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

Profile and Settings

• Name (Japanese)

  Uchida

• Name (Kana)

  Takeshi

• Name

  200901018713222865

Alternate Names

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

Achievement

Research Interests

• シトクロムc   転写制御   ラマン分光法   ヘム   酸化還元   病原菌   共鳴ラマン   水素結合   時計蛋白質   チオエーテル結合   チオエーテル結   ヘムタンパク質   翻訳後修飾   DNA結合   鉄   蛋白質   NPAS2   NPAS   酵素   生体分子   シグナル伝達   シトクロム酸化酵素   センサー蛋白質   酸化修飾   ヘム生合成   共鳴ラマン分光   ナノディスク   ROS   酸素センサー蛋白   回転半径   生物無機化学   

Research Areas

• Nanotechnology/Materials / Basic physical chemistry
• Nanotechnology/Materials / Biochemistry
• Life sciences / Biophysics
• Life sciences / Functional biochemistry
• Life sciences / Structural biochemistry

Research Experience

• 2013/06 Hokkaido University
• 2001/04 - 2001/10 岡崎国立共同研究機構分子科学研究所 学術振興会特別研究員
• 1999/10 - 2001/03 アルバートアインシュタイン医科大学 博士研究員
• 1998/04 - 1999/09 岡崎国立共同研究機構分子科学研究所 大学等非常勤研究員
• 1998 - 1999 Part-time researcher for university or other academic organization

Education

•        - 1998  Kyoto University
•        - 1998  Kyoto University  Graduate School, Division of Engineering
•        - 1995  Kyoto University
•        - 1995  Kyoto University  Graduate School, Division of Engineering
•        - 1993  Kyoto University  Faculty of Engineering
•        - 1993  Kyoto University  Faculty of Engineering

Awards

• 2009 日本化学会北海道支部奨励賞
  

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
• Converting cytochrome c into a DyP-like metalloenzyme

  Issei Omura, Koichiro Ishimori, Takeshi Uchida

  Dalton Transactions 51 (33) 12641 - 12649 1477-9226 2022/09 [Refereed]
   

  Dye-decolorizing peroxidase (DyP), which can degrade anthraquinone dyes using H₂O₂, is an attractive prospect for potential biotechnological applications for environmental purification.
• 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 61 (34) 13543 - 13553 0020-1669 2022/08/12 [Refereed][Not invited]
  
• Heme binding to cold shock protein D, CspD, from Vibrio cholerae

  Dayeon Nam, Wataru Motegi, Koichiro Ishimori, Takeshi Uchida

  Biochemical and Biophysical Research Communications 624 151 - 156 0006-291X 2022/07 [Refereed]
  
• 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]
  
• 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 111422 - 111422 0162-0134 2021/06 [Refereed][Not invited]
   

  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.
• 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

  Journal of Biological Chemistry 295 (32) 11316 - 11325 0021-9258 2020/08/07 [Refereed][Not invited]
   

  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.
• 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 2020/04/30 [Refereed][Not invited]
   

  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.
• 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.
• A single mutation converts Alr5027 from cyanobacteria Nostoc sp. PCC 7120 to a heme-binding protein with heme-degrading ability

  Dojun, N, Muranishi, K, Ishimori, K, Uchida, T

  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.
• 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 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 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.
• Specific heme binding to heme regulatory motifs in iron regulatory proteins and its functional significance

  Yudai Nishitani, Hirotaka Okutani, Yukiko Takeda, Takeshi Uchida, Kazuhiro Iwai, Koichiro Ishimori

  Journal of Inorganic Biochemistry 198 110726 - 110726 0162-0134 2019/09 [Refereed][Not invited]
  
• Heme uptake and biosynthesis in Vibrio cholerae

  UCHIDA Takeshi

  Journal of Japanese Biochemical Society 91 (3) 404 - 408 2019/06 [Refereed][Not invited]
  
• 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.
• 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.
• 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 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) 829  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 11030  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.
• 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.
• 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 [Not 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.
• 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 1873-3344 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.
• 3P101 Functional significance of homotrimer formation in the Nanodisc-embedded Halorhodopsin(03. Membrane proteins,Poster,The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))

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

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 54 (1) S265  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
• 2SBP-03 Heme-iron uptake proteins from Vibrio cholerae(New Development of Structural Cell Biology in Signal Transduction,Symposium,The 52th Annual Meeting of the Biophysical Society of Japan(BSJ2014))

  Uchida Takeshi

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 54 (1) S131  2014
• 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
• 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][Not 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][Not 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 characterizations of a novel globin-coupled histidine kinase from Anaeromixobacter sp. Fw109-5

