, indicating that the biochemical requirements for such genetic exchanges are minimal. However, the deficiencies in recombinational DNA repair suggest that the biochemical requirements for this function are more exacting. (C) 1996 Academic Press Limited
• Catalytic roles of the distal site asparagine-histidine couple in peroxidases

  S Nagano, M Tanaka, K Ishimori, Y Watanabe, Morishima, I

  BIOCHEMISTRY 35 (45) 14251 - 14258 0006-2960 1996/11 [Refereed][Not invited]
   

  There are highly conserved hydrogen bonds between the distal His and the adjacent Asn in many peroxidases. Although the crystal structure of horseradish peroxidase C (HRP) is not available, comparison of the amino acid sequence with cytochrome c peroxidase indicates that Asn70 is making the hydrogen bond with the distal His in the active site of HRP. To investigate the catalytic roles of the hydrogen bond, Asn70 in HRP was replaced with Val (N70V) or Asp (N70D), Though UV-vis, CD, and H-1-NMR spectra of native (plant enzyme), wild-type (recombinant enzyme), and mutant HRPs suggest that the active site and secondary structure are very similar even after the mutation, the mutants exhibit low V-max values for the hydroquinone oxidation (native, 281; wild-type, 283; N70V, 18; and N70D, 33 mu M . min(-1)). The rates of compound I formation were decreased to less than 10% of that of the native enzyme. The reduction rates of compounds I and II by guaiacol also were reduced to less than 10% of that of the native enzyme. Substituent effects of various phenol derivatives on the reduction of native, wild-type, and mutant compound I were examined. Large negative Hammett rho values (rho(N70V:fast) = -4.0, rho(N70V:slow) = -3.6, rho(N70D) = -3.8, rho(native) = -6.9, and rho(wild-type) = -6.8) are an indication of electron transfer being the rate-determining step in the phenol oxidation. However, these results also indicate the participation of the deprotonation step in the compound I reduction process. The proton abstraction from phenol must be harder for the mutants due to the decrease of basicity of the distal His upon mutation. Contrary to phenol oxidation, ABTS [2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] oxidation activity was substantially increased by the mutations (native, 73; wild-type, 71; N70V, 217; and N70D, 234 mu M . s(-1)). The redox potentials of N70V and N70D compounds II are 957 and 970 mV (vs NHE), which are 95 and 108 mV higher than that of native compound II (862 mV), respectively, Therefore, the high ABTS oxidation activities of mutants are attributed to these high redox potentials of compound II.
• The distal glutamic acid as an acid-base catalyst in the distal site of horseradish peroxidase

  M Tanaka, K Ishimori, Morishima, I

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 227 (2) 393 - 399 0006-291X 1996/10 [Refereed][Not invited]
   

  The distal His is an essential amino acid residue as a general acid-base catalyst for peroxidase reaction cycle. However, the x-ray structure of chloroperoxidase revealed that Glu is located near the heme, suggesting that the carboxyl group also assists cleavage of O-O bond in peroxides. In this paper, we examined functional and structural properties of a horseradish peroxidase mutant having Glu instead of the distal His. Although this amino acid replacement depressed reaction rate with H2O2 and oxidation activity for guaiacol, the mutant still exhibited much higher activity than mutants in which the distal His was replaced by hydrophobic amino acid. Kinetic measurements suggest that the proton abstraction is decelerated in the mutant due to large fluctuation of the carboxyl group of the distal Glu. Therefore, we can conclude that Gla can be a potent acid-base catalyst for peroxidase reaction cycle, if the carboxyl group can be fixed at the optimum position. (C) 1996 Academic Press, Inc.
• Preparation and reactions of myoglobin mutants bearing both proximal cysteine ligand and hydrophobic distal cavity: Protein models for the active site of P-450

  T Matsui, S Nagano, K Ishimori, Y Watanabe, Morishima, I

  BIOCHEMISTRY 35 (40) 13118 - 13124 0006-2960 1996/10 [Refereed][Not invited]
   

  We have reported that H93C human myoglobin (Mb), in which proximal histidine (His93, F8) was replaced by cysteine, gave nearly identical spectroscopic features of P-450 [Adachi, S., Nagano, S., Ishimori, K., Watanabe, Y., Morishima, I., Egawa, T., Kitagawa, T., & Makino R. (1993) Biochemistry, 32, 241-252]. More importantly, the thiolate ligand enhanced its oxygenation activities when supported by H2O2 due to the exclusive encouragement of heterolytic O-O bond cleavage of peroxides. While we have attributed the enhanced heterolysis to the electron donation from the thiolate ligand, possible participation of the distal histidine (H64, E7) in H93C Mb cannot be eliminated. In addition, the racemic product formation catalyzed by H93C Mb implied that its distal cavity could prevent substrates from accessing to the heme and the reactions may proceed other than by the P-450 type mechanism (ferryl oxygen transfer). In order to clarify whether the distal histidine is involved in the O-O bond cleavage step and to improve accessibility of substrates, the distal histidine of H93C Mb is replaced by smaller and nonpolar residues, glycine (H64G/H93C Mb) and valine (H64V/H93C Mb), by site-directed mutagenesis. Various spectroscopic studies on these double-mutated Mbs revealed the ligation of cysteine to the ferric heme as a thiolate form. In the reaction with cumene hydroperoxide, the anionic nature of the proximal cysteine in H64G/H93C and H64V/H93C Mbs was found to encourage the heterolytic O-O bond cleavage as observed for H93C Mb, The results clearly demonstrate that the distal histidine of H93C Mb is hardly involved in the O-O bond cleavage step and are in good agreement with the role of thiolate ligation for the formation of the reactive intermediate, equivalent to compound I, in the catalytic cycle of P-450 reactions. In the oxygenation of methyl p-tolyl sulfide, the ratios of ferryl oxygen transfer increased in H64G/H93C Mb (58%) and H64V/H93C Mb (78%) as compared to H93C Mb (53%). The increased ratios of ferryl oxygen transfer imply the active site of H64G/H93C and H64V/H93C Mbs being more accessible for substrates; however, the sulfoxidation by the ferric mutant Mbs/H2O2 system was much slower than that by H93C Mb. The poor activities of these mutant Mbs are attributed to the significantly discouraged binding of H2O2.
• NMR studies of recombinant cytochrome P450cam mutants

  K Wakasugi, K Ishimori, Morishima, I

  BIOCHIMIE 78 (8-9) 763 - 770 0300-9084 1996 [Refereed][Not invited]
   

  In the active center of cytochrome P450cam, Thr-252 is one of the conserved amino acid residues in the cytochrome P450 superfamily and plays a key role in hydroxylation of camphor. T252A mutant, in which Thr-252 is replaced by alanine, consumed O-2 at a rate comparable to that of the wild-type enzyme, whereas the amount of exo-5-hydroxycamphor formed was less than 10% of that formed by the wild-type enzyme and H2O2 is the main product in the hydroxylation reaction. H2O2 was also yielded by the valine mutant and the consumption rate of O-2 was much lower than that for the wild-type enzyme (Imai et al (1989) Pi-oc Natl Acad Sci USA 86, 7823-7827). On the basis of the H-1- and N-15-NMR spectra. it was revealed that the anionic nature of the axial thiolate and the heme-environmental structures were substantially affected in the absence of d-camphor by the amino acid substitution at 252 Thr. In T252A mutant, however, the binding of camphor reduced these conformational alterations in the heme vicinity, probably due to the formation of interactions between camphor and enzyme. On the other hand, T252V mutant still exhibited large reduction of the anionic nature of the axial ligand in the presence of d-camphor and structural changes around heme were also enhanced, since the affinity of the valine mutant to d-camphor was low. These results imply that the hydrophobic and/or steric effects of the valine residue at 252 interfere with the interactions around heme and camphor binding sites, which correspond to the larger functional defects for T252V mutant.
• Structure and function of module-substituted hemoproteins

  Keisuke Wakasugi, Kenhi Inaba, Koichiro Ishimori, Isao Morishima

  Journal of Inorganic Biochemistry Elsevier {BV} 59 (2-3) 435  1995/08 [Refereed][Not invited]
  
• Module substitution in globins: Preparation and association characteristics of chimeric hemoglobin subunits and myoglobin

  K Wakasugi, K Ishimori, Morishima, I

  TRACING BIOLOGICAL EVOLUTION IN PROTEIN AND GENE STRUCTURES 283 - 295 1995 [Refereed][Not invited]
  
• HIGH-PRESSURE FLASH-PHOTOLYSIS STUDY OF HEMOPROTEIN - EFFECTS OF SUBSTRATE-ANALOGS ON THE RECOMBINATION OF CARBON-MONOXIDE TO CYTOCHROME P450(CAM)

  M UNNO, K ISHIMORI, Y ISHIMURA, MORISHIMA, I

  BIOCHEMISTRY 33 (32) 9762 - 9768 0006-2960 1994/08 [Refereed][Not invited]
   

  The effects of camphor and camphor analogues on the CO recombination kinetics of ferrous cytochrome P450(CAM) (P450(CAM)) at 293 K have been studied as a function of hydrostatic pressure (0.1-200 MPa) by means of flash photolysis. At 0.1 MPa, the association rate constant (k(on)) for substrate-free P450(CAM) is 8.5 x 10(6) M(-1) s(-1). Measurements as a function of pressure lead to a determination of the activation volume (Delta V-double dagger) of +4 cm(3) mol(-1) for substrate-free protein. This positive Delta V-double dagger is interesting because the CO association reaction of various hemoproteins, such as myoglobin and hemoglobin, exhibit negative Delta V-double dagger values [Adachi, S., & Morishima, I. (1989) J. Biol. Chem. 264, 18896-18901; Unno, M., Ishimori, K., & Morishima, I. (1990) Biochemistry 29, 10199-10205]. The binding of d-camphor and some camphor analogues (d-fenchone, 3-endo-bromocamphor, and 3,3,5,5-tetramethylcyclohexanone) into the heme pocket strongly influences the kinetics, i.e., k(on) is reduced ((1-10) x 10(5) M(-1) s(-1)) and Delta V-double dagger is altered to a negative value (-14 to -32 cm(3) mol(-1)). The negative Delta V-double dagger suggests that the effects of camphor and these camphor analogues are due to an increase in the iron-ligand bond formation barrier. On the other hand, the binding of adamantane and norcamphor does not affect the kinetics. This result is particularly surprising because both substrate analogues are located in the immediate vicinity of the CO binding site. Since both adamantane and norcamphor show high mobility in the heme active site, we conclude that a substrate fluctuation at the heme active site is an important determinant of the rate of the bond formation process.
• HIGH-PRESSURE FLASH-PHOTOLYSIS STUDY OF HEMOPROTEIN - EFFECTS OF SUBSTRATE-ANALOGS ON THE RECOMBINATION OF CARBON-MONOXIDE TO CYTOCHROME P450(CAM)

  M UNNO, K ISHIMORI, Y ISHIMURA, MORISHIMA, I

  BIOCHEMISTRY 33 (32) 9762 - 9768 0006-2960 1994/08 [Refereed][Not invited]
   

  The effects of camphor and camphor analogues on the CO recombination kinetics of ferrous cytochrome P450(CAM) (P450(CAM)) at 293 K have been studied as a function of hydrostatic pressure (0.1-200 MPa) by means of flash photolysis. At 0.1 MPa, the association rate constant (k(on)) for substrate-free P450(CAM) is 8.5 x 10(6) M(-1) s(-1). Measurements as a function of pressure lead to a determination of the activation volume (Delta V-double dagger) of +4 cm(3) mol(-1) for substrate-free protein. This positive Delta V-double dagger is interesting because the CO association reaction of various hemoproteins, such as myoglobin and hemoglobin, exhibit negative Delta V-double dagger values [Adachi, S., & Morishima, I. (1989) J. Biol. Chem. 264, 18896-18901; Unno, M., Ishimori, K., & Morishima, I. (1990) Biochemistry 29, 10199-10205]. The binding of d-camphor and some camphor analogues (d-fenchone, 3-endo-bromocamphor, and 3,3,5,5-tetramethylcyclohexanone) into the heme pocket strongly influences the kinetics, i.e., k(on) is reduced ((1-10) x 10(5) M(-1) s(-1)) and Delta V-double dagger is altered to a negative value (-14 to -32 cm(3) mol(-1)). The negative Delta V-double dagger suggests that the effects of camphor and these camphor analogues are due to an increase in the iron-ligand bond formation barrier. On the other hand, the binding of adamantane and norcamphor does not affect the kinetics. This result is particularly surprising because both substrate analogues are located in the immediate vicinity of the CO binding site. Since both adamantane and norcamphor show high mobility in the heme active site, we conclude that a substrate fluctuation at the heme active site is an important determinant of the rate of the bond formation process.
• MODULE SUBSTITUTION IN HEMOGLOBIN SUBUNITS - PREPARATION AND CHARACTERIZATION OF A CHIMERA BETA-ALPHA-SUBUNIT

  K WAKASUGI, K ISHIMORI, K IMAI, Y WADA, MORISHIMA, I

  JOURNAL OF BIOLOGICAL CHEMISTRY 269 (29) 18750 - 18756 0021-9258 1994/07 [Refereed][Not invited]
   

  In the genes of alpha- and beta-subunits of hemoglobin, Go showed that modules F1, F2 + F3, and F4 correspond to exons 1, 2, and 3, respectively (Go, M. (1981) Nature 291, 90). The analysis of the correlation of function with its exon pattern showed that the residues associated with the defined function are concentrated in the specific exons encoding the ''module'' (Eaten, W.A. (1980) Nature 284, 183). To investigate the functional and structural significance of the ''modular structure,'' we engineered a ''chimera'' subunit, in which module F4 of the beta-subunit was replaced by that of the alpha-subunit by use of mutagenesis. The NMR and resonance Raman spectra of the isolated ''chimera beta alpha-subunit'' have revealed that it has a beta-subunit-like heme environmental structure. However, the gel chromatography and NMR spectra of mixtures of the chimera and native subunits clearly showed that the chimera beta alpha-subunit binds specifically to the beta-subunit to form a heterotetramer, not to the alpha-subunit. These results led us to conclude that the predominant role of the module F4 is the subunit association and suggest that the modules are structural and functional units that have advantages in producing stable functional proteins.
• SITE-DIRECTED MUTAGENESIS IN HEMOGLOBIN - FUNCTIONAL AND STRUCTURAL ROLE OF THE PENULTIMATE TYROSINE IN THE ALPHA-SUBUNIT

  K ISHIMORI, M HASHIMOTO, K IMAI, K FUSHITANI, G MIYAZAKI, H MORIMOTO, Y WADA, MORISHIMA, I

  BIOCHEMISTRY 33 (9) 2546 - 2553 0006-2960 1994/03 [Refereed][Not invited]
   

  The penultimate tyrosine in the hemoglobin subunit is considered to be one of the most important residues for the normal structure and function of hemoglobin. To elucidate the functional and structural role of the penultimate residue in the alpha-subunit, we prepared new artificial mutants; Hb Y140 alpha Q, in which Tyr-140 alpha is replaced by a nonaromatic residue, Gln, and Hb Y140 alpha F, which loses its hydrogen bond to Val-93 alpha by the substitution of Phe for Tyr. HB Y140 alpha Q exhibited a markedly increased oxygen affinity and almost completely diminished cooperativity, whereas Hb Y140 alpha F showed similar but less extensively impaired function, indicating that the aromatic residue at the penultimate position in the cr-subunit contributes to the stabilization of the T-quaternary structure as does the corresponding residue in the beta-subunit. However, the deoxygenated forms of these mutants bear significant T-state character in their spectroscopic properties observed at high protein concentrations. The tetramer-dimer equilibrium data of the mutants suggested that a significant part of the functional alterations observed for dilute solution appears to result from partial dissociation into alpha beta dimers rather than direct destabilization of the T-quaternary structure in the deoxygenated form. Therefore, we can conclude that the penultimate tyrosine in the alpha-chain plays a key role not only in the stabilization of the T-state but also in the subunit assembly. Such ease of dissociation from tetramer to dimers by amino acid substitution at the penultimate position did not occur in the beta-subunit mutant, implying different structural and functional roles of the penultimate tyrosine between the alpha- and P-subunits.
• SITE-DIRECTED MUTAGENESIS IN HEMOGLOBIN - FUNCTIONAL AND STRUCTURAL STUDY OF THE INTERSUBUNIT HYDROGEN-BOND OF THREONINE-38(C3)ALPHA AT THE ALPHA-1-BETA-2 INTERFACE IN HUMAN HEMOGLOBIN