  KITANISHI Kenichi, KOBAYASHI Kazuo, UCHIDA Takeshi, ISHIMORI Koichiro, IGARASHI Jotaro, SHIMIZU Toru

  Journal of Biological Chemistry 286 (41) 35522 - 35534 0021-9258 2011/10 [Refereed][Not invited]
  
• 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.
• 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.
• 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.
• 2SC0933 Heme transfer mechanism of heme chaperone protein(2SC Structure and Function of Weak Interaction in Metalloproteins-Protein Field and Highly Organized Specific Reaction,The 48th Annual Meeting of the Biophysical Society of Japan)

  Uchida Takeshi

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 50 (2) S10  2010
• 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.
• 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
• 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.
• Reactive complexes in myoglobin and nitric oxide synthase

  Hans-Petter Hersleth, Armelle Varnier, Espen Harbitz, Asmund Kjendseth Rohr, Peter P. Schmidt, Morten Sorlie, F. Henning Cederkvist, Stephane Marchal, Antonius C. F. Gorren, Bernd Mayer, Takeshi Uchida, Volker Schuenemann, Teizo Kitagawa, Alfred X. Trautwein, Toru Shimizu, Reinhard Lange, Carl Henrik Gorbitz, K. Kristoffer Andersson

  INORGANICA CHIMICA ACTA 361 (4) 831 - 843 0020-1693 2008/03 [Refereed][Not invited]
   

  In this overview some of our crystallographic and spectroscopic studies on reactive complexes in myoglobin and nitric oxide synthase are summarised. Myoglobin and nitric oxide synthase are both haemoproteins with some similar reaction intermediates. For myoglobin we have studied different intermediates generated in the reaction with hydrogen peroxide by X-ray diffraction, single-crystal microspectrophotometry, electron paramagnetic resonance spectroscopy, Mossbauer spectroscopy, resonance Raman spectroscopy and quantum refinement. Several of these myoglobin states are quite susceptible to radiation-induced changes during crystallographic data collection, and we have observed a radiation-induced change of the ferric resting myoglobin to aqua ferrous myoglobin, of myoglobin compound II to a proposed intermediate H, and of myoglobin compound III to peroxy myoglobin. For the myoglobin compound II/ intermediate H we observe a single-bonded Fe-IV - O species, which is probably protonated. The long Fe - O bond seen in the crystal structure can be supported by the observation of a new O-18- sensitive resonance Raman mode at 687 cm(-1). For nitric oxide synthase we detected with cryobiochemical methods in electron paramagnetic resonance spectra the first biopterin radical serving as electron donor to the ferrous-oxy complex, and that biopterin serves as a proton donor as well, in addition we could observe formation of the Fe( NO) complex with a amino-pterin cofactor capable to form a reactive radical. (c) 2007 Published by Elsevier B. V.
• 1S1-6 Heme Transfer Mechanism of Heme Chaperone Protein -CcmE- to Apocytochrome c Revealed by Resonance Raman Spectroscopy(1S1 Biophysics of Transition Metals: Challenges of Young Researchers,The 46th Annual Meeting of the Biophysical Society of Japan)

  Uchida Takeshi

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 48 S1  2008
• Construction of a subnanosecond time-resolved, high-resolution ultraviolet resonance Raman measurement system and its application to reveal the dynamic structures of proteins

  Minoru Kubo, Takeshi Uchida, Satoru Nakashima, Teizo Kitagawa

  APPLIED SPECTROSCOPY 62 (1) 30 - 37 0003-7028 2008/01 [Refereed][Not invited]
   

  A subnanosecond time-resolved ultraviolet (UV) resonance Raman system has been developed to study protein structural dynamics. The system is based on a 1 kHz Nd:YLF-pumped Ti:Sapphire regenerative amplifier with harmonic generation that can deliver visible (412, 440, 458, and 488 nm) and UV (206, 220, 229, and 244 nm) pulses. A subnanosecond (0.2 ns) tunable near-infrared pulse from a custom-made Ti:Sapphire oscillator is used to seed the regenerative amplifier. A narrow linewidth of the subnanosecond pulse offers the advantage of high resolution of UV resonance Raman spectra, which is critical to obtain site-specific information on protein structures. By combination with a I m single spectrograph equipped with a 3600 grooves/mm holographic grating and a custom-made prism prefilter, the present system achieves excellent spectral (<10 cm(-1)) and frequency (similar to 1 cm(-1)) resolutions with a relatively high temporal resolution (<0.5 ns). We also report the application of this system to two heme proteins, hemoglobin A and CooA, with the 4,40 nm pump and 220 nm probe wavelengths. For hemoglobin A, a structural change during the transition to the earliest intermediate upon CO photodissociation is successfully observed, specifically, nanosecond cleavage of the A-E interhelical hydrogen bonds within each subunit at Trp alpha 14 and Trp beta 15 residues. For CooA, on the other hand, rapid structural distortion (<0.5 ns) by CO photodissociation and nanosecond structural relaxation following CO geminate recombination are observed through the Raman bands of Phe and Trp residues located near the heme. These results demonstrate the high potential of this instrument to detect local protein motions subsequent to photoreactions in their active sites.
• Crystallographic and spectroscopic studies of peroxide-derived myoglobin compound II and occurrence of protonated Fe(IV)-O

  Hans-Petter Hersleth, Takeshi Uchida, Asmund K. Rohr, Thomas Teschner, Volker Schuenemann, Teizo Kitagawa, Alfred X. Trautwein, Carl Henrik Gorbitz, K. Kristoffer Andersson

  JOURNAL OF BIOLOGICAL CHEMISTRY 282 (32) 23372 - 23386 0021-9258 2007/08 [Refereed][Not invited]
   

  High resolution crystal structures of myoglobin in the pH range 5.2-8.7 have been used as models for the peroxide-derived compound II intermediates in heme peroxidases and oxygenases. The observed Fe-O bond length (1.86-1.90 angstrom) is consistent with that of a single bond. The compound II state of myoglobin in crystals was controlled by single-crystal microspectrophotometry before and after synchrotron data collection. We observe some radiation-induced changes in both compound II (resulting in intermediate H) and in the resting ferric state of myoglobin. These radiation-induced states are quite unstable, and compound II and ferric myoglobin are immediately regenerated through a short heating above the glass transition temperature (< 1 s) of the crystals. It is unclear how this influences our compound II structures compared with the unaffected compound II, but some crystallographic data suggest that the influence on the Fe-O bond distance is minimal. Based on our crystallographic and spectroscopic data we suggest that for myoglobin the compound II intermediate consists of an Fe(IV)-O species with a single bond. The presence of Fe(IV) is indicated by a small isomer shift of delta = 0.07 mm/s from Mossbauer spectroscopy. Earlier quantum refinements (crystallographic refinement where the molecular-mechanics potential is replaced by a quantum chemical calculation) and density functional theory calculations suggest that this intermediate H species is protonated.
• 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.
• Structure and Function of Cytochrome c Maturation Proteins

  UCHIDA Takeshi

  Seibutsu Butsuri 一般社団法人日本生物物理学会 47 (2) 112 - 117 0582-4052 2007/03/25 

  c-type cytochromes are electron transfer proteins that are essential for the life of virtually all organisms. They characteristically carry covalently-bound heme via thioether bonds to two cysteines in the protein. In Gram-negative bacteria, biogenesis of c-type cytochrome is conducted by a multiprotein complex system known as the cytochrome c maturation (Ccm) system. This system is consisted of 8 gene products (CcmA-CcmH). CcmE, which is called as a heme chaperon, binds heme and delivers it to apocytochrome c. In this article, I discuss the structure of CcmE and how it works as a heme chaperone protein.
  
• 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
• Similarities and differences between cyclobutane pyrimidine dimer photolyase and (6-4) photolyase as revealed by resonance Raman spectroscopy - Electron transfer from the FAD cofactor to ultraviolet-damaged DNA

  Jiang Li, Takeshi Uchida, Takeshi Todo, Teizo Kitagawa

  JOURNAL OF BIOLOGICAL CHEMISTRY 281 (35) 25551 - 25559 0021-9258 2006/09 [Refereed][Not invited]
   