  M HASHIMOTO, K ISHIMORI, K IMAI, G MIYAZAKI, H MORIMOTO, Y WADA, MORISHIMA, I

  BIOCHEMISTRY 32 (49) 13688 - 13695 0006-2960 1993/12 [Refereed][Not invited]
   

  To clarify the functional and structural roles of Thr-38alpha at the alpha1-beta2 interface, two artificial alpha-chain mutants, in which Thr-38alpha is replaced by Ser (Hb T38alphaS) or Val (Hb T38alphaV), were prepared. Thr-38alpha is one of the highly conserved amino acid residues in hemoglobins and forms a hydrogen bond to Asp-99beta, which is a crucial residue to stabilize the T state, via a water molecule in the deoxygenated form. We investigated their oxygen binding properties together with structural consequences of the mutations by using various spectroscopic probes. Their oxygen equilibrium curves showed small changes in the oxygen binding properties. Structural probes such as ultraviolet-region derivative and oxy-minus-deoxy difference spectra, rcsonance Raman scattering, and H-1-NMR spectra also indicated that the oxy and deoxy forms of these mutants show spectra characteristic of the R and T states, respectively, and the R-T transition is not very disturbed. The present structural and functional data of the mutants imply that the hydrogen bond between Thr-38alpha and Asp-99beta does not play a key role in stabilizing the deoxy T structure, which is in sharp contrast to the role of the hydrogen bond between Tyr-42alpha and Asp-99beta, and suggest that the interactions via the intersubunit hydrogen bonds are highly site-specific, depending on the amino acid residue which participates in them.
• NMR-STUDY OF HEME EXCHANGE-REACTION OF NATIVE MYOGLOBIN AND HEMOGLOBIN

  K ISHIMORI, MORISHIMA, I

  MAGNETIC RESONANCE IN CHEMISTRY 31 (13) S113 - S117 0749-1581 1993/12 [Refereed][Not invited]
   

  A proton NMR study of the haem exchange reactions of native myoglobin and haemoglobin with the various haems revealed that the haem displacement reaction occurs in native haemoproteins as has been detected in the reconstituted haemoproteins. In the presence of an excess amount of the modified haem, protohaem was excluded from a native haemoprotein to yield a reconstituted haemoprotein containing the modified haem, suggesting that the conformational fluctuations in native haemoprotein are large enough to dissociate its haem from the haem crevice. Although the haemoglobin subunits and myoglobin exhibit very similar three-dimensional structures as revealed by x-ray diffraction studies, the rates of the haem exchange reactions in myoglobin were larger than those in the haemoglobin beta-subunit. Such differences in the dynamic properties suggest that these haemoproteins have different interactions between the haem and amino acid residues inside the haem pocket.
• EFFECTS OF INTRASUBUNIT AND INTERSUBUNIT HYDROGEN-BONDS ON THE R-T TRANSITION IN HUMAN HEMOGLOBIN AS STUDIED WITH ALPHA-42(C7) AND BETA-145(HC2) MUTATIONS

  A TOGI, K ISHIMORI, M UNNO, T KONNO, MORISHIMA, I, G MIYAZAKI, K IMAI

  BIOCHEMISTRY 32 (38) 10165 - 10169 0006-2960 1993/09 [Refereed][Not invited]
   

  To clarify the effects of specific inter- and intrasubunit hydrogen bonds on the R-T transition in human hemoglobin (Hb A), the recombination reaction of carbon monoxide with artificial mutant Hbs was measured and analyzed. One of the hydrogen bonds we focused on is formed between Tyr-42alpha and Asp-99beta in the alpha1-beta2 interface of Hb A, which is one of the hydrogen bonds characteristic of the T state. Hb His-42alpha, in which Tyr-42alpha is replaced by His to perturb this hydrogen bond, showed that the ligand-free R to T transition rate was decreased by 20-fold compared with that for Hb A. This mutation caused the destabilization of the transition state in the R to T quaternary structure change by about 7 kJ mol-1, indicating that the hydrogen bond between Tyr-42alpha and Asp-99beta plays a definite role in the R-T transition as well as in stabilization of the equilibrium T state. Hb Phe-145beta, in which Tyr-145beta is replaced by Phe and the intrasubunit hydrogen bond between Tyr-145beta and Val-98beta is lacking, also showed a slow R-T transition rate as observed in Hb His-42alpha. The published crystallographic data suggest that this intrasubunit hydrogen bond stabilizes the transition state by reducing the freedom of motion of the C-terminus of the beta subunit and, thereby, facilitates the R-T transition.
• ROLES OF PROXIMAL LIGAND IN HEME-PROTEINS - REPLACEMENT OF PROXIMAL HISTIDINE OF HUMAN MYOGLOBIN WITH CYSTEINE AND TYROSINE BY SITE-DIRECTED MUTAGENESIS AS MODELS FOR P-450, CHLOROPEROXIDASE, AND CATALASE

  S ADACHI, S NAGANO, K ISHIMORI, Y WATANABE, MORISHIMA, I, T EGAWA, T KITAGAWA, R MAKINO

  BIOCHEMISTRY 32 (1) 241 - 252 0006-2960 1993/01 [Refereed][Not invited]
   

  Histidine-93(F8) in human myoglobin (Mb), which is the proximal ligand of the heme iron, has been replaced with cysteine or tyrosine by site-directed mutagenesis. The resultant proximal cysteine and tyrosine mutant Mbs (H93C and H93Y Mbs, respectively) exhibit the altered axial ligation analogous to P-450, chloroperoxidase, and catalase. Coordination of cysteine or tyrosine to the ferric heme iron is confirmed by spectroscopic measurements including electronic absorption, hyperfine-shifted H-1-NMR, EPR, resonance Raman spectroscopies, and redox potential measurements of ferric/ferrous couple. H93C Mb is five-coordinate ferric high-spin with the proximal cysteine. H93Y Mb bearing the proximal tyrosine ligated to the iron is also in a ferric high-spin, five-coordinate state. The reactions of the mutants with cumene hydroperoxide show that the thiolate ligand enhances heterolytic O-O bond cleavage of the oxidant, while the phenolate ligand hardly affects the heterolysis/homolysis ratio for O-O bond scission in comparison with wild-type Mb. Monooxygenase activities such as epoxidation of styrene and N-demethylation of N,N-dimethylaniline, and catalase activity (dismutation of hydrogen peroxide) by wild-type Mb and the mutants, are examined by using H2O2. The increase of the catalytic activities by the mutation was, at most, 5-fold in the epoxidation reaction.
• Characterization and Molecular Design of Hemoproteins by Protein Engineering.

  Koichiro ISHIMORI, Isao MORISHIMA

  Seibutsu Butsuri Biophysical Society of Japan 33 (4) 212 - 28 0582-4052 1993 [Refereed][Not invited]
   

  Hemoproteins are a class of biologically important proteins which have three distinct functions: reversible oxygen transport (myoglobin and hemoglobin), reversible electron transfer (cytochrome b's and c's), and catalytic activity (cytochrome P-450's and peroxidases). Recent protein engineering techniques have played important roles in understanding the regulation mechanism of the various functions in hemoproteins. In this review, we discuss the regulation mechanism of enzymatic oxidation reactions, modular structure, and cooperative oxygen binding in hemoproteins by using amino acid substituted proteins.
• ROLES OF PROXIMAL LIGAND IN HEME-PROTEINS - REPLACEMENT OF PROXIMAL HISTIDINE OF HUMAN MYOGLOBIN WITH CYSTEINE AND TYROSINE BY SITE-DIRECTED MUTAGENESIS AS MODELS FOR P-450, CHLOROPEROXIDASE, AND CATALASE

  S ADACHI, S NAGANO, K ISHIMORI, Y WATANABE, MORISHIMA, I, T EGAWA, T KITAGAWA, R MAKINO

  BIOCHEMISTRY 32 (1) 241 - 252 0006-2960 1993/01 [Refereed][Not invited]
   

  Histidine-93(F8) in human myoglobin (Mb), which is the proximal ligand of the heme iron, has been replaced with cysteine or tyrosine by site-directed mutagenesis. The resultant proximal cysteine and tyrosine mutant Mbs (H93C and H93Y Mbs, respectively) exhibit the altered axial ligation analogous to P-450, chloroperoxidase, and catalase. Coordination of cysteine or tyrosine to the ferric heme iron is confirmed by spectroscopic measurements including electronic absorption, hyperfine-shifted H-1-NMR, EPR, resonance Raman spectroscopies, and redox potential measurements of ferric/ferrous couple. H93C Mb is five-coordinate ferric high-spin with the proximal cysteine. H93Y Mb bearing the proximal tyrosine ligated to the iron is also in a ferric high-spin, five-coordinate state. The reactions of the mutants with cumene hydroperoxide show that the thiolate ligand enhances heterolytic O-O bond cleavage of the oxidant, while the phenolate ligand hardly affects the heterolysis/homolysis ratio for O-O bond scission in comparison with wild-type Mb. Monooxygenase activities such as epoxidation of styrene and N-demethylation of N,N-dimethylaniline, and catalase activity (dismutation of hydrogen peroxide) by wild-type Mb and the mutants, are examined by using H2O2. The increase of the catalytic activities by the mutation was, at most, 5-fold in the epoxidation reaction.
• STRUCTURE AND LIGAND-BINDING PROPERTIES OF LEUCINE-29(B10) MUTANTS OF HUMAN MYOGLOBIN

  S ADACHI, N SUNOHARA, K ISHIMORI, MORISHIMA, I

  JOURNAL OF BIOLOGICAL CHEMISTRY 267 (18) 12614 - 12621 0021-9258 1992/06 [Refereed][Not invited]
   

  Site-specific mutants of human myoglobin (Mb) have been prepared, in which Leu29(B10) is replaced by Ala(L29A) or Ile(L29I), in order to examine the influence of this highly conserved residue in the hydrophobic clusters of the heme distal site on the heme environmental structure and ligand binding properties of Mb. Structural characterizations of these recombinant Mbs are studied by electronic absorption, infrared (IR), one- and two-dimensional proton nuclear magnetic resonance spectroscopies, and ligand-binding kinetics by laser photolysis measurements under ambient and high pressures (up to 2000 bar). Multiple split carbon monoxide (CO) stretch bands in the IR spectra of mutant Mbs exhibit a relative decrease of the 1945 cm-1 band (approximately 50%) which is associated with an upright binding geometry of CO, accompanied by an increase of the tilted CO conformer at 1932 cm-1. On the basis of these results, replacement of Leu29(B10) by Ala or Ile appears to allow bound CO to rotate from a conformation pointing toward the beta-meso carbon of the heme group to the one pointing toward the alpha-meso carbon atom, presumably filling the space left by removal of the delta-2 carbon atom of Leu29(B10). These substitutions cause the rate constants for CO and O2 association to decrease almost 3-5-fold. Present results show that CO and O2 bindings to the heme iron of Mb are controlled by Leu29(B10) by influencing the structure of close vicinity of the heme and the geometry of iron-bound ligand. Further, mutant Mbs (Leu72(E15)-->Ala and Leu104 (G5)-->Ala) which have altered residues in another hydrophobic clusters around proximal and distal site are also examined.
• STRUCTURE AND LIGAND-BINDING PROPERTIES OF LEUCINE-29(B10) MUTANTS OF HUMAN MYOGLOBIN

  S ADACHI, N SUNOHARA, K ISHIMORI, MORISHIMA, I

  JOURNAL OF BIOLOGICAL CHEMISTRY 267 (18) 12614 - 12621 0021-9258 1992/06 [Refereed][Not invited]
   

  Site-specific mutants of human myoglobin (Mb) have been prepared, in which Leu29(B10) is replaced by Ala(L29A) or Ile(L29I), in order to examine the influence of this highly conserved residue in the hydrophobic clusters of the heme distal site on the heme environmental structure and ligand binding properties of Mb. Structural characterizations of these recombinant Mbs are studied by electronic absorption, infrared (IR), one- and two-dimensional proton nuclear magnetic resonance spectroscopies, and ligand-binding kinetics by laser photolysis measurements under ambient and high pressures (up to 2000 bar). Multiple split carbon monoxide (CO) stretch bands in the IR spectra of mutant Mbs exhibit a relative decrease of the 1945 cm-1 band (approximately 50%) which is associated with an upright binding geometry of CO, accompanied by an increase of the tilted CO conformer at 1932 cm-1. On the basis of these results, replacement of Leu29(B10) by Ala or Ile appears to allow bound CO to rotate from a conformation pointing toward the beta-meso carbon of the heme group to the one pointing toward the alpha-meso carbon atom, presumably filling the space left by removal of the delta-2 carbon atom of Leu29(B10). These substitutions cause the rate constants for CO and O2 association to decrease almost 3-5-fold. Present results show that CO and O2 bindings to the heme iron of Mb are controlled by Leu29(B10) by influencing the structure of close vicinity of the heme and the geometry of iron-bound ligand. Further, mutant Mbs (Leu72(E15)-->Ala and Leu104 (G5)-->Ala) which have altered residues in another hydrophobic clusters around proximal and distal site are also examined.
• SITE-DIRECTED MUTAGENESIS IN HEMOGLOBIN - FUNCTIONAL AND STRUCTURAL ROLE OF INTERSUBUNIT AND INTRASUBUNIT HYDROGEN-BONDS AS STUDIED WITH 37-BETA AND 145-BETA MUTATIONS

  K ISHIMORI, K IMAI, G MIYAZAKI, T KITAGAWA, Y WADA, H MORIMOTO, MORISHIMA, I

  BIOCHEMISTRY 31 (12) 3256 - 3264 0006-2960 1992/03 [Refereed][Not invited]
   