  The cyclobutane pyrimidine dimer (CPD) and (6-4) photoproduct, two major types of DNA damage caused by UV light, are repaired under illumination with near UV-visible light by CPD and (6-4) photolyases, respectively. To understand the mechanism of DNA repair, we examined the resonance Raman spectra of complexes between damaged DNA and the neutral semiquinoid and oxidized forms of (6-4) and CPD photolyases. The marker band for a neutral semiquinoid flavin and band I of the oxidized flavin, which are derived from the vibrations of the benzene ring of FAD, were shifted to lower frequencies upon binding of damaged DNA by CPD photolyase but not by ( 6-4) photolyase, indicating that CPD interacts with the benzene ring of FAD directly but that the (6-4) photoproduct does not. Bands II and VII of the oxidized flavin and the 1398/1391 cm(-1) bands of the neutral semiquinoid flavin, which may reflect the bending of U-shaped FAD, were altered upon substrate binding, suggesting that CPD and the (6-4) photoproduct interact with the adenine ring of FAD. When substrate was bound, there was an upshifted 1528 cm(-1) band of the neutral semiquinoid flavin in CPD photolyase, indicating a weakened hydrogen bond at N(5)-H of FAD, and band X seemed to be downshifted in (6-4) photolyase, indicating a weakened hydrogen bond at N(3)-H of FAD. These Raman spectra led us to conclude that the two photolyases have different electron transfer mechanisms as well as different hydrogen bonding environments, which account for the higher redox potential of CPD photolyase.
• Characteristic structure and environment in FAD cofactor of (6-4) photolyase along function revealed by resonance Raman spectroscopy

  Jiang Li, Takeshi Uchida, Takehiro Ohta, Takeshi Todo, Teizo Kitagawa

  JOURNAL OF PHYSICAL CHEMISTRY B 110 (33) 16724 - 16732 1520-6106 2006/08 [Refereed][Not invited]
   

  A pyrimidine-pyrimidone (6-4) photoproduct and a cyclobutane pyrimidine dimer (CPD) are major DNA lesions induced by ultraviolet irradiation, and (6-4) photolyase, an enzyme with flavin adenine dinucleotide (FAD) as a cofactor, repairs the former specifically by light illumination. We investigated resonance Raman spectra of (6-4) photolyase from Arabidopsis thaliana having neutral semiquinoid and oxidized forms of FAD, which were selectively intensity enhanced by excitations at 568.2 and 488.0 nm, respectively. DFT calculations were carried out for the first time on the neutral semiquinone. The marker band of a neutral semiquinone at 1606 cm(-1) in H2O, whose frequency is the lowest among various flavoenzymes, apparently splits into two comparable bands at 1594 and 1608 cm(-1) in D2O, and similarly, that at 1522 cm(-1) in H2O does into three bands at 1456, 1508, and 1536 cm(-1) in D2O. This D2O effect was recognized only after being oxidized once and photoreduced to form a semiquinone again, but not by simple H/D exchange of solvent. Some Raman bands of the oxidized form were observed at significantly low frequencies (1621, 1576 cm(-1)) and with band splittings (1508/1493, 1346/1320 cm(-1)). These Raman spectral characteristics indicate strong H-bonding interactions (at N5-H, N1), a fairly hydrophobic environment, and an electron-lacking feature in benzene ring of the FAD cofactor, which seems to specifically control the reactivity of (6-4) photolyase.
• Specific hydrogen-bonding networks responsible for selective O-2 sensing of the oxygen sensor protein HemAT from Bacillus subtilis

  H Yoshimura, S Yoshioka, K Kobayashi, T Ohta, T Uchida, M Kubo, T Kitagawa, S Aono

  BIOCHEMISTRY 45 (27) 8301 - 8307 0006-2960 2006/07 [Refereed][Not invited]
   

  HemAT from Bacillus subtilis (HemAT-Bs) is a heme-based O-2 sensor protein that acts as a signal transducer responsible for aerotaxis. HemAT-Bs discriminates its physiological effector, O-2, from other gas molecules to generate the aerotactic signal, but the detailed mechanism of the selective O-2 sensing is not obvious. In this study, we measured electronic absorption, electron paramagnetic resonance (EPR), and resonance Raman spectra of HemAT-Bs to elucidate the mechanism of selective O-2 sensing by HemAT-Bs. Resonance Raman spectroscopy revealed the presence of a hydrogen bond between His86 and the heme propionate only in the O-2-bound form, in addition to that between Thr95 and the heme-bound O-2. The disruption of this hydrogen bond by the mutation of His86 caused the disappearance of a conformer with a direct hydrogen bond between Thr95 and the heme-bound O-2 that is present in WT HemAT-Bs. On the basis of these results, we propose a model for selective O-2 sensing by HemAT-Bs as follows. The formation of the hydrogen bond between His86 and the heme propionate induces a conformational change of the CE-loop and the E-helix by which Thr95 is located at the proper position to form the hydrogen bond with the heme-bound O-2. This stepwise conformational change would be essential to selective O-2 sensing and signal transduction by HemAT-Bs.
• Spectroscopic and DNA-binding characterization of the isolated heme-bound basic helix-loop-helix-PAS-A domain of neuronal PAS protein 2 (NPAS2), a transcription activator protein associated with circadian rhythms

  Y Mukaiyama, T Uchida, E Sato, A Sasaki, Y Sato, J Igarashi, H Kurokawa, Sagami, I, T Kitagawa, T Shimizu

  FEBS JOURNAL 273 (11) 2528 - 2539 1742-464X 2006/06 [Refereed][Not invited]
   

  Neuronal PAS domain protein 2 (NPAS2) is a circadian rhythm-associated transcription factor with two heme-binding sites on two PAS domains. In the present study, we compared the optical absorption spectra, resonance Raman spectra, heme-binding kinetics and DNA-binding characteristics of the isolated fragment containing the N-terminal basic helix-loop-helix (bHLH) of the first PAS (PAS-A) domain of NPAS2 with those of the PAS-A domain alone. We found that the heme-bound bHLH-PAS-A domain mainly exists as a dimer in solution. The Soret absorption peak of the Fe(III) complex for bHLH-PAS-A (421 nm) was located at a wavelength 9 nm higher than for isolated PAS-A (412 nm). The axial ligand trans to CO in bHLH-PAS-A appears to be His, based on the resonance Raman spectra. In addition, the rate constant for heme association with apo-bHLH-PAS (3.3 x 10(7) Mol(-1)center dot s(-1)) was more than two orders of magnitude higher than for association with apo-PAS-A (< 10(5) mol(-1)center dot s(-1)). These results suggest that the bHLH domain assists in stable heme binding to NPAS2. Both optical and resonance Raman spectra indicated that the Fe(II)-NO heme complex is five-coordinated. Using the quartz-crystal microbalance method, we found that the bHLH-PAS-A domain binds specifically to the E-box DNA sequence in the presence, but not in the absence, of heme. On the basis of these results, we discuss the mode of heme binding by bHLH-PAS-A and its potential role in regulating DNA binding.
• On the relationship of coral allene oxide synthase to catalase - A single active site mutation that induces catalase activity in coral allene oxide synthase

  T Tosha, T Uchida, AR Brash, T Kitagawa

  JOURNAL OF BIOLOGICAL CHEMISTRY 281 (18) 12610 - 12617 0021-9258 2006/05 [Refereed][Not invited]
   

  A heme domain of coral allene oxide synthase (cAOS) catalyzes the formation of allene oxide from fatty acid hydroperoxide. Although cAOS has a similar heme active site to that of catalase, cAOS is completely lacking in catalase activity. A close look at the hydrogen-bonding possibilities around the distal His in cAOS suggested that the imidazole ring is rotated by 180 degrees relative to that of catalase because of the hydrogen bond between Thr-66 and the distal His-67. This could contribute to the functional differences between cAOS and catalase, and to examine this possibility, we mutated Thr-66 in cAOS to Val, the corresponding residue in catalase. In contrast to the complete absence of catalase activity in wild type (WT) cAOS, T66V had a modest catalase activity. On the other hand, the mutation suppressed the native enzymatic activity of the formation of allene oxide to 14% of that of WT cAOS. In the resonance Raman spectrum, whereas WT cAOS has only a 6-coordinate/high spin heme, T66V has a 5-coordinate/ high spin heme as a minor species. Because catalase adopts a 5-coordinate/ high spin structure, probably the 5-coordinate/ high spin portion of T66V showed the catalase activity. Furthermore, in accord with the fact that the CN affinity of catalase is higher than that of WTc AOS, the CN affinity of T66V was 8-fold higher than that of WT cAOS, indicating that the mutation could mimic the heme active site in catalase. We, therefore, propose that the hydrogen bond between Thr-66 and distal His-67 could modulate the orientation of distal His, thereby regulating the enzymatic activity in cAOS.
• Evidence for displacements of the C-helix by CO Ligation and DNA binding to CooA revealed by UV resonance Raman spectroscopy

  M Kubo, S Inagaki, S Yoshioka, T Uchida, Y Mizutani, S Aono, T Kitagawa

  JOURNAL OF BIOLOGICAL CHEMISTRY 281 (16) 11271 - 11278 0021-9258 2006/04 [Refereed][Not invited]
   