  In order to clarify the functional and structural role of intra- and intersubunit hydrogen bonds in human hemoglobin (Hb A), we prepared two artificial beta-chain mutant hemoglobins by site-directed mutagenesis. The mutant Hb Phe-37-beta, in which Trp-37-beta is replaced by Phe to remove the intersubunit hydrogen bond between Asp-94-alpha and Trp-37-beta at the alpha-1-beta-2 interface in deoxy Hb A, showed a markedly increased oxygen affinity and almost completely diminished Bohr effect and cooperativity. However, H-1-NMR data indicated that the structure of deoxy Hb Phe-37-beta is rather similar to that of deoxy Hb A. The enhanced tetramer-to-dimer dissociation previously observed in Hb Hirose (Trp-37-beta --> Ser) together with our observation of the effects of organic phosphate on the structure and function of Hb Phe-37-beta suggested that a large part of the abnormal properties of Hb Phe-37-beta observed for dilute solutions appears to result from partial dissociation into alpha-beta-dimers rather than direct destabilization of the T-quaternary structure in the deoxygenated state. Thus, the primary and direct role of the hydrogen bond between Asp-94-alpha and Trp-37-beta is to stabilize the tetrameric assembly, and thereby this hydrogen bond indirectly contributes to stabilization of the T-quaternary structure. The other mutant Hb Phe-145-beta has a Phe residue at the 145-beta site and lacks the intrasubunit hydrogen bond formed between Tyr-145-beta and the carbonyl group of Val-98-beta in deoxy Hb A. Although this hydrogen bond has been considered to fix the phenolic group of Tyr-145-beta in a pocket between the F and H helices, to strengthen the salt bridges formed by His-146-beta, and thereby to stabilize the T-quaternary structure, Hb Phe-145-beta exhibited only mild functional and structural alterations. This result led us to conclude that the van der Waals contacts between the benzene ring of Tyr-145-beta and the tyrosine pocket, rather than the hydrogen bond between Tyr-145-beta and Val-98-beta, make a major contribution to the stabilization of the T-quaternary structure. The present NMR spectra of deoxygenated Hb Phe-37-beta and Hb Phe-145-beta further showed that the exchangeable proton resonance observed at 6.4 ppm for deoxy Hb A originates from the intersubunit hydrogen bond between Asp-94-alpha and Trp-37-beta, although it has previously been assigned to the intrasubunit hydrogen bond between Val-98-beta and Try-145-beta.
• PRESSURE EFFECTS ON CARBON-MONOXIDE REBINDING TO THE ISOLATED ALPHA-CHAINS AND BETA-CHAINS OF HUMAN HEMOGLOBIN

  M UNNO, K ISHIMORI, MORISHIMA, I, T NAKAYAMA, K HAMANOUE

  BIOCHEMISTRY 30 (44) 10679 - 10685 0006-2960 1991/11 [Refereed][Not invited]
   

  The effects of pressure on the recombination kinetics of carbon monoxide binding to the isolated alpha and beta-chains of human adult hemoglobin at pH 7, approximately 20-degrees-C, were studied by the use of millisecond and nanosecond laser photolyses. The kinetic data were analyzed on the basis of a simple three-species model, which assumes two elementary reaction processes of bond formation and ligand migration steps. The activation volume for each elementary step was obtained from the pressure dependence of the rate constants. A pressure-dependent activation volume change from negative to positive values in the bimolecular carbon monoxide association reaction was observed for both of the isolated chains. This finding is attributed to a change of the rate-limiting step from the bond formation step to the ligand migration step. For both of the isolated chains, the activation volumes for ligand migration into and from the protein were estimated as +12-16 and +7-11 cm3 mol-1, respectively. These positive activation volumes for the ligand migration process may be caused by conformational fluctuations of proteins, that is, the conformational changes from "closed" to "open" structure. In the iron-ligand bond formation process, the activation volumes are -15 to -22 cm3 mol-1, which are almost identical to that for the model heme complexes [Taube, D. J., Projahn, H.-D., van Eldik, R., Magde, D., & Traylor, T. G. (1990) J. Am. Chem. Soc. 112, 6880-68861. Accordingly, the surrounding protein contributions to the activation volumes for the bond formation process could be small. A slight difference in the activation volumes between the isolated chains was found for each elementary step. This is discussed in relation to characteristic features of the dynamic aspects of the isolated alpha and beta-chains.
• ALTERATION OF HUMAN MYOGLOBIN PROXIMAL HISTIDINE TO CYSTEINE OR TYROSINE BY SITE-DIRECTED MUTAGENESIS - CHARACTERIZATION AND THEIR CATALYTIC ACTIVITIES

  S ADACHI, S NAGANO, Y WATANABE, K ISHIMORI, MORISHIMA, I

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 180 (1) 138 - 144 0006-291X 1991/10 [Refereed][Not invited]
  
• SITE-DIRECTED MUTAGENESIS IN HEMOGLOBIN - FUNCTIONAL-ROLE OF TYROSINE-42(C7)ALPHA AT THE ALPHA-1-BETA-2 INTERFACE

  K IMAI, K FUSHITANI, G MIYAZAKI, K ISHIMORI, T KITAGAWA, Y WADA, H MORIMOTO, MORISHIMA, I, DT SHIH, J TAME

  JOURNAL OF MOLECULAR BIOLOGY 218 (4) 769 - 778 0022-2836 1991/04 [Refereed][Not invited]
  
• HIGH-PRESSURE LASER PHOTOLYSIS STUDY OF HEMOPROTEINS - EFFECTS OF PRESSURE ON CARBON-MONOXIDE BINDING-DYNAMICS FOR R-STATE AND T-STATE HEMOGLOBINS

  M UNNO, K ISHIMORI, MORISHIMA, I

  BIOCHEMISTRY 29 (44) 10199 - 10205 0006-2960 1990/11 [Refereed][Not invited]
  
• CEREBRAL OXYGEN UTILIZATION ANALYZED BY THE USE OF O-17 AND ITS NUCLEAR-MAGNETIC-RESONANCE

  T ARAI, SI NAKAO, K MORI, K ISHIMORI, MORISHIMA, I, T MIYAZAWA, B FRITZZIEROTH

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 169 (1) 153 - 158 0006-291X 1990/05 [Refereed][Not invited]
  
• SITE-DIRECTED MUTAGENIC STUDIES ON THE MOLECULAR MECHANISM OF OXYGEN BINDING COOPERATIVITY IN HEMOGLOBIN

  K IMAI, K ISHIMORI, K FUSHITANI, G MIYAZAKI, T KITAGAWA, Y WADA, H MORIMOTO, MORISHIMA, I, D SHIH, J TAME, K NAGAI

  PROTEIN ENGINEERING // 213 - 218 1990 [Refereed][Not invited]
  
• RUTHENIUM IRON HYBRID HEMOGLOBINS AS A MODEL FOR PARTIALLY LIGANDED HEMOGLOBIN - OXYGEN EQUILIBRIUM CURVES AND RESONANCE RAMAN-SPECTRA

  K ISHIMORI, A TSUNESHIGE, K IMAI, MORISHIMA, I

  BIOCHEMISTRY 28 (21) 8603 - 8609 0006-2960 1989/10 [Refereed][Not invited]
  
• RUTHENIUM IRON HYBRID HEMOGLOBINS AS A MODEL FOR PARTIALLY LIGANDED HEMOGLOBIN - OXYGEN EQUILIBRIUM CURVES AND RESONANCE RAMAN-SPECTRA

  K ISHIMORI, A TSUNESHIGE, K IMAI, MORISHIMA, I

  BIOCHEMISTRY 28 (21) 8603 - 8609 0006-2960 1989/10 [Refereed][Not invited]
  
• NMR-STUDY OF HUMAN MUTANT HEMOGLOBINS SYNTHESIZED IN ESCHERICHIA-COLI - CONSEQUENCES OF TYROSINE-ALPHA-42 SUBSTITUTIONS

  K ISHIMORI, MORISHIMA, I, K IMAI, K FUSHITANI, G MIYAZAKI, D SHIH, J TAME, J PEGNIER, K NIGAI

  JOURNAL OF BIOLOGICAL CHEMISTRY 264 (25) 14624 - 14626 0021-9258 1989/09 [Refereed][Not invited]
   

  Ishimori K, Morishima I, Imai K, Fushitani K, Miyazaki G, Shih D, Tame J, Pegnier J, Nigai K, The Journal of biological chemistry, 1989, vol. 264, no. 25, pp. 14624-14626, 1989
• STUDY OF THE SPECIFIC HEME ORIENTATION IN RECONSTITUTED HEMOGLOBINS

  K ISHIMORI, MORISHIMA, I

  BIOCHEMISTRY 27 (13) 4747 - 4753 0006-2960 1988/06 [Refereed][Not invited]
  
• RUTHENIUM IRON HYBRID HEMOGLOBINS AS A MODEL FOR PARTIALLY LIGANDED HEMOGLOBIN - NMR-STUDIES OF THEIR TERTIARY AND QUATERNARY STRUCTURES

  K ISHIMORI, MORISHIMA, I

  BIOCHEMISTRY 27 (11) 4060 - 4066 0006-2960 1988/05 [Refereed][Not invited]
  
• DECREASED INTRACELLULAR FREE MAGNESIUM IN ERYTHROCYTES OF SPONTANEOUSLY HYPERTENSIVE RATS

  T MATUURA, M KOHNO, Y KANAYAMA, K YASUNARI, K MURAKAWA, T TAKEDA, K ISHIMORI, MORISHIMA, I, T YONEZAWA

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 143 (3) 1012 - 1017 0006-291X 1987/03 [Refereed][Not invited]
  
• INTERACTION OF FULLY LIGANDED VALENCY HYBRID HEMOGLOBIN WITH INOSITOL HEXAPHOSPHATE - IMPLICATION OF THE IHP-INDUCED T-STATE OF HUMAN ADULT METHEMOGLOBIN IN THE LOW-SPIN STATE

  MORISHIMA, I, M HARA, K ISHIMORI

  BIOCHEMISTRY 25 (22) 7243 - 7250 0006-2960 1986/11 [Refereed][Not invited]
  
• INTERACTION OF FULLY LIGANDED VALENCY HYBRID HEMOGLOBIN WITH INOSITOL HEXAPHOSPHATE - IMPLICATION OF THE IHP-INDUCED T-STATE OF HUMAN ADULT METHEMOGLOBIN IN THE LOW-SPIN STATE

  MORISHIMA, I, M HARA, K ISHIMORI

  BIOCHEMISTRY 25 (22) 7243 - 7250 0006-2960 1986/11 [Refereed][Not invited]
  
• NMR-STUDY OF HYBRID HEMOGLOBINS CONTAINING UNNATURAL HEME - EFFECT OF HEME MODIFICATION ON THEIR TERTIARY AND QUATERNARY STRUCTURES

  K ISHIMORI, MORISHIMA, I

  BIOCHEMISTRY 25 (17) 4892 - 4898 0006-2960 1986/08 [Refereed][Not invited]
  

MISC

• Structural Dynamics of the Trigger Factor Chaperone Regulating Proline cis/trans Isomerization in Protein Folding

  KAWAGOE Soichiro, KAWAGOE Soichiro, NAKAGAWA Hiroshi, KUMETA Hiroyuki, ISHIMORI Koichiro, SAIO Tomohide  電子スピンサイエンス学会年会講演要旨集  60th (CD-ROM)-  2021
• 電子伝達タンパク質シトクロムcを用いた難分解性色素分解酵素の設計

  大村翼世, 石森浩一郎, 内田毅  生体分子科学討論会講演要旨集  47th-  2021
• Creating an Artificial Dye-Decolorizing Peroxidase Rationally Designed Using Cytochrome c

  大村翼世, 石森浩一郎, 内田毅  酸化反応討論会講演要旨集  53rd-  2020
• 筋萎縮性側索硬化症原因タンパク質Fused in sarcoma(FUS)における液-液相分離制御機構の解明

  川向ほの香, 齋尾智英, 石森浩一郎  日本分子生物学会年会プログラム・要旨集(Web)  43rd-  2020
• The Catalytic Mechanism of a PPIase Chaperone Investigated by the Structural Analysis at the Transition State Using NMR and MD Simulation

  KAWAGOE Soichiro, SAIO Tomohide, NAKAGAWA Hiroshi, KUMETA Hiroyuki, ISHIMORI Koichiro  日本蛋白質科学会年会プログラム・要旨集  20th (Web)-  2020
• Exploiting paramagnetic metal ions for protein structural study in solution

  SAIO Tomohide, ISHIMORI Koichiro  生物物理(Web)  60-  (Supplement 1-2)  2020
• Mechanistic insight into dysregulation of FUS liquid-liquid phase separation by toxic repeat peptide

  KAWAMUKAI Honoka, SAIO Tomohide, ISHIMORI Koichiro  日本蛋白質科学会年会プログラム・要旨集  20th (Web)-  2020
• コレラ菌由来DyP型ペルオキシダーゼ(VcDyP)の色素分解反応の機構解明と至適pHの変換

  大村翼世, 石森浩一郎, 内田毅  酸化反応討論会講演要旨集  52nd-  2019
• ランタノイドイオンを用いたマルチドメインタンパク質MurDの構造分布解析

  斉尾智英, 平松蒼野, 浅田瑞枝, 瀧下俊平, 中川洋, 中村敏和, 石森浩一郎  日本細胞生物学会大会(Web)  71st-  2019
• タンパク質フォールディングにおける分子シャペロンの機能とメカニズム:立体構造と速度論からのアプローチ

  斉尾智英, 石森浩一郎  日本生化学会大会(Web)  92nd-  2019
• Structural Basis for the Concerted Action of Trigger Factor and ClpX Chaperones in Protein Unwinding

  ZHU Haojie, SAIO Tomohide, HOURY WalidA, ISHIMORI Koichiro  Abstracts. Annual Meeting of the NMR Society of Japan  58th (CD-ROM)-  2019
• 重金属イオンを利用したマルチドメインタンパク質におけるドメイン間距離の測定

  川向ほの香, 瀧下俊平, 齋尾智英, 齋尾智英, 石森浩一郎, 石森浩一郎  日本生化学会大会(Web)  92nd-  2019
• The activity control of dye-decolorizing peroxidase (DyP) by conversion of the optimal pH

  大村翼世, 石森浩一郎, 内田毅  金属の関与する生体関連反応シンポジウム講演要旨集  28th (Web)-  2018
• Dynamics analysis of conformational change of a multi-domain protein MurD using pulsed EPR with Gd³⁺

  平松蒼野, 齋尾智英, 浅田瑞枝, 瀧下俊平, 中村敏和, 石森浩一郎  電子スピンサイエンス学会年会講演要旨集  57th-  2018
• Complex Network Approach for Characterization of Protein Secondary Structure

  Shohei Konno, Takao Namiki, Koichiro Ishimori  Chem-Bio Informatics Society(CBI) Annual Meeting 2017 Abstracts  P05-02  2017/10  [Refereed][Not invited]
  
• マルチドメインタンパク質における構造アンサンブルと各構造の存在比の解析

  瀧下俊平, 齋尾智英, 齋尾智英, 齋尾智英, 平松蒼野, 久米田博之, 久米田博之, 清水和美, 石森浩一郎, 石森浩一郎  Abstracts. Annual Meeting of the NMR Society of Japan  56th-  2017
• パルスEPRとGd³⁺を用いたマルチドメイン蛋白質MurDの構造変化解析

  平松蒼野, 齋尾智英, 齋尾智英, 浅田瑞枝, 瀧下俊平, 中村敏和, 石森浩一郎  日本蛋白質科学会年会プログラム・要旨集  17th-  2017
• ヘム生合成制御因子Irrにおけるヘムによる標的DNAへの結合制御機構

  南多娟, 内田毅, 齋尾智英, O’BRIAN Mark R., 石森浩一郎  日本生化学会大会(Web)  90th-  2017
• ヘムを制御分子とする細胞内鉄濃度制御機構の構造化学的解明

  小倉麻梨子, 齋尾智英, 内田毅, 田中秀明, 田中秀明, 栗栖源嗣, 栗栖源嗣, 岩井一宏, 石森浩一郎  日本蛋白質科学会年会プログラム・要旨集  17th-  2017
• Amorphous Aggregation of Cytochrome C with Inherently low Amyloidogenicity is Characterized by the Metastability of Supersaturation and the Phase Diagram

  Yuxi Lin, Jozsef Kardos, Koichiro Ishimori, Yuji Goto, Young-Ho Lee  BIOPHYSICAL JOURNAL  110-  (3)  399A  -399A  2016/02  [Not refereed][Not invited]
  
• ヘム分解酵素HutZへのヘム輸送タンパク質HutXの構造・機能解析

  関根由可里, 石森浩一郎, 内田毅  日本化学会春季年会講演予稿集(CD-ROM)  96th-  2016
• ヘム分解酵素HutZにおけるヘム分解反応で生じた鉄による酵素活性の自己制御