  The UV and visible resonance Raman spectra are reported for CooA from Rhodospirillum rubrum, which is a transcriptional regulator activated by growth in a CO atmosphere. CO binding to heme in its sensor domain causes rearrangement of its DNA-binding domain, allowing binding of DNA with a specific sequence. The sensor and DNA-binding domains are linked by a hinge region that follows a long C-helix. UV resonance Raman bands arising from Trp-110 in the C-helix revealed local movement around Trp-110 upon CO binding. The indole side chain of Trp-110, which is exposed to solvent in the CO-free ferrous state, becomes buried in the CO-bound state with a slight change in its orientation but maintains a hydrogen bond with a water molecule at the indole nitrogen. This is the first experimental data supporting a previously proposed model involving displacement of the C-helix and heme sliding. The UV resonance Raman spectra for the CooA-DNA complex indicated that binding of DNA to CooA induces a further displacement of the C-helix in the same direction during transition to the complete active conformation. The Fe-CO and C-O stretching bands showed frequency shifts upon DNA binding, but the Fe-His stretching band did not. Moreover, CO-geminate recombination was more efficient in the DNA-bound state. These results suggest that the C-helix displacement in the DNA-bound form causes the CO binding pocket to narrow and become more negative.
• Dynamic Ligation properties of the Escherichia coli heme chaperone CcmE to non-covalently bound heme

  JM Stevens, T Uchida, O Daltrop, T Kitagawa, SJ Ferguson

  JOURNAL OF BIOLOGICAL CHEMISTRY 281 (10) 6144 - 6151 0021-9258 2006/03 [Refereed][Not invited]
   

  The cytochrome c maturation protein CcmE is an essential membrane-anchored heme chaperone involved in the post-translational covalent attachment of heme to c-type cytochromes in Gram-negative bacteria such as Escherichia coli. Previous in vitro studies have shown that CcmE can bind heme both covalently ( via a histidine residue) and non-covalently. In this work we present results on the latter form of heme binding to a soluble form of CcmE. Examination of a number of site-directed mutants of E. coli CcmE by resonance Raman spectroscopy has identified ligands of the heme iron and provided insight into the initial steps of heme binding by CcmE before it binds the heme covalently. The heme binding histidine (His-130) appears to ligate the heme iron in the ferric oxidation state, but two other residues ligate the iron in the ferrous form, thereby freeing His-130 to undergo covalent attachment to a heme vinyl group. It appears that the heme ligation in the non-covalent form is different from that in the holo-form, suggesting that a change in ligation could act as a trigger for the formation of the covalent bond and showing the dynamic and oxidation state-sensitive ligation properties of CcmE.
• Biophysical properties of a c-type heme in chemotaxis signal transducer protein DcrA

  Shiro Yoshioka, Katsuaki Kobayashi, Hideaki Yoshimura, Takeshi Uchida, Teizo Kitagawa, Shigetoshi Aono

  Biochemistry 44 (46) 15406 - 15413 0006-2960 2005/11/22 [Refereed][Not invited]
   

  Chemotaxis signal transducer protein Dcr A from a sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough was previously shown to contain a c-type heme in its periplasmic domain (DcrA-N) for sensing redox and/or oxygen [Fu et al. (1994) J. Bacteriol. 176, 344-350], which is the first example of a heme-based sensor protein containing a c-type heme as a prosthetic group. Optical absorption and resonance Raman spectroscopies indicates that heme c in DcrA-N shows a redox-dependent ligand exchange. Upon reduction, a water molecule that may be the sixth ligand of the ferric heme c is replaced by an endogenous amino acid. Although the reduced heme in DcrA-N is six-coordinated with two endogenous axial ligands, CO can easily bind to the reduced heme to form CO-bound DcrA-N. Reaction of the reduced DcrA-N with molecular oxygen results in autoxidation to form a ferric state without forming any stable oxygen-bound form probably due to the extremely low redox potential of DcrA-N (-250 mV). Our study supports the initial idea by Fu et al. that DcrA would act as a redox and/or oxygen sensor, in which the ligand exchange between water and an endogenous amino acid is a trigger for signal transduction. While the affinity of CO to DcrA-N (Kd = 138 μM) is significantly weak compared to those of other heme proteins, we suggest that CO might be another physiological effector molecule. © 2005 American Chemical Society.
• Mechanism for transduction of the ligand-binding signal in heme-based gas sensory proteins revealed by resonance Raman spectroscopy

  T Uchida, T Kitagawa

  ACCOUNTS OF CHEMICAL RESEARCH 38 (8) 662 - 670 0001-4842 2005/08 [Refereed][Not invited]
   