  道順暢彦, 関根由可里, 石森浩一郎, 内田毅  日本化学会春季年会講演予稿集(CD-ROM)  96th-  2016
• コレラ菌由来ヘム分解酵素HutZのヘム分解反応機構

  関根由可里, 道順暢彦, 石森浩一郎, 内田毅  生体分子科学討論会講演要旨集  43rd-  2016
• ランタノイドイオンを用いたマルチドメインタンパク質の動的構造解析

  瀧下俊平, 齋尾智英, 齋尾智英, 久米田博之, 稲垣冬彦, 石森浩一郎  日本蛋白質科学会年会プログラム・要旨集  16th-  2016
• 鉄キレート剤の添加によるコレラ菌由来ヘム分解酵素HutZの活性抑制機構

  道順暢彦, 関根由可里, 松井敏高, 石森浩一郎, 石森浩一郎, 内田毅, 内田毅  東北大学多元物質科学研究所研究発表会講演予稿集  15th-  2015
• Demetallation of Heme by Dye-Decolorizing Peroxidase from Vibrio cholerae

  T. Uchida, K. Ishimori, T. Uchida, M. Sasaki, K. Ishimori  JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY  19-  S295  -S295  2014/03  [Not refereed][Not invited]
  
• HutZの活性中心近傍に存在する水素結合によるヘム分解活性の制御

  関根由可里, 内田毅, 松井敏高, 齋藤正男, 石森浩一郎  日本化学会講演予稿集  93rd-  (3)  2013
• ナノディスクを用いた膜タンパク質ハロロドプシンの機能解析

  山本愛弓, 塚本卓, 小橋川敬博, 内田毅, 出村誠, 稲垣冬彦, 石森浩一郎  日本蛋白質科学会年会プログラム・要旨集  12th-  63  2012/05/31  [Not refereed][Not invited]
  
• Function of Heme Regulatory Motif in the Oxidative Modification for Transcriptional Regulation

  TATSUKAWA Taira, UCHIDA Takeshi, ISHIMORI Koichiro  Peptide science : proceedings of the ... Japanese Peptide Symposium  2011-  191  -192  2012/03/01
• ステロール生合成を触媒するタンパク質CYP51のPGRMC1による活性化機構の解明

  畦地翔, 内田毅, 加部泰明, 末松誠, 石森浩一郎  化学系学協会北海道支部冬季研究発表会講演要旨集  2012-  2012
• COによるGAPDHを介した神経細胞防御機構の解明

  加部泰明, 山本竜也, 内田毅, 石森浩一郎, 半田宏, 末松誠  日本分子生物学会年会プログラム・要旨集(Web)  35th-  2012
• コレラ菌由来ヘム分解酵素HutZのヘム周辺環境とその機能

  関根由可里, 内田毅, 石森浩一郎  日本化学会講演予稿集  92nd-  (3)  2012
• コレラ毒素産生性Vibrio cholerae由来HutZにおけるヘム分解機構の解明

  関根由可里, 内田毅, 石森浩一郎  化学系学協会北海道支部冬季研究発表会講演要旨集  2012-  2012
• コレラ菌の鉄取り込み機構

  内田毅, 関根由可里, 宗田壮一朗, 石森浩一郎  生体分子科学討論会講演要旨集  39th-  2012
• コレラ菌由来HutZによるヘム分解反応とその構造化学的知見

  関根由可里, 内田毅, 石森浩一郎  日本蛋白質科学会年会プログラム・要旨集  12th-  2012
• コレラ菌由来HutZによるヘム分解活性と水素結合の役割

  関根由可里, 内田毅, 松井敏高, 齋藤正男, 石森浩一郎  酸化反応討論会講演要旨集  45th-  2012
• コレラ菌由来に存在するヘムから鉄を引き抜く酵素の反応機構と構造

  内田毅, 佐々木美穂, 関根由可里, 石森浩一郎  酸化反応討論会講演要旨集  45th-  2012
• ヘム/COによるGAPDHを介した細胞防御機構の解明

  加部泰明, 内田毅, 山本竜也, 石森浩一郎, 半田宏, 末松誠  生体分子科学討論会講演要旨集  38th-  2011
• ミトコンドリアの呼吸鎖におけるシトクロムcと活性酸素の反応機構の解明

  関根由可里, 内田毅, 石森浩一郎  日本化学会講演予稿集  91st-  (3)  2011
• ヘムチオレート錯体の構造と酸化還元機能に及ぼすNH-S水素結合の特異な効果

  鈴木潤, 山崎訓由, 泉康雄, 奥園希美子, 内田毅, 白川慶典, 山根健浩, 関根由可里, 加藤信樹, 石森浩一郎, 樋口恒彦  生体分子科学討論会講演要旨集  38th-  2011
• ミトコンドリア呼吸鎖でのシトクロムcによる新規の過酸化水素除去サイクルの検討

  関根由可里, 内田毅, 石森浩一郎  日本化学会北海道支部夏季研究発表会講演要旨集  2011-  2011
• コレラ毒素産生性Vibrio cholerae由来HutZによるヘムの分解機構

  関根由可里, 内田毅, 石森浩一郎  日本化学会バイオテクノロジー部会シンポジウム講演要旨集  14th-  2011
• Report of Symposium on Gender Equality in the 45th Annual Meeting of the Biophysical Society of Japan

  Koichiro ISHIMORI  Seibutsu Butsuri  48-  (1)  056  -57  2008  [Not refereed][Invited]
  
• グロビン型センサードメインを有するセカンドメッセンジャー(c-di-GMP)合成酵素の酸素による活性制御

  澤井仁美, 吉岡資郎, 内田毅, 兵藤守, 早川芳宏, 石森浩一郎, 青野重利  生体分子科学討論会講演要旨集  34th-  2007
• セカンドメッセンジャー(c-di-GMP)合成を制御する新規な酸素センサータンパク質の構造と性質

  吉岡資郎, 澤井仁美, 内田毅, 兵藤守, 早川芳宏, 石森浩一郎, 青野重利  酸化反応討論会講演要旨集  40th-  2007
• Early Research in Biophysics Award

  Shigeki MITAKU, Akinori KIDERA, Koichiro ISHIMORI  Seibutsu Butsuri  47-  (1)  059  -61  2007  [Not refereed][Invited]
  
• サブミリ秒H/D交換法とNMRによって観測されたアポミオグロビンの折り畳み中間体の構造ゆらぎ

  鵜澤尊規, 西村千秋, 秋山修志, 石森浩一郎, 高橋聡, DYSON H. Jane, WRIGHT Peter E  Abstracts. Annual Meeting of the NMR Society of Japan  45th-  38  -39  2006/11/20  [Not refereed][Not invited]
  
• Analysis of Stabilization by Substitution of Cyclohexylalanine for Phe341 in Hydrophobic Core of p53 Tetramer

  NOMURA Takao, SAKAMOTO Koichi, KASAI Yusuke, CHUMAN Yoshiro, ISHIMORI Koichiro, SAKAGUCHI Kazuyasu  Peptide science : proceedings of the ... Japanese Peptide Symposium  2005-  399  -402  2006/03/01
• 3P065 Helix formation in apomyoglobin folding revealed by submillisecond H/D exchange & NMR

  Uzawa T, Nishimura C, Akiyama S, Ishimori K, Takahashi S, Dyson Jane, Wright Peter  Seibutsu Butsuri  45-  (0)  S220  2005  [Not refereed][Not invited]
  
• Direct observation of the multistep helix formation of poly-L-glutamic acids using microsecond-resolved FTIR and CD spectroscopies.

  T Kimura, S Takahashi, S Akiyama, T Uzawa, K Ishimori, Morishima, I  BIOPHYSICAL JOURNAL  84-  (2)  162A  -162A  2003/02  [Not refereed][Not invited]
  
• Regulation Mechanism of a Novel Myoglobin with a Metal Binding Loop

  Taniguchi S, Ihara M, Takahashi S, K. Koichiro, Morishima I  Seibutsu Butsuri  43-  (0)  S77  2003  [Not refereed][Not invited]
  
• 1M1700 A Strategy for the Stabilization of Artificial-Protein Structure by Phage Display

  SUgi T, Ihara M, Ishimori K, Morishima I  Seibutsu Butsuri  42-  (2)  S79  2002  [Not refereed][Not invited]
  
• Local interaction is mainly important for the formation of apomyoglobin folding intermediate.

  M Kondo, S Akiyama, S Takahashi, Y Harada, K Ishimori, Morishima, I  BIOPHYSICAL JOURNAL  80-  (1)  557A  -558A  2001/01  [Not refereed][Not invited]
  
• Design of a New Myoglobin by Introducing a Metal Binding Loop

  Nishiyama A, Ihara M, Takahashi S, Ishimori K, Morishima I  Seibutsu Butsuri  40-  (0)  S187  2000  [Not refereed][Not invited]
  
• Studies for the design of artificial proteins by the module substitution between heterogenous proteins

  Ihara M, Takahashi S, Ishimori K, Morisima I  Seibutsu Butsuri  40-  (0)  S187  2000  [Not refereed][Not invited]
  
• Axial thiolate in cytochrome P450cam promotes the O-O bond scission by controlling the protonation site in the putative compound 0

  S Yoshioka, S Takahashi, K Ishimori, Morishima, I  JOURNAL OF INORGANIC BIOCHEMISTRY  74-  (1-4)  346  -346  1999/04  [Not refereed][Not invited]
  
• Characterization of the heme environment in CooA by H-1 NMR spectroscopy

  H Ishikawa, K Yamamoto, H Harada, K Ishimori, Morishima, I, H Nakajima, S Aono  JOURNAL OF INORGANIC BIOCHEMISTRY  74-  (1-4)  176  -176  1999/04  [Not refereed][Not invited]
  
• Properties of Mutant Hemoglobins with a Substitution at Tyr35-β

  Nakatsukasa T., Nomura N., Miyazaki G., Imai K., Wada Y., Morimoto H., Ishimori K., Morishima I.  Biophysics  38-  (2)  S38  1998/09/07
• The pressure Effects on the Intramolecular Electron Transfer Reactions in Hemoproteins

  Furukawa Y., Takahashi S., Ishimori K., Morishima I.  Biophysics  38-  (2)  S38  1998/09/07
• Structure of the folding intermediate of apomyolobin studied by fluorescence quenching.

  Tanioka H., Akiyama S., Inaba K., Takahashi S., Ishimori K., Morishima I.  Biophysics  38-  (2)  S161  1998/09/07
• Observation of structural fluctuation of Zn-substituted hemeprotein by time-resolved transient-hole-burning spectroscopy:individuality of protein

  SHIBATA Y., KANEKO R., KURITA A., KUSHIDA T., GOTO Y., FURUKAWA Y., ISHIMORI K., MORISHIMA I.  Meeting abstracts of the Physical Society of Japan  53-  (2)  854  -854  1998/09/05
• Heme environmental structure and CO binding reaction of CO-dependent transcriptional regulator under presence and absence of DNA

  UCHIDA T., ISHIKAWA H., TAKAHASHI S., ISHIMORI K., MORISHIMA I., OHKUBO K., NAKAJIMA H., SHIMONO T., AONO S.  Biophysics  37-  S175  1997/10
• Structures and Functions of the artificial globins designed by the substitution of the heme binding module

  INABA K., ISHIMORI K., MORISHIMA I.  Biophysics  37-  S98  1997/10
• Crystal structure analyses of module substituted chimera hemoglobins

  SHIRAI T., TANAKA M., FUJIKAKE M., INABA K., ISHIMORI K., MORISHIMA I.  Biophysics  37-  S97  1997/10
• Effects of intersubunit hydrogen bonds on association property of hemoglobin

  KONDO S., INABA K., ISHIMORI K., MORISHIMA I.  Biophysics  37-  S96  1997/10
• Catalytic and structural roles of Glu64 in the distal site of horseradish peroxidase

  TANAKA M., ISHIMORI K., MORISHIMA I.  Biophysics  37-  S177  1997/10
• The evolution of allostericity in vertevrate Hemoglobin

  KATAOKA H., TAKAHASI S., ISIMORI K., MORISIMA I.  Biophysics  37-  S177  1997/10
• Pressure Dependence of Intramolecular Electron Transfer Rate in Cytochrome b_5

  FURUKAWA Y., ISHIMORI K., MORISHIMA I.  Biophysics  37-  S180  1997/10
• Site-directed mutagenetics studies on molecular recognition of the heme orientational disorder in Myoglobin

  ISHIKAWA H., UCHIDA T., ISHIMORI K., MORISHIMA I.  Biophysics  37-  S176  1997/10
• The structure and function of Cytochrome b_5 mutant H39L

  IHARA M, TAKAHASI S, ISIMORI K, MORISHIMA I  Biophysics  37-  S181  1997/10  [Not refereed][Not invited]
  
• Effects of Concerted Hydrogen Bonding of Distal Histidine on Active Site Structures of Horseradish Peroxidase ; Resonance Raman Studies with Asn-70 Mutants

  NAGANO Shingo, MUKAI Masahiro, TANAKA Motomasa, ISHIMORI Koichiro, MORISHIMA Isao, OGURA Takashi, WATANABE Yoshihito, KITAGAWA Teizo  Keio journal of medicine  45-  S67  1996/12/01
• Design and engineering of module-substituted hemoproteins based on the exon-shuffling hypothesis

  Morishima, I, K Wakasugi, K Inaba, K Ishimori  PROGRESS IN BIOPHYSICS & MOLECULAR BIOLOGY  65-  SA503  -SA503  1996  [Not refereed][Not invited]
  
• Preparation and characterization of novel hemoproteins by module substitution in hemoglobin subunits

  K Inaba, K Wakasugi, K Ishimori, Morishima, I  PROTEIN ENGINEERING  8-  (9)  50  -50  1995/09  [Not refereed][Not invited]
  
• Preparation and characterization of novel hemoproteins by module substitution in myoglobin and hemoglobin subunits

  K Wakasugi, K Ishimori, Morishima, I  PROTEIN ENGINEERING  8-  (9)  39  -39  1995/09  [Not refereed][Not invited]
  
• 分子生物学的手法 (生物無機化学の新展開) -- (生物無機化学における研究手法の新展開)

  石森 浩一郎, 森島 績  季刊化学総説  (24)  p18  -23  1995/03
• ヘム酵素の機能発現と構造因子 (生物無機化学の新展開) -- (生物無機化学におけるモデル系は生体系にどこまで迫ることができるか)

  渡辺 芳人, 石森 浩一郎, 森島 績  季刊化学総説  (24)  p61  -86  1995/03
• Protein Molecular Engineering- Structure and Function of Mutant Hemoproteins

  ISHIMORI Koichiro  化学と工業 = Chemistry and chemical industry  45-  (2)  289  -294  1992/02/01

Books etc

• タンパク質科学

  化学同人 2005
• Protein Science

  Kagaku Dojin 2005

Association Memberships

• アメリカ生化学会   アメリカ化学会   日本生物物理学会   日本化学会   日本生化学会   American Society of Biochemistry and Molecular Biology   American Society of Chemistry   Biophysical Society of Japan   Chemical Society of Japan   Biochemical Society of Japan   

Research Projects

• Molecular Mechanism of Signal Transduction System Regulating Biometal Dynamics

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2019/06 -2024/03 

  Author : 石森 浩一郎

   