  Gene analysis has revealed a variety of new heme-containing gas sensory proteins in organisms ranging from bacteria to mammals. These proteins are composed of sensor, communication, and functional domains. The sensor domain contains a heme that binds effector molecules such as NO, O-2, or CO. Ligand binding by the sensor domain modulates the physiological role of the protein, such as DNA binding in the case of transcriptional factors or the catalytic reaction rate in the case of enzymes. This Account summarizes resonance Raman (RR) studies, including static and time-resolved measurements, which have enabled elucidation of the mechanisms by which binding of specific target molecule by the sensor domain is transduced to alteration of the functional domain. These studies have shown that signals can be conveyed from the heme to the functional domain via three different pathways: (i) a distal pathway, (ii) a proximal pathway, and (iii) a heme peripheral pathway.
• Covalent cofactor attachment to proteins: cytochrome c biogenesis

  JM Stevens, T Uchida, O Daltrop, SJ Ferguson

  BIOCHEMICAL SOCIETY TRANSACTIONS 33 792 - 795 0300-5127 2005/08 [Refereed][Not invited]
   

  Haem (Fe-protoporphyrin IX) is a cofactor found in a wide variety of proteins. it confers diverse functions, including electron transfer, the binding and sensing of gases, and many types of catalysis. The majority of cofactors are non-covalently attached to proteins. There are, however, some proteins in which the cofactor binds covalently and one of the major protein classes characterized by covalent cofactor attachment is the c-type cytochromes. The characteristic haem-binding mode of c-type cytochromes requires the formation of two covalent bonds between two cysteine residues in the protein and the two vinyl groups of haem. Haem attachment is a complex post-translational process that, in bacteria such as Escherichia coli, occurs in the periplasmic space and involves the participation of many proteins. Unexpectedly, it has been found that the haem chaperone CcmE (cytochrome c maturation), which is an essential intermediate in the process, also binds haem covalently before transferring the haem to apocytochromes. A single covalent bond is involved and occurs between a haem vinyl group and a histidine residue of CcmE. Several in vitro and in vivo studies have provided insight into the function of this protein and into the overall process of cytochrome c biogenesis.
• Spectroscopic characterization of the isolated heme-bound PAS-B domain of neuronal PAS domain protein 2 associated with circadian rhythms

  R Koudo, H Kurokawa, E Sato, J Igarashi, T Uchida, Sagami, I, T Kitagawa, T Shimizu

  FEBS JOURNAL 272 (16) 4153 - 4162 1742-464X 2005/08 [Refereed][Not invited]
   

  Neuronal PAS domain protein 2 (NPAS2) is an important transcription factor associated with circadian rhythms. This protein forms a heterodimer with BMAL1, which binds to the E-box sequence to mediate circadian rhythm-regulated transcription. NPAS2 has two PAS domains with heme-binding sites in the N-terminal portion. In this study, we overexpressed wild-type and His mutants of the PAS-B domain (residues 241-416) of mouse NPAS2 and then purified and characterized the isolated heme-bound proteins. Optical absorption spectra of the wild-type protein showed that the Fe(III), Fe(II) and Fe(II)-CO complexes are 6-co-ordinated low-spin complexes. On the other hand, resonance Raman spectra indicated that both the Fe(III) and Fe(II) complexes contain mixtures of 5-co-ordinated high-spin and 6-co-ordinated low-spin complexes. Based on inverse correlation between nu(Fe-CO) and nu(C-O) of the resonance Raman spectra, it appeared that the axial ligand trans to CO of the heme-bound PAS-B is His. Six His mutants (His266Ala, His289Ala, His300Ala, His302Ala, His329Ala, and His335Ala) were generated, and their optical absorption spectra were compared. The spectrum of the His335Ala mutant indicated that its Fe(III) complex is the 5-co-ordinated high-spin complex, whereas, like the wild-type, the complexes for the five other His mutants were 6-co-ordinated low-spin complexes. Thus, our results suggest that one of the axial ligands of Fe(III) in PAS-B is His335. Also, binding kinetics suggest that heme binding to the PAS-B domain of NPAS2 is relatively weak compared with that of sperm whale myoglobin.
• CO-dependent activity-controlling mechanism of heme-containing CO-sensor protein, neuronal PAS domain protein

  T Uchida, E Sato, A Sato, Sagami, I, T Shimizu, T Kitagawa

  JOURNAL OF BIOLOGICAL CHEMISTRY 280 (22) 21358 - 21368 0021-9258 2005/06 [Refereed][Not invited]
   