  生命金属科学研究基盤の確立に向けての研究 昨年度に引き続きヘム生合成の鍵酵素アミノレブリン酸合成酵素（ALAS1）の機能発現制御機構と、細胞内鉄代謝制御機構における鉄制御蛋白質（IRP）の機能制御機構について、以下の研究を行う。 １．細胞内鉄代謝制御機構におけるIRPの機能制御機構解析 細胞内鉄濃度恒常性は、鉄の細胞内への取り込みや鉄を貯蔵する蛋白質を翻訳段階で制御するIRPによって維持されている。本研究課題により、このIRPの翻訳機能を制御するシグナル伝達因子として、ヘムが同定されたが、その結合部位については、アミノ酸変異によって部位が異なることが示唆され、野生型におけるヘム結合部位については確定させることができていなかった。今年度は公募班の岐阜薬科大学の平山が開発したヘム修飾プローブを適用することで、IRPのホモログ蛋白質の一つであるIRP1におけるヘム結合部位が、これまでもそのヘム結合が示唆されていたヘム結合モチーフ部位であると決定することができた。 ２．鉄応答転写因子Furの新たな機能とその生物学的意義 多くのバクテリアに存在している鉄応答性転写因子Furは、細胞内で利用できる鉄量に応じて鉄取り込みなどの鉄代謝に関する蛋白質の転写制御を行うことで、細胞内鉄恒常性を維持している。しかし、コレラ菌のFurは、ニッケル代謝に関連する蛋白質群をコードしているnikオペロンを制御していることが見出され、その転写機能について、精製蛋白質を用いて検討したところ、ニッケルではなく鉄によって標的DNA配列であるFur boxへの結合が制御されることを見出した。さらに、このnikオペロンを構成する蛋白質の一つであるVC1098はヘムを結合し、その親和性はヘムを活性中心として結合する蛋白質に比べ低く、これまで未同定であったシトクロムc生合成におけるヘムシャペロンである可能性が示唆された。
• Integrated Biometal Science: Research to Explore Dynamics of Metals in Cellular System

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2019/06 -2024/03 

  Author : 津本 浩平, 石森 浩一郎, 小椋 康光, 古川 良明, 青野 重利, 城 宜嗣, 高野 順平, 神戸 大朋

   

  第1期の公募研究が参画したことから、ホームページを更新、毎月のニュースレター発行を継続、充実させた。前年度構築した連携研究・共同研究の積極的な実施体制を浸透させた。 総括班会議については、COVID-19の感染状況を鑑みてオンライン実施1回、対面とオンラインのハイブリッド実施2回の他、メール会議1回実施した。領域会議は、オンライン実施1回、ハイブリッド実施1回とした。領域会議地方巡業と銘打って、対面とオンラインでのハイブリッド方式での実施を東北大にて準備した（COVID-19感染状況からオンライン実施）。 また、領域の班会議やシンポジウムとは別に、生命金属科学関連分野の若手研究者を集めて「若手会」を設け、若い世代の人的交流を活発にする試みを開始した（3月にオンライン若手会を実施）。また、オンライン実施等となった、生化学会、化学会、薬学会等各種学術集会においても、合計11回のワークショップ、シンポジウムを共催したほか、生命金属に関連する合同年会（ConMetal2021）を共催し、研究成果を国内外に発信した。前年度の研究成果報告書を冊子としてまとめ、生命金属に関連する研究者に送付、御意見を頂き、領域運営に積極的に活用した。また、生命金属研究各領域の魅力について語り、融合研究を加速させるほか、一般社会にも広くアピールするため、IBmSウエブセミナーを領域内で合計12回にわたって実施した。特に公開可能な内容について順次ホームページで公開している。また、班員が中心となって進めている研究の最先端の内容をまとめた動画の作製に着手、領域内での十分な議論を踏まえ、公開を開始した。
• Molecular Design and Functional Regulation of Integrated Membrane-bound Protein Using Nono-disc

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2019/06 -2021/03 

  Author : Ishimori Koichiro

   

  We focused on nonodisc-reconstituted halorhodopsin (HR), pumping chloride ion into the cell in response to light, and bacterial cytochrome oxidase (cbb3), promoting four-electron reduction of molecular oxygen in the respiratory chain. Their structures and enzymatic activities were investigated by various kinds of spectroscopies, and the applications of these nanodisc-reconstituted proteins were examined. To facilitate the effective chloride pumping in HR, the interactions between HRs, effects of charges on the membrane, and flexibility of the membrane to tolerate the conformational changes associated with the chloride binding and releasing were found to be essential. We successfully immobilized cbb3 on the electrode and found that immobilized cbb3 can electrochemically mediate the four-electron reduction of molecular oxygen on the electrode. Further experiments are required to improve the efficiency of the reduction of molecular oxygen by using the nanodisc-reconstituted enzyme.
• Development and Application of Analysis for Physico-chemical Properties of Proteins in Cellular Multimolecular Crowding Biosystems

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2018/04 -2020/03 

  Author : 石森 浩一郎

   

  令和元年度も平成30年度に引き続き以下の項目について研究を進めたが、新学術領域研究「生命金属科学」の計画研究「生命金属動態を制御するシグナル伝達の分子機構」が採択されたため、年度途中で本研究課題は廃止となった。 ①細胞場における蛋白質構造の「構造揺らぎ」の部位特異的，定量的評価 平成30年度に引き続き、蛋白質表面にTrpを導入したミオグロビンとシトクロムcの作成を試みた。いずれも安定なTrp導入蛋白質を得るために、そのTrp導入部位について検討したが、実際の変異体を作成する以前に課題が廃止となった。 ②蛋白質立体構造形成反応 令和元年度はこれまでの研究で明らかになった脱水和に影響を及ぼすアミノ酸残基として、変性状態でヘムが配位するHis26に注目した。このHis26をヘムが配位しないGlnに置換した変異体を作成し、その立体構造過程における脱水和が細胞場環境においては野生型に比べどのように変化するのか追跡を試みたが、予備的な実験が終了する前に課題が廃止となった。 ③蛋白質間電子伝達複合体形成反応 令和元年度はNMRの緩和解析により、Cyt cにおける構造揺らぎの大きな部位を特定したところ、CcOとの相互作用部位とは離れたHis33に構造揺らぎを反映した化学交換が観測された。このHis33はAsn103と水素結合を形成していることから、HisをPheに置換することでこの水素結合切断したところ、化学交換が消失し、構造揺らぎが抑制されたことが示された。さらに、このような構造揺らぎの抑制はCcOへの電子伝達速度の低下をもたらし、相互作用部位から離れた部位での構造揺らぎがCyt cの電子伝達活性を制御していることが示された。このような構造揺らぎが細胞場類似環境でどのように変化するのか明らかにすることを試みたが、NMRの測定条件を検討するところで本課題が廃止となった。
• Dynamic Structural Characterization of Electron Transfer Complex in Respiratory Chain of Mitochondria and Its Electron Transfer Regulation Mechanism

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2016/04 -2019/03 

  Author : Ishimori koichiro

   

  One of the essential biological processes, the four-electron reduction of molecular oxygen to water molecules in the mitochondrial respiratory chain, is promoted by the electron transfer from cytochrome c, a typical heme-containing electron transfer protein, to membrane-bound cytochrome c oxidase. In this study, we examined the electron transfer reaction under the physiological conditions and revealed that the interactions between the proteins and lipids in the membrane, structural fluctuations, transient structural changes, of the proteins, the specific protein-protein interactions mediated by a few amino acid residues, and hydrophobic environment in the electron transfer pathways are the crucial factors to effectively promote the electron transfer reaction from cytochrome c to cytochrome c oxidase. These observations and discussion would contribute to the understanding of the molecular regulation mechanism for the inter-protein electron transfer reaction in vivo.
• 複合化活性部位を有する金属酸化酵素における感応性化学種の制御とその設計

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2015/04 -2017/03 

  Author : 石森 浩一郎

   

  平成28年度の研究計画の各項目における研究成果は以下の通りである。
  １．活性化Irrにおける活性部位の構造解析 昨年度に引き続き，NMRによるIrrの構造解析を進めるため、15N、13Cによるラベル化Irrの発現、単離、精製を行い、再現性良く高純度の安定同位体ラベルIrr収量を得ることができる手法を確立することができた。この安定同位体ラベルIrrを用いて、その15N-1H HSQCスペクトルを測定したが、NMRシグナルの分散が十分ではなく、立体構造解析可能なスペクトルを得ることができなかった。これは、これまでのIrr精製時には添加していた2価のマンガンイオンを、その常磁性によるNMR信号への影響を除くため添加しなかったことによって、Irrが安定な立体構造を形成できなかったためと考えられた。そこでこの常磁性の2価マンガンイオンの代わりに、反磁性の2価マグネシウムを加えて精製し、そのNMR測定を行ったが、NMR信号の分散は変化せず、マグネシウムイオンはIrrに結合しないと考えられた。そこで、同様に反磁性の2価亜鉛イオンを結合させることを試みたところ、分散したNMRスペクトルが得られ、その立体構造解析の指針が得られた。
  ２．アミノ酸置換による酸化反応の制御 Irrにおける酸化反応を制御するため、ヘム結合部位に位置するCys29をAlaに置換したところ、その酸化活性は大きく低下し、このCys29はIrrの酸化反応に重要な寄与をすることが明らかとなった。さらに、野生型では二量体を形成するIrrが、この変異により単量体化することが明らかとなった。以上の結果より、このヘム近傍に位置するCys29は酸化反応だけではなく、Irrにおける蛋白質間の会合状態についても大きな役割を果たしていることが新たに示された。
• Studies on the proton pump mechanism of the terminal oxidase by time resolved analyses at the hydrogen atom level resolution

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2014/04 -2017/03 

  Author : Yoshikawa Shinya, ISHIMORI Koichiro, TATENO Masaru, SHINZAWA Kyoko, KUBO Minoru, TAMADA Tarou, MISAKI Tomonori

   

  Cytochrome c oxidase, which is one of the most important enzymes for preserving the Life, reduces molecular oxygen (O2) coupled with proton pumping which creates the proton gradient for driving ATP synthesis by FoF1 ATPase. By using the X-ray free electron laser facility, we have succeeded in (i) determination of the O2 reduction site and (ii) showing that the pumping proton back leak is blocked by O2 binding to a copper ion site included in the O2 reduction site. The O2 reduction site structure has been yet to be determined for the last 50 years. The latter accomplishment together with the former set a mile stone for elucidation of the mechanism of this enzyme at the hydrogen atom level resolution which is a dream of most of Life Scientist.
• Functional and Structural Characterization of Electron Transfer Reactions in Mitochondria Respiratory Chain Utilizing Nanodisc

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2013/04 -2016/03 

  Author : Ishimori Koichiro, Takeshi Uchida, Tomohide Saio

   

  We reconstituted cytochrome c oxidase (CcO) into a biomembrane model, nanodisc, to characterize its functions in the absence of detergents. The resonance Raman spectra revealed that the reconstitution of CcO into the nanodisc increases the oxygen affinity, leading to efficient reduction of dioxygen to water molecules. To discuss interactions regulating the electron transfer (ET) reactions, we examined the cytochrome c (Cyt c) interaction site on CcO by docking simulation. Unexpectedly, electrostatic interactions do not contribute to the stabilization of the complex, but regulate the binding orientation of Cyt c on CcO. Instead, hydrophobic interactions are the primary factors to stabilize the complex, and the dehydration associated with the formation of the hydrophobic interactions is the key process to facilitate the complex formation. On the other hand, the structural fluctuations are suppressed in the CcO interaction site on Cyt c, which would also characterize the ET reaction.
• ヘムをシグナリング分子とする情報伝達システムの構造化学的基盤

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2013/04 -2015/03 

  Author : 石森 浩一郎

   

  IRP制御複合体の蛋白質間相互作用解析 IRPと複合体を形成すると考えられるmRNAのIREに対する結合特性，および，ヘムによるIRE結合阻害効果を検討した。その結果，ゲルシフトアッセイでは，その蛋白質としての安定性が低いため，明確な結果が得られなかったIRP2のIRE親和性について，その解離定数はIRP1のおよそ50％程度の0.013 mMであることが示された。さらに，そのヘムによる阻害効果はついては，1当量のヘムの添加で明確な阻害効果が観測され，阻害効果が現れるまでに数当量のヘムが必要なIRP1に比べ，IRP2のほうが低濃度のヘムによって阻害がかかることが示された。このことは細胞内においてIRP2が主な鉄濃度センサーとして機能していることとも対応している。さらに，IRPのIREからの解離に関しては，精製蛋白質を用いた実験では，数十当量のヘムの添加が必要であり，細胞内においてはヘムシャペロンによるIRPへのヘムの輸送および添加と，そのヘム結合によるIRPのIREからの解離が示唆された。一方，細胞内において，鉄高濃度時にIRPと相互作用することでIRPをユビキチン化し，最終的にはその蛋白質分解を促進するFBXL-5蛋白質についても，その大量精製系の確立を試みた。種々のプロモーターの検討の結果，大腸菌としてRosetta (DE3)pLysSを用い，MBPとの融合蛋白質として発現するのが最も収量が多いことを見出し，FBXL-5の大量精製系を確立することに成功した。 制御系に関連した蛋白質の探索とその機能解析 IRPへヘムを輸送，供与する蛋白質を同定するため，IRPと相互作用する蛋白質の探索を試みた。探索系としては，酵母菌を用いたtwo-hybrid systemと，ヘムを磁気ビーズに結合させた手法について検討を行った。
• 活性部位の複合化による感応性化学種の制御を利用した酸化酵素の設計と創製

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2013/04 -2015/03 

  Author : 石森 浩一郎

   

  活性化Irrの立体構造決定と酸素活性化機構の解明 昨年度に引き続き活性型Irrの構造決定を試み，本年度は特に，そのシグナル伝達分子としてのヘムの結合部位の立体構造について詳細に検討を行った。まず，これまでの研究から，その二段階酸素活性化の第一段階である分子状酸素の過酸化水素の活性化部位については，ラマンスペクトルの解析から，ヘモグロビンなど分子状酸素を安定に結合するタンパク質と異なり，分子状酸素を安定化する水素結合等の相互作用がないことが示され，容易にスーパーオキサイドアニオンが生成しすること，また，ヘム結合部位が疎水的なへムポケットではなく，容易に酸素分子へのプロトン化が起こり，過酸化水素の生成が誘起されることが示された。一方，もう一方のヘム結合部位であるヒスチジンクラスター領域は，昨年度の研究からヘムの分解と非ヘム鉄結合部位が形成されることが示され，さらに活性化Irrの質量分析の結果から，非ヘム鉄結合のアミノ酸としてHis63，His37等を同定することができた。これらの結果から，この非ヘム鉄結合部位は，過酸化水素を水酸ラジカルに活性化することで，自らのアミノ酸の酸化修飾を行うストレスセンサータンパク質PerRと同様な機能を有していることが示された。 酸化反応酵素としての利用を目指した分子設計 これまでの精製タンパク質を用いた実験から，Irrはタンパク質としての安定性が低く，また，溶存酸素と反応し自己酸化修飾反応が容易に誘起されることが明らかとなった。そこで，Irrを酸化反応に利用する環境として，Irrが安定に存在できる菌体内を検討し，in vivoの条件下で酸化反応を進行させることを試みるため，大腸菌内でIrrを発現する系を構築した。その結果，大腸菌内でIrrの発現を確認し，ヘム添加による酸化反応についてもその活性を検討できる系を構築することができた。
• Structural Characterization of Subunit-Specific Isotope labelled Membrane Bound Protein

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2013/04 -2015/03 

  Author : ISHIMORI Koichiro, UCHIDA Takeshi

   

  To discuss the electron transfer mechanism in the respiratory chain in mitochondria, the reconstitution of an isotope labelled key enzyme, cytochrome c oxidase, into the nano-disc, a mimic of membrane in cells, has been examined, which allows us to measure the high resolution NMR spectra of the membrane-bound proteins under the physiological condition. The expression, purification and reconstitution of the bacterial cytochrome c oxidase into the nano-disc were confirmed, leading to the high resolution structural analysis of high molecular weight membrane bound proteins.
• ヘムをシグナリング分子とする情報伝達システムの構造化学的基盤

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2011/04 -2013/03 

  Author : 石森 浩一郎

   

  Irr，IRPの標的DNA，RNAへの親和性 IrrやIRPの標的DNA，RNAへの親和性は，そのIrr-DNA，およびIRP-RNA複合体の結晶作成の上で重要であるが，これまではゲルシフトアッセイによる解離定数が見積もられただけで，その定量性は十分ではなかった．今年度は蛍光ラベルした標的DNAおよびRNAを用い，そのIrrやIRPへの結合による蛍光の偏光解消度から解離定数をより正確に見積もることを試み，その結果，それぞれ，220 ± 57 nM，6.85 ± 0.26 nMと決定することができた．さらにこの反応溶液中にヘムを添加することにより，蛍光の偏光解消は観測されず，ヘムの添加によって標的DNAやRNAへの結合が完全に阻害されることが確認できた． IRPへおけるヘム結合ドメインの決定 完全長のIRPでは，発現に昆虫細胞系を用いるほかなく，その限られた精製蛋白質量と蛋白質の構造不安定性から，結晶形成の条件検討は非常に困難であった．そこで，完全長のIRPの結晶化と並行して，そのヘム結合ドメインのみの結晶構造解析を行うことで，ヘム結合の蛋白質構造に与える影響の解明を試みた．まず，IRP1におけるヘム結合部位の同定を目指して，ヘム結合部位と想定されるヘム制御モチーフ中のCys118とCys300の変異体を作成することで検討を行った．その結果，いずれの変異体においても，ヘム制御蛋白質に特徴的なヘム鉄とその軸配位子Cys間の伸縮振動のラマン線が観測され，ヘムはCys118とCys300に配位することが確認できた．さらに，これらのCys残基はそれぞれドメイン1（1-240）とドメイン2（241-367）にあることから，IRP1におけるヘム結合ドメインは，ドメイン1とドメイン2と決定でき，これらの発現系を大量発現・精製が可能な大腸菌で確立させることにより，その結晶化が期待できる．
• Analysis of erythroid differentiation by HSP27

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2011 -2013 

  Author : KABE Yasuaki, ISHIMORI Koichiro

   

  During erythropoiesis, haem synthesis is significantly induced, and abundant haem is utilized as hemoglobin in erythroid cells. However, excessive haem results in cell damages by membrane oxidation. In the present study, we found that haem directly bound to heat shock protein 27 (HSP27) by affinity purification. HSP27 is a small heat shock family protein, which plays an important role for cytoprotection, stress tolerance, cellular differentiation or tissue development. It has been known that the multimerized HSP27 dissociates by several stimuli. Interestingly, exposure of haem directly dissociated the multimerized HSP27 in vivo and in vitro. Knockdown of HSP27 expression resulted in the haem-induced apoptosis and reduced the haemin-induced erythroid differentiation. Furthermore, we also found that HSP27 is essential for erythroid differentiation by using HSP27 knockdown mice.
• Analysis of Interactions in High Molecular Weight Protein Complexes by Using Segment Label and Cutting-edge NMR Technologies

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2011 -2012 

  Author : ISHIMORI Koichiro, UCHIDA Takeshi

   

  To develop new methodologies for structural analysis of biologically important high-molecular-weight protein complexes, applications of the segment label and new NMR techniques such as the transfer cross-saturation (TCS) and residual dipole coupling (RDC) were examined. By using TCS for the final electron transfer complex in the respiratory chain, the complex between cytochrome c and cytochrome c oxidase, we successfully determined the amino acid residues of cytochrome c directly interacting with cytochrome c oxidase.
• Structural Characterization of Heme-mediated Signaling Mechanism in Protein Regulation System

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2009 -2011 

  Author : ISHIMORI Koichiro, UCHIDA Takeshi

   

  In this research project, we characterized the heme binding in Iron Response Regulator (Irr) and Iron Regulatory Protein (IRP) and examined their functional significance. We spectroscopically identified the heme binding sites of Irr and, based on the structural information, a molecular mechanism of heme-induced oxidative modification in Irr was proposed. The heme binding sites of IRP were also determined and a new mechanism for the translational regulation by binding of heme was suggested in IRP. These new findings in this research project shed new light on the functional significance of heme in vivo.
• 高圧下蛍光分光システムによる蛋白質の構造的揺らぎの定量的解析

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2009 -2010 

  Author : 石森 浩一郎

   

  本年度の主な研究成果は以下のとおりである. FRETを用いた蛋白質における局所的線圧縮率の算出とその意義の検討 マッコウクジラミオグロビン(Mb)において,そのTrp14をPheに置換し,Eヘリックス末端近くのLys63をCysに置換することで,蛍光団であるAEDANSを63位に結合させたAEDANS修飾変異ミオグロビン,W14F/L63C-AEDNAS Mbを作成することに成功した.このAEDANS修飾変異ミオグロビンにおけるTrp14と,Cys63に結合したAEDANSとの間のFRET効率の圧力依存性について,昨年度に作成した高圧下蛍光分光システムを用いて検討した.この変異Mbは,295nmの励起光で発光する340nm付近のTrp残基に由来する蛍光強度が減少し,逆にAEDANS由来の460nm付近の蛍光を発するようになったことから,Trp14と63位に結合したAEDANSとの間にFRETが起こっていることが示された.これらの蛍光団間のFRET効率は,加圧に従い0.769(50気圧)から0.802(1500気圧)に上昇し,このことはTrp14とCys63位のAEDANS間の距離が加圧により短縮したことを示している.さらにこのFRET効率の定量的解析から,これら2点間の線圧縮率を求めると,3.1×10^<-10>m^2N^<-1>と見積られた.この値は,Mbの等温圧縮率から求めた3×10^<-11>m^2N^<-1>よりも大きく,また,ヘムと蛋白質表面に結合した金属錯体間の電子移動反応の圧力依存性から求めた3×10^<-11>～2×10^<-10>m^2N^<-1>に比べても大きいことから,Trp14とLys63の間の構造揺らぎは大きいと考えられた.このLys63が位置しているEヘリックスはミオグロビンにおける酸素結合部位を形成しており,ヘムに結合した酸素分子と水素結合しているHis64もその隣のアミノ酸残基として位置していることを考慮すると,本研究から得られた結果は,酸素分子が効率よくヘム鉄に結合するために,Eヘリックスの構造的な揺らぎは大きいことを意味している.
• 電子伝達蛋白質複合体の構造化学的解明とその会合・解離の分子機構

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2008 -2009 

  Author : 石森 浩一郎, 内田 毅

   

  本年度に得られた研究成果の概要は以下のとおりである. 1.シトクロム酸化酵素(CcO)の結合により誘起されるシトクロムc(Cytc)における構造変化の解明Cyt cまその電子受容体であるCcOと結合し,CcO-Cytc電子伝達複合体を形成する際には,その立体構造が変化することで電子伝達反応を制御していると考えられる.一般にはこのような高分子量の膜蛋白質であるCcOを含む蛋白質複合体の構造解析は困難であるが,^<15>NラベルしたCyt cを用いて3D-^1H-^<15>N NOESYHSQC(^<15>N-edited NOESY)を用いることで,CcO結合によるCytcの構造変化を検出することに成功した.特に,本研究者等のこれまでの研究から推定されたCyt cのCcOに対する相互作用部位周辺がCcOの結合に伴い有意な構造変化が観測され,Cyt cはCcOと電子伝達複合体を形成する際にはその相互作用部位付近に特異的な構造変化を起こすことが示唆された.今後さらに定量的な構造解析を進めることにより,構造変化による電子伝達の制御機構が明らかになると期待できる. 2.ドッキングシミュレーションによるCcO側の会合部位の同定 東京大学の北尾准教授の研究グループと共同で,CcO-Cytc電子伝達複合体形成の際のCcO側の相互作用部位について検討を行った.剛体モデルを用いたドッキングシミュレーションの結果,Cyt c側の相互作用部位は本研究者らがNMRを用いて実験的に明らかにした部位と一致し,一方,CcO側も予想通り,負電荷と疎水性のアミノ酸残基による相互作用部位の形成が認められた.さらにここで得られた結果についてMDを適用することにより,さらに詳細にCcO)側の相互作用部位を明らかにすることで,CcO側からもCcO-Cytc間の電子伝達機構を解明できると期待できる.
• 高圧分光法を用いた蛋白質における構造的揺らぎの解析

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2007 -2007 

  Author : 石森 浩一郎, 内田 毅, 竹内 浩

   

  蛋白質構造の構造的揺らぎを定量的に解明するため,人工的な分子内電子伝達蛋白質を設計し,その電子伝達速度の圧力依存性から算出した蛋白質構造における特定の2点問の線圧縮率と,多核多次元NMR法によるdistance geometryや緩和測定から得られる構造的揺らぎの結果を比較した.人工的な分子内電子伝達蛋白質であるルテニウム置換亜鉛ミオグロビン(48,81,83位にそれぞれRu錯体を修飾)の光励起によるZnからRu,あるいはその逆の電子移動過程の反応速度を,常圧から2000気圧程度までの圧力で追跡し,その圧力依存性から,その亜鉛ポルフィリンの亜鉛イオンと蛋白質表面に特異的に修飾したRu錯体間の距離は,Ru錯体の修飾位置(48,81,83位)によって,加圧により0.1から2Å程度,距離が短縮される場合(48位と伸張される場合(81,83位)が観測された.このような異方的な蛋白質構造の短縮・伸張は,緩和測定の結果から得られた局所的な運動性や,distance geometryとは相関がみられず,従来想定されていたように,アミノ酸残基の局部的な運動性や主鎖構造のずれが大きい部位で,必ずしも蛋白質構造の大きな揺らぎが起こっているのではないということを示すことができた.さらに,酸素結合蛋白質であるミオグロビンに比べ,外部からの配位子の結合がなく,そのヘム鉄が6配位構造であるシトクロムcについてもNMRによる緩和時間測定を行ない,主鎖構造の運動性について検討した.その結果,主鎖末端領域やループ領域にやや運動性の高い領域が観測されたものの,全体的にミオグロビンに比べ運動性が制限されている領域が多く,シトクロムcは,ミオグロビンに比べ,蛋白質構造上の揺らぎが小さいことを示唆している.
• ヘム依存性転写制御複合体の構造と機能

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2006 -2007 

  Author : 石森 浩一郎, 内田 毅

   

  ヘム依存性転写因子Irrにおける酸化修飾機構の詳細を検討するため,典型的なペプチド鎖の酸化修飾様式であるカルボニル化を認識する「Oxyblot」法を用いて検討したところ,過酸化水素のスカベンジャー試薬であるカタラーゼを添加した際に酸化修飾が大きく阻害されることを見出した.一方,OHラジカルやO_2-のスカベンジャー試薬の添加ではその阻害効果が見られなかったことから,Irrはヘムと分子状酸素の存在下で過酸化水素を産生し,この過酸化水素によってペプチド鎖の酸化修飾反応が引き起こされることが示された.さらに,以上のような過酸化水素の産生部位を同定するために,ヘムの軸配位子と想定されるCysやHisをそれぞれAlaに置換し,その酸化修飾反応を追跡したところ,His残基が連続しているHis117,His118,His119の置換により酸化修飾反応が大きく阻害されることが示された.しかし,この変異体において産生する過酸化水素の定量を行ったところ,野生型同様の産生能を示し,過酸化水素は酸化修飾には必須であるものの,ペプチド鎖への酸化修飾反応の直接的な活性種ではないことが示唆された.づまり,Irrによって産生された過酸化水素は,再びHis117,His118,His119付近の酸化活性化部位によって,さらに活性な酸化活性種に変換されることを示唆している.以上の結果からIrrにおける酸化修飾反応は,分子状酸素から過酸化水素を経た二段階の活性化反応で進行し,そこで生成した酸化活性種が蛋白質分解の端緒となることが考えられる.
• 圧力を用いた分子体積プロファイル解析による蛋白質立体構造形成過程での水分子の寄与

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2006 -2007 

  Author : 石森 浩一郎, 内田 毅

   

  本年度の研究実績の概要は以下のとおりである。 1.疎水性アミノ酸残基からの脱水和の分子体積に対する寄与: 疎水性部位からの脱水和による部分分子体積減少の実験的確証を得るため、Cyt cの蛋白質表面に位置する親水性のAsp93を疎水性のLeuに置換し、この変異による立体構造形成に伴う体積変化を追跡した。その結果、この変異により、その立体構造形成による体積の減少量は、約5mLmol^<-1>程度増加した。 2.高圧下時分割蛍光観測システムの構築疎水性アミノ酸残基からの脱水和の分子体積に対する寄与: 蛋白質の立体構造形成機構を考える上で重要な遷移状態における水分子の挙動を解明するためには、活性化体積ΔV^≠を見積もる必要がある。従来、Cyt cでは、このΔV^≠を見積もるため、種々の圧力下におけるヘムの紫外可視吸収を利用してきたが、この手法では蛋白質部分の構造変化が直接には反映されない。そこで、立体構造形成により、ヘムに近接することでその蛍光強度が減少するTrpの蛍光に注目し、蛋白質部分の変化を直接観察することを試みた。高圧下での立体構造形成反応の追跡については、還元型CytcのCO結合体が非結合体に比べその安定性が低く、レーザー光照射によるヘム鉄からCO分子の解離より、立体構造形成反応が開始可能であることを利用した。その結果、高濃度の塩酸グアニジン存在下のCO結合還元型Cyt cや、COが結合しない酸化型Cyt cでは光照射により、有意な蛍光変化は観測されないが、3.6M塩酸グアニジン存在下のCO結合還元型型Cyt cでは光照射に伴い、蛍光強度の低下が観測された。これはCOの解離によって立体構造形成反応が進行したと考えられ、本装置を用いて種々の圧力下における蛋白質立体構造形成反応をその蛍光変化により、追跡できることが示された。現状では観測される蛍光の強度が弱いが、集光レンズの装着などにより、その強度を上げることで、再現性の良い定量的な測定が期待できる。
• 圧力を用いた蛋白質立体構造形成の遷移過程における水分子の寄与の解明

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2004 -2005 

  Author : 石森 浩一郎, 木村 哲哉

   

  本研究では、水和水の挙動を反映する蛋白質の部分体積に注目し、チトクロムc(Cyt c)の立体構造形成過程の圧力依存性を測定することで、蛋白質立体構造形成における水の挙動について、以下の成果を得た。 1.塩酸グアニンジン存在下、天然状態と変性状態の平衡状態にあるCyt cについて、その平衡定数の圧力依存性から、部分モル体積の差(ΔV_)をより正確に求めたところ、天然状態の方が約25ml/mol小さいことが示された。Cyt cの立体構造形成におけるこのような負の体積変化は、疎水性部位からの水和水の解離(脱水和)による体積減少が顕著であることを示しており、その原因として疎水性の高い分子団であるヘム周りからの脱水和の寄与が示唆された。このような脱水和は、大きな正のエントロピー変化を伴い、変性状態から天然状態への立体構造形成に伴うペプチド鎖が失う莫大な構造エントロピーを相殺することで、蛋白質の立体構造形成をエネルギー的に有利にしていると考えられた。 2.初期収縮状態(collapsed state)から天然状態への構造形成のための活性化体積、つまりcollapsed stateから遷移状態までの部分体積の差(ΔV^*_)(-14ml/mol)と1.の結果を比較すると、その体積差は同程度であった。このことは、Cyt cにおいては、その変性状態から天然状態への立体構造形成の全過程で排出される水和水の個数と同程度の個数の水和水が、この遷移状態形成に脱水和することを意味している。つまり、Cyt cにおけるcollapsed stateから天然状態に至る遷移状態の段階で、既に天然状態に近い構造が形成されている可能性を示している。
• Structure and Function of Heme-regulated Proteins and Their Molecular Mechanisms

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2003 -2005 

  Author : ISHIMORI Koichiro, TAKAHASHI Satoshi, WAKASUGI Keisuke

   

  The major results we have obtained in this research project are as follows: 1. Ligation of Cys to Ferric Heme in Irr and IRP2. Based on the resonance Raman spectra, we successfully identified the Fe-Cys stretching modes in ferric heme-bound Irr and IRP2. These Fe-Cys stretching modes were downshifted, compared with that in conventional Cys-ligated hemoproteins such as P450cam. The downshifted Fe-Cys stretching modes correspond to the lower affinities of these proteins to ferric heme, which is also supported by fluorescence heme titration in Irr. Such weak affinities of these proteins to heme would be one of the characteristics of heme-regulated proteins having "Heme Regulatory Motif' 2. Redox-dependent Replacement of Axial Ligands. We confirmed the ligation of Cys to ferric heme in Irr and IRP2. By reduction of the heme iron, however, the absorption spectra of heme-bound Irr and IRP2 were drastically changed and the resultant spectra were quite different from ferrous P450cam, which were rather similar to bis-His ligated hemoproteins like cytochrome b_5. The spectral similarity to His-ligated hemoprotein was more evident in the CO adducts of heme-bound Irr and IRP2. The resonance Raman measurements. clearly showed the Fe-His stretching modes in ferrous heme bound lrr and IRP2 and the Fe-C and FeC-O stretching modes in the CO adducts, confirming that axial Cys is replaced with His by reduction of the heme iron. Considering that molecular oxygen can bind to ferrous heme, not ferric heme, the His ligated species in these proteins would be active species to generate reactive oxygen species, which leads to the oxidative modification of the peptide and protein degradation.
• 急速凍結分光法を使った金属酵素およびセンサー蛋白質の反応機構の研究

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2003 -2004 

  Author : 石森 浩一郎, 高橋 聡, 堀 洋, 若杉 桂輔

   

  本研究課題で得られた成果は以下のとおりである. 1.P450酵素における活性阻害剤の結合様式の検討 ヘム酵素であるP450はバクテリアから高等動物まで広く分布し,種々の薬物の代謝において重要な役割を担っている.したがって,感染症の原因となる真菌類のP450の機能を選択的に阻害できれば抗菌剤として有望な薬剤の開発が期待できる.そこで本研究課題では結核菌とヒトのP450(CYP51)に対する活性阻害剤であるいくつかのアゾール化合物について,その活性阻害様式を共鳴ラマンスペクトルやEPRを用いることで,構造化学的に検討した.その結果,アゾール環の置換基の立体障害とその疎水性度を制御することによって結核菌のP450のみ選択的に結合するアゾール化合物の分子設計が可能であることを示した. 2.ヘム酸化酵素中間体におけるラジカル位置の制御 ヘムを含む酸化酵素であるペルオキシターゼ類は多くの動植物に存在し,種々の酸化反応を触媒している.これらの酵素はその反応中間体としてラジカル種を形成するが,そのラジカルの位置については基質の大きさに依存して異なることが知られている.つまり,小さな基質の場合は蛋白質に埋め込まれたポルフィリン環上に,大きな基質の場合は蛋白質表面に露出したアミノ酸上に形成される.われわれは既に小さな基質に対する酸化酵素である西洋わさびペルオキシターゼ(HRP)について,蛋白質表面に芳香族アミノ酸を導入することで、ラジカル位置をポルフィリン環上から導入した芳香族アミノ酸に移動できることを報告してきた.今回,その移動したラジカル種による活性を検証するために,野生型のHRPでは反応性が低い立体障害の大きな基質を用いて検討したところ,蛋白質表面にラジカル種を有する変異体ではその活性が数十倍に増大し,ラジカル位置の制御によってヘム酵素の基質特異性が制御できることが示された.
• 金属が関与するセンサーとスイッチのケミカルバイオロジー

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2000 -2004 

  Author : 西野 武士, 石浜 明, 斎藤 正男, 石森 浩一郎, 岩崎 俊雄, 岡本 研, 新井 賢一, 三浦 謹一郎, 京極 好正

   

  研究組織は総括班(研究代表者、その他の計画研究代表者2名、評価委員3名および事務担当その他の分担者)および計画研究代表者全体会議である。 1)平成16年6月19日(午後1時より)に日本医科大学において、計画研究代表者全員および分担研究者,評価委員2名の出席で全体会議を持つた。会議では、それぞれの計画研究代表者の今までの成果と今後の計画を発表し、班員の連係および最終年度のまとめおよび成果公開の具体的計画を立て確認した。 とくに成果公開を重点におくことそのための3回の公開および関連学会でのシンポジウム開催が確認された。 2)平成16年度公開シンポジウムを横浜で開催した。生化学会と連動させ実質二日間の国際シンポジウムとなった。公開シンポジウムは生化学会直前にもかかわらず海外からの参加者5名に加え、総数70名を超えた。 3)また生化学会は初日午前であったが会場はほぼ満席に近く、成果については科学新聞でも大きく取り上げられた。 4)生物物理学会において代表である西野および斉藤が中心となり金属蛋白質に関するシンポジウムを計画した。 5)平成17年1月7日(午後1時より7時まで)および8日(午前9時より12時まで)に日本医科大学において、計画研究代表者全員および分担研究者、評価委員1名の出席で全体会議を持った。総括班会議では、それぞれの計画研究の進展状況および各種シンポジウムの報告がなされ、研究の進展状況と問題点が検討された。全体会議では代表者の1年間の成果を発表し、詳しく討論された。さらに最終年度でもあり5年間の研究成果の集約にむけ研究のまとめをと今後の計画の討論を行なった。
• 構造及び機能単位としてのモジュールを組み合わせた新規蛋白質の分子設計と創製

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2000 -2003 

  Author : 森島 績, 若杉 桂輔, 高橋 聡, 石森 浩一郎

   

  1.モジュール間相互作用の再生を目指した方法論の開発 ヘモグロビンα鎖のヘム結合モジュールをシトクロムb_5の対応する領域に導入したb_5αb_5を作製し、ランダム変異導入後、ヘム親和性を指標に構造力安定化している蛋白質のスクリーニングを行ったところ、ヘム結合モジュールに近接する特定の部位に変異が集中していた。そこで、構造上の歪みがかかっていると考えられるこれら残基に範囲をしぼってランダム変異を導入した変異体集団を作製し、さらに、スクリーニング過程に、崩れた構造の変異蛋白質を蛋白質分解酵素により除去するプロセスを新たに加え方法論の改良を行ったところ、モジュール置換蛋白質の安定性の向上に成功した。 2.新規機能性蛋白質の創製に成功 ヒトのトリプトファニルtRNA合成酵素(TrpRS)と相互作用する蛋白質を探索したところ、解糖系の酵素であるグリセルアルデヒド3-リン酸デヒドロゲナーゼ(GapDH)がTrpRSと結合することが明らかになった。TrpRSと結合するGapDHの結合部位の特定を試みるために、様々なキメラ蛋白質を作製した。ミオグロビン(Mb)のN末端側にGapDHのあるモジュールを融合したキメラ蛋白質は、野生型Mb同様、酸素を可逆的に配位でき、またGAPDH同様にTrpRSと会合する安定な新規蛋白質であることが明らかになった。 3.酸化ストレス応答性新規グロビン蛋白質の分子機構の解明とその知見に基づく人工蛋白質の設計 「ニューログロビン(Ngb)」には酸化ストレスに伴う神経細胞死を抑制する働きがあると指摘されている。今回、このNgbが脳神経系においてシグナル伝達系を制御する分子として機能しているという仮説を立て、Ngbが関与する脳神経シグナル伝達系を明らかにすることを目指した。その結果、酸化ストレス下で生成する鉄3価Ngbが細胞内シグナル伝達蛋白質であるGαと特異的に結合すること、他方、通常の酸素正常状態の鉄2価NgbはGαとは相互作用しないことを発見した。また、Ngbは酸化ストレス応答性のセンサー蛋白質として働き、酸化ストレスを受けた時のみGαと結合し、GαのGDP/GTP交換反応抑制蛋白質として機能することにより、神経細胞死を抑制することを明らかにした。さらに、Ngbに関しモジュール置換した種々のキメラ蛋白質を作製し、それらの解析を行うことにより、制御メカニズムを分子レベルで明らかにすることに成功した。
• セグメント特異的同位体ラベルP450を用いた電子伝達相互作用部位の解明

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2002 -2002 

  Author : 石森 浩一郎

   

  チトクロムP450は炭化水素の特定部位を水酸化する酵素で,その温和な反応条件と反応位置の特異性から酵素化学的だけではなく,実用的,工業的にもその応用が注目されている.この酵素の水酸化反応においての解明するべき点の一つは,分子状酸素の活性化に必要な電子の供給機構であり,この電子伝達機構が解明され,その人工的制御が可能になればこの酵素の応用範囲が大きく広がることが期待される.本研究課題ではこのチトクロムP450における電子伝達機構の解明とその制御を目指して,NMRとペプチド鎖の同位体置換を組み合わせることで,代表的なチトクロムP450であるd-カンファーを基質とする緑膿菌のチトクロムP450(P450cam)と,その電子供与体であるプチダレドキシン(Pd)の電子伝達相互部位の決定,およびその電子伝達過程の制御機構について検討を行った.本研究で得られた主な結果は以下のとおりである. 1.P450camのNMRスペクトルを詳細に検討し,基質であるd-カンファーやヘム近傍に位置するスレオニン252に由来するNMRシグナルの帰属に成功した. 2.今回帰属したNMRシグナルを用いて電子供与体であるPdの結合によるP450camのヘム近傍の構造変化を検討し,Pd結合によりヘム面が傾き,ヘム鉄とd-カンファーとの距離が短くなることを見出した.このことは,ヘム鉄上で生成する活性酸素を効率的にd-カンファーに転移させる上で有利であると考えられた. 3.電子伝達相互作用部位と想定される領域を含むP450camのアミノ酸配列が無細胞系でも合成できることを示し,セグメント特異的同位体ラベルがP450camでも可能であると考えられた.しかし,実際のセグメント特異的同位体ラベルP450camの作製のためには,この配列の収率は低く,更なる反応条件の検討が必要であった.
• Development of time-resolved spectroscopic observation systems for the fast dynamics of protein folding

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2000 -2001 

  Author : TAKAHASHI Satoshi, ISHIMORI Koichiro, HARADA Yoshiyuki

   

  We developed the following original instruments to observe the early dynamics of protein folding. 1) The rapid mixing device that can initiate protein folding within 50 μs. The previous mixing devices has the mixing time of 300〜400 μs. Thus, we achieved to reduce the mixing times to nearly an order shorter. 2) We constructed to the observation system for the CD spectroscopy based on the prism polychrometer and a CCD detector. We resolved several problems associated with the new ideas of spectroscopic observation system, such as the effect of optical retardation. The developed system enabled us to observe the circular dichroism spectra with a high S/N ratio based on the multichannel detection. 3) We tried to combine the developped flow cell system and the spectroscopic observation system ; however, faild to observe reliable data for the repid-mixing cell. We are currently working to reduce the optical retardations of the cells. Using the developed flow cell systems, we obtained the following results that were already reported in scientific journals. 1) We observed time-resolved small-angle X-ray scattering profiles for the protein folding dynamics of cytochrome c. The developed cell enabled us to obtain information on the early submillisecond phase of cytohrome c folding that were inaccessible by the conventional devices. We conclude that the protein compaction and the secondary structure formations proceed simultaneously in the folding process of cytochrome c. 2) We observed the helix formation processes of polyglutamic acid using the developed rapid-mixing cell. We demonstrated that a short helix formation precedes the longer helix formation process in the PGA helix formation. This is a new dynamical event observed in the helix formation of polypeptides. 3) The helix-formation and the collapse process of apomyoglobin were observed using the developed rapid mixing systems in the submillisecond time domain. We clarified that the apomyoglobin folding proceed as a stepeise process that is similar to the observation for cytochrome c.
• 金属酵素の不安定中間体を観測するための高速溶液混合実験システムの開発

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 1999 -2000 

  Author : 森島 績, 高橋 聡, 石森 浩一郎

   

  平成12年度はペルオキシターゼの活性中間体であるCompound Iの生成機構を検討するため,独自に開発した高速混合装置を用いて,西洋ワサビペルオキシターゼ(HRP)と過酸化水素との反応を追跡した。装置としては,本研究者らが開発した高速混合装置を改良することにより,時間分解能50マイクロ秒で可視紫外吸収スペクトルことに成功した。この装置を用いて,上記反応を追跡したところ,HRPの休止状態からCompound Iへのスペクトル変化が,ほぼ等吸収点をとりながら観測できた。このことは休止状態からCompound Iの変化において中間体が存在しないことを意味しているが,過酸化水素の濃度に対するCompound I生成速度の依存性は,速度論的な中間体が存在する明らかに飽和現象を示し,Michaelis-Menten型の解析をすることで,その寿命(半減期)が約10μ秒であることが明らかとなった。さらにこの値をもとに,このような寿命を持つ中間体を仮定したモデルを考え,反応後50マイクロ秒後の中間体の存在割合を求めたところ,約15%と見積もられ,その推定される紫外可視スペクトルは休止状態に類似した鉄3価高スピン型であった。このことは,Compound I生成の中間体として,従来想定されていた3価のヘム鉄にアニオン化した過酸化水素(HOO^-)が結合した鉄3価低スピン型ではなく,中性の過酸化水素(HOOH)が結合した状態であると推定できた。このような中間体を形成する反応機構として,中性の過酸化水素がヘム鉄に結合できるように,ヘム近傍の遠位ヒスチジンと遠位アルギニンが同時に過酸化水素と相互作用しているモデルを新たに提出した。
• 蛋白質構造の「揺らぎ」による電子移動過程の制御

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 1999 -2000 

  Author : 森島 績, 若杉 桂輔, 高橋 聡, 石森 浩一郎

   

  平成12年度は電子伝達蛋白質のモデル系として,亜鉛置換ミオグロビンとチトクロムb_5の系を構築し,レーザー光の照射による光誘起電子移動反応を検討することで,この電子移動反応における電子移動複合体に構造について考察を行った。従来,この系は電子移動反応を元にした会合定数が,等温適定実験からの会合定数よりはるかに小さいことが予想されており,電子移動を行える会合体は全会合体の一部であると考えられてきた。しかし,この系では,その会合が弱いことから電子移動反応を元にした正確な会合定数は,報告されておらず,実際に一部の会合体しか電子移動を行えないのかどうかは明確ではない。そこで,本研究ではこの系の会合における相互作用が静電的相互作用であることに注目し,イオン強度を下げることにより,Michaelis-Menten型の解析から,その会合定数を8.0x10^4M^<-1>と決定できた。一方,すべての会合体に対する結合定数を求めるために,亜鉛ミオグロビンの蛍光がチトクロムb_5により消光されることに注目した。その結果,会合定数は4.0x10^4M^<-1>と求めることができ,この値は,電子移動反応から求められた値とほぼ同程度であった。以上の結果は,亜鉛置換ミオグロビンとチトクロムb_5における電子移動はほとんどすべての会合体で起こっていることになり,従来の予想を否定する結果となった。さらに,電子移動反応のイオン強度依存性の結果から,この会合体は両蛋白質のヘム面が向かい合った状態であることが示唆され,これはマーカスの式から予想される電子移動反応の距離ともほぼ一致した。
• Protein structure, comparison, prediction, and design

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1995 -1999 

  Author : GO Nobuhiro, YURA Kei, NISHIKAWA Ken, WAKO Hiroshi, YOMO Tetsuya, ISHIMORI Koichiro

   

  Natural history group (Go, Yura, Wako, Nishikawa, Mitaku, and Umeyama) From a classification of the spatial arrangements of the secondary structure elements, Go discovered 'the symmetry rule', whish states that apair of similar protein folds, having no common ancestor, tend to possessan internal symmetry in their fold. Yura found two types of 'modules', a phosphate binding module observed commonly in polymerases and transcription factors, and a substrate-specificity determining module of peroxidases. A newly developed method for detecting similarity in the atomic level revealed a lot of similar ATP binding structures in totally different folds (Go). Wako developed an efficient algorithm of expressing the local environment by a code representation. A new secondary structure prediction method was developed by Nishikawa, who applied the threading method for this purpose. As for the structural prediction for membrane bound proteins, Mitaku improved his method to predict the structure of bacteriorhodopsin correctly. Umeyama established a fully automated algorithm of homology modeling, which is estimated to give a sufficient accuracy. Design group (Yomo and Ishimori) Yomo found in random mutagenesis experiments on catalase that the thermal stability and activity of the protein is extremely robust against mutations, and that a possibility of the functional optimization by the evolutional engineering technique can enormously enhanced by the elongation of the chain length. The concept of 'module' was applied to produce chimera hemoglobin, which was synthesized by exchanging its modules each other. From such chimera proteins, Ishimori confirmed that the structural unit, module, works not only as the unit of maintaining the protein stability, but also as a unit for realizing the function of hemoglobin.
• Protein Engineering and Reactivity Control in Multi-functional Chimeric Metalloproteins

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1996 -1997 

  Author : ISHIMORI Koichiro

   

  This research project includes the following two subjects. (1)Structural Characterization of Natural Chimeric Protein, CooA Protein, and Its Molecular Mechanism To investigate structural characterization of CooA protein, which has a myoglobin-like heme binding domain and helix-turn-helix type DNA binding domain, NMR, resonance Raman spectra and ligand binding were utilized. The resonance Raman spectrum for the ferrous CO adduct of CooA protein confirmed the ligation of a histidine residue to the heme iron, while, in the absence of CO, the ligation of an arginine or lysine residue instead of CO were suggested by the NMR spectrum, which is quite unusual in hemoproteins. Although the association rate for the CO rebinding in CooA protein was comparable to that in myoglobin or hemoglobin, it was characterized by its large fraction of the very fast (
• Developement and Application of High Pressure Multi-Dimensional NMR Spectroscopy

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1995 -1997 

  Author : MORISHIMA Isao, TAKAHASHI Satoshi, ISHIMORI Koichiro

   

  The primary results in this research project are as follows : (1)Development of High Pressure Multi-Dimensional NMR Spectroscopy. We have tried to measure 2-dimensional proton NMR spectrum of hemoprotein under high pressure (up to 2000 bar). Our preliminary measurement in normal pressure has revealed that the very high sample concentration (more than 10 mM) and 40-60 hours accumulation would be required to obtain the 2D NMR spectrum by using a glass capillary as sample tube. Although the glass capillary needs enough space below the detection coil in the NMR probe, the bottom part of the our probe was used for the temperature control unit, which severely limits the size of the glass capillary. We final concluded that our NMR probe must be modified to use the glass capillary. (2)Application of High Pressure Laser Flash Photolysis to Dynamic Properties of Hemoproteins. Since pressure has been considered to perturb thermal fluctuation in protein structure, we examined the effects of the thermal fluctuation on dynamic properties of hemoprotein. One of the dynamic properties we focused on in this research project was the ligand binding in hemoproteins. Systematic combination of mutant myoglobins and kinetic measurement under high pressure has clearly shown that the some of the hydrophobic amino acid residues play a key role in controlling the ligand binding by maintaining the high hydrophobic environments in the heme pocket. Another dynamic property is the electron transfer reaction in hemoproteins. Basied on the measurements of the reaction rates for electron transfer in hemoprotein (myoglobin) under high pressure, we can pointed out that the electron transfer pathway and free energy difference for the reaction would highly depend on the thermal fluctuation in protein structure, and the thermal fluctuation is one of the critical factors of the molecular mechanism for the electron transfer in proteins.
• Protein Engineering for New Functional Hemoproteins Based on Module Substitution

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1995 -1997 

  Author : MORISHIMA Isao, TAKAHASHI Satoshi, ISHIMORI Koichiro

   

  The primary results in this research project are as followa : (1)Module Substitution in Globins and Peroxidases. The structural and functional properties of the module-substituted globins and peroxidases have been characterized. Most of the module-substituted proteins exhibited highly destabilized protein structure, probably due to the missing or severe perturbation in the key interactions between the modules. The destabilized protein structure also interfered the formation of the specific dimers or tetramers in globins and enzymatic activity in peroxidases. It can be, therefore, concluded that the module-module interactions are also essential to design new stable and functional proteins as well as the module substitution. (2)Identification of New Module Structure in Globins. The amino acid substituteions based on the structural unit of wchic boundaries are located at the middle of the conventional modules have revealed that the module previously proposed can be divided into new and smaller structural units. In this research project, I focused upon one of the newly identified structural units, which includes the iron-liganded histidine and about 20 amino acid residues in the proximal site, "sub-module m6". The substitution of the sub-module m6 in globins drastically affect the electronic state of heme and configuration of the iron-liganded histidine, indicating that the sub-module m6 would be a "heme binding modules" respondible for the heme binding structure.
• Protein Engineering for New Functional Hemoproteins Based on Module Substitution

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1994 -1995 

  Author : ISHIMORI Koichiro

   

  In this research project, the structural and functional properties of the module-substituted globins have extensively been characterized in order to establish a new strategy for the protein design based on the molecular evolution in nature. The module substitutions between the alpha-subunit of human hemoglobin and myoglobin have revealed that the implantation of the module M4 in the hemoglobin alpha-subunit into myoglobin produced a fairly stable dimeric globin protein, MbMbalpha-globin. The formation of dimers in the monomeric myoglobin-based globin strongly suggest that the specific module substitution can be a potent strategy to add the new function to proteins. In the module-substituted globins between the hemoglobin a-subunit and myoglobin except for MbMbalpha-globin, however, their structure were highly destabilized and did anot show any specific subunit association. Some of the module-substituted globins were not expressed in Escherichia coli due to the unstable protein structure. Such destructive effects on the globin structure imply that the intramolecular interactions essential for the stable globin formation are severely perturbed by the module substitution and additional mutations would be required to produce stable functional proteins as well as the module substitution.
• モジュール置換による新規金属蛋白質の分子設計

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 1993 -1993 

  Author : 石森 浩一郎

   

  蛋白質の立体構造の詳細な検討から、多くの蛋白質は、モジュールと呼ばれる構造的にほぼ独立した部分の集合体であることが明らかになってきている。しかし、そのモジュール構造と蛋白質全体の構造と機能との相関についての実験的研究はほとんど報告されていない。本研究では金属蛋白質のうち、モジュール構造をとることが知られているミオロビン、ヘモグロビンなどのグロビン蛋白質に注目し、そのモジュール置換により、新規な構造や機能の分子設計、とくに多量形成能と酸素親和性の制御について検討をおこなった。 天然のミオロビンやヘモグロビンの単離鎖(alpha,beta)は単独では安定な多量体構造をとれないのに対し、2種のヘモグロビンの単離鎖を混合することによりalpha2beta2型の安定な四量体構造が形成される。立体構造とアミノ酸配列からモジュールF4がサブユニット会合に関与していることが示唆されており、実際、我々の従来の研究から天然のbeta鎖のモジュールF4をalpha鎖のものに置換したChimera-betaalphaグロビンは、天然のbeta鎖と特異的に結合し、Chimera-betaalpha2beta2型四量体が生成することを見いだしてきた。今回、これらモジュール置換蛋白質の機能を詳細に検討することにより、Chimera-betaalpha2beta2型四量体が生成する際には、天然での四量体形成時(alpha2beta2)と同様に、酸素親和性が低下することが確認でき、このことはモジュールF4は単に蛋白質を会合させるだけでなく、会合体の機能をも制御することが明かとなった。さらに、単量体でしか存在しないミオグロビンを多量化するため、ヘモグロビンのモジュールF4を含むモジュール置換ミオグロビン2種(Chimera-Mbalpha,Chimera-Mbbeta)を設計し、単離、生成することに成功した。現在、その会合特性と機能変化を検討している。
• Structural and Functional Analysis of Hemoproteins Under High Pressure by Las Photolysis Measurements

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1991 -1992 

  Author : MORISHIMA Isao, ISHIMORI Koichiro, WATANABE Yoshihito

   

  1. Several site-specific mutants of human myoglobin(Mb) have been prepared in order to examine the influence of highly conserved leucine residues located in the hydrophobic clusters in the heme distal and proximal site on the heme environmental structures and ligand binding properties. Structural and ligand binding characterization of these recombinant proteins were studied by NMR, IR and laser photolysis measurements under different pressures. The leucine 29 mutants exhibited unusual pressure dependence of CO binding rate constant and the resulting pressure dependence of the activation volume implies that pressurization affects the fractional distributions of multi-conformers of Mb so that the fast CO rebinding conformers is increased under pressure. 2. The effects of pressure on the recombination kinetics of CO binding to the isolated alpha and beta chains of human hemoglobin(Hb) were studied. Pressure dependent activation volume change from negative to positive values in the bimolecular CO association reaction was observed for both isolated chains. This finding was attributed to a change in the rate-determining step from the bond formation to the ligand migration process. 3. We have also studied pressure effects on the CO association reaction for cytochrome P-450. It was found that the activation volume is quite sensitive to the presence or absence of the substrate, the molecular structure of the substrate. 4. Finally, we have made a preliminary study on the pressure effect on the intraprotein electron transfer reaction rate.
• 色素置換ヘム蛋白質における光電子移動反応の動的制御

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 1990 -1990 

  Author : 森島 績, 石森 浩一郎

   

  本年度は亜鉛置換ミオグロビンにおける光誘起電子移動反応について検討を行なった。電子移動反応はアミノ酸に部位特異的に結合させたルテニウム錯体と亜鉛ポルフィリンの間で行なわせ、その電子移動過程の速度と活性化体積を求めた。ポルフィリン中心から約15オングストロ-ム離れたヒスチジン48にルテニウム錯体を結合させたときの電子移動速度は約5_x10^4s^<ー1>、活性化体積は約1cm^3mol^<ー1>であるのに対し、20オングストロ-ム離れたヒスチジン81にルテニウム錯体を結合させたときの電子移動速度は約50s^<ー1>と非常に遅くなり、一方、活性化体積は約11cm^3mol^<ー1>と非常に大きな値を示した。電子移動過程の速度は電子移動反応の起こりやすさ、つまり、電子移動に適した蛋白質構造への変化のしやすさを示しており、速度の遅い遠距離間の電子移動では電子移動に適した蛋白質構造になりにくいことを示唆している。活性化体積は一般には蛋白質の動的な構造変化を反映しており、本研究の結果は電子移動の距離や位置環境によって電子移動の際の蛋白質の動的構造変化が大きく異なることを示している。特に、電子移動時の活性化体積が正の符号を持つことは、電子移動に伴う蛋白質の構造変化が体積の増加する方向にあることを示しており、電子伝達のメカニズムを考えるうえで非常に興味深い。今回用いたミオグロビンはウマ由来のものであるが同時にアミノ酸置換を施したヒトのミオグロビンについても同様な実験を行なっており、さまざまな電子移動距離、位置環境にあるヒスチジン残基を導入し、その電子移動速度と活性化体積を評価することにより蛋白質内における電子移動反応のメカニズムに対して検討する予定である。
• Studies on the Mechanism of Physiological Function of Hemoglobin by Using Artificial Mutants

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1989 -1990 

  Author : IMAI Kiyohiro, ISHIMORI Koichiro, MIYAZAKI Gentaro, WATANABE Manabu

   

  Artificial hemoglobin mutants which contained single amino acid substitutions at particular sites were synthesized by site-directed mutagenesis using recombinant DNA and their oxygen binding function, light absorption spectra, proton nuclear magnetic resonance (NMR) spectra and resonance Raman scattering were measured to explore the roles of amino acid residues which are considered to be a key residue for the allosteric properties of hemoglobin. Four mutants were synthesized : Hb Y145betaF in which Phe substitutes for Tyr-145beta which is considered to induce a tertiary structure change in the beta subunit ; Hb W37betaF in which Phe substitutes for Trp-37beta which forms a hydrogen bond with Asp-94alpha at the alpha1-beta2 interface ; Hb H92betaV and Hb H92betaD in which the proximal His at 92beta is replaced by Val or Asp. The M13 Phase-E. coli system was used to introduce the mutations into human globin gene and to express the globin gene. The changes in oxygen binding functions (oxygen affinity, the Bohr effect, co-operativity, effect of inositol hexaphosphate) of Hb Y145betaF were moderate while those of Hb W37betaF were drastic. The tertiary and quaternary structure data acquired from UV light absorption, NMR, and resonance Raman spectra were nearly consistent with the functional data. It was noted that the important role of Tyr-145beta is that it has a side chain whose size is appropriate to fit to the tyrosine pocket rather than that it forms a hydrogen bond with Asp-94beta. The drastic functional changes in Hb W37betaF may partly be attributed to partial dissociation into alphabeta dimers. Hb H92betaV and Hb H92betaD showed drastic functional changes. The replacement of the proximal His by neutral or negatively charged residue caused disapearance of allosteric effects whereas the heme iron of the mutant chains were maintained in a ferrous state, different from the M-type hemoglobins.
• 高圧下におけるNMR、分子分光併用測定法の開発とその応用

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 1987 -1989 

  Author : 森島 績, 石森 浩一郎

   

  本年度は本研究の最終年度として、試作に成功した四ツ窓付きレ-ザ-フォトリシス高圧セルを用いて、ヘム蛋白質の酸素、一酸化炭素の再結合反応速度の圧力依存性に関する詳細な測定実験を行った。本年度は特に、単離したα鎖、β鎖(ヘモグロビン)とCOの結合反応の圧力依存性を詳細に検討した。α、β鎖のCO結合体にレ-ザ-光を照射すると、COが解離した後ヘム鉄に再結合する。このCO再結合反応はナノ秒およびミリ秒の二つの時間領域で観測され、それぞれCOが蛋白質内部再結合する反応、COが蛋白質外部(溶媒)から再結合する反応に対応している。先ず加圧に併ってCOは蛋白質内部で再結合する割合が大きくなった。各素過程の圧力依存性からこれら素反応過程についての活性化体積が見積られた。これより、α、β鎖ともにCOが蛋白質内部に入る際には一旦体積が増加した後、減少することが分かった。この反応に伴う体積変化は、昨年度行ったミオグロビンと酸素の結合反応にも観測されており、ヘモグロビン、ミオグロビンに共通した挙動と考えられる。また、α鎖とβ鎖を比較するとの鎖の方は1モル当り16cm^3だけ体積が増加した後、11cm^3減少する。それに対し、β鎖の方は12cm^3増加後、7cm^3減少とα鎖の方が変化量が大きいことが分かった。このようなα鎖とβ鎖の違いはヘム近傍構造の違いを反映していると考えられた。
• Structural and Functional Characterization of Metalloproteins and Its Molecula Design

  Date (from‐to) : 1983