  Neuronal PAS domain protein 2, which was recently established to be a heme protein, acts as a CO-dependent transcription factor. The protein consists of the basic helix-loop-helix domain and two heme- containing PAS domains (PAS-A and PAS-B). In this study, we prepared wild type and mutants of the isolated PAS-A domain and measured resonance Raman spectra of these proteins. Upon excitation of the Raman spectrum at 363.8 nm, a band assignable to Fe3+-S stretching was observed at 334 cm(-1) for the ferric wild type protein; in contrast, this band was drastically weaker in the spectrum of C170A, suggesting that Cys(170) is an axial ligand of the ferric heme. The Raman spectrum of the reduced form of wild type was mainly of six-coordinate low spin, and the v(11) band, which is sensitive to the donor strength of the axial ligand, was lower than that of reduced cytochrome c(3), suggesting coordination of a strong ligand and thus a deprotonated His. In the reduced forms of H119A and H171A, the five-coordinate species became more prevalent, whereas no such changes were observed for C170A, indicating that His(119) and His(171), but not Cys(170), are axial ligands in the ferrous heme. This means that ligand replacement from Cys to His occurs upon heme reduction. The v(Fe-CO) versus v(C-O) correlation indicates that a neutral His is a trans ligand of CO. Our results support a mechanism in which CO binding disrupts the hydrogen bonding of His(171) with surrounding amino acids, which induces conformational changes in the His(171)- Cys(170) moiety, leading to physiological signaling.
• 2SB02 CO Sensory Protein Containing PAS Domains

  Uchida T.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 45 S18  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
• 3P097 Contribution of the heme-PAS domain of NPAS2 to DNA binding activity

  Ishida M., Uchida T., Kitagawa T., Sagami I.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 45 S228  2005
• 3P090 Origin of Functional Difference in Tyrosinate-ligated Homologous Enzymes, Coral Allene Oxide Synthase and Catalase

  Tosha T., Uchida T., Brash Alan R., Kitagawa T.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 45 S226  2005
• 3P091 Structural change of the heme-containing transcriptional activator CooA on DNA binding: A resonance Raman spectroscopic study

  Kubo M., Inagaki S., Uchida T., Aono S., Kitagawa T.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 45 S226  2005
• 2P299 Resonance Raman Study on the Active Site of (6-4) Photolyase : Electron Density Localization in FAD and Strong H-bonding Environment

  Li, Uchida T., Todo T., Kitagawa T.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 45 S194  2005
• Role of Tyr-288 at the dioxygen reduction site of cytochrome bo studied by stable isotope labeling and resonance Raman spectroscopy

  T Uchida, T Mogi, H Nakamura, T Kitagawa

  JOURNAL OF BIOLOGICAL CHEMISTRY 279 (51) 53613 - 53620 0021-9258 2004/12 [Refereed][Not invited]
   

  To explore the role of a cross-link between side chains of Tyr-288 and His-284 at the heme-copper binuclear center, we prepared cytochrome bo where d(4)-Tyr, 1-[C-13]Tyr, or 4-[C-13]Tyr has been biosynthetically incorporated. Unexpectedly, the d(4)-Tyr-labeled enzyme showed a large decrease in the ubiquinol-1 oxidase and CO binding activities. Optical absorption and resonance Raman spectra identified the defect in the distal side of the heme-copper binuclear center. In the CO-bound d(4)-Tyr-labeled enzyme, a large fraction of the nu((Fe-C)) mode was shifted from the normal 520-cm(-1) band to a broad band centered around 491 cm(-1), as found for the Y288F mutant. Our results suggested that the substitution of ring hydrogens of Tyr-288 with deuteriums slows down the formation of the His-Tyr cross-link essential for dioxygen reduction at the binuclear center.
• The interaction of covalently bound heme with the cytochrome c maturation protein CcmE

  T Uchida, JM Stevens, O Daltrop, EM Harvat, L Hong, SJ Ferguson, T Kitagawa

  JOURNAL OF BIOLOGICAL CHEMISTRY 279 (50) 51981 - 51988 0021-9258 2004/12 [Refereed][Not invited]
   

  The heme chaperone CcmE is a novel protein that binds heme covalently via a histidine residue as part of its essential function in the process of cytochrome c biogenesis in many bacteria as well as plant mitochondria. In the continued absence of a structure of the holoform of CcmE, identification of the heme ligands is an important step in understanding the molecular function of this protein and the role of covalent heme binding to CcmE during the maturation of c-type cytochromes. In this work, we present spectroscopic data that provide insight into the ligation of the heme iron in the soluble domain of CcmE from Escherichia coli. Resonance Raman spectra demonstrated that one of the heme axial ligands is a histidine residue and that the other is likely to be Tyr(134). In addition, the properties of the heme resonances of the holo-protein as compared with those of a form of CcmE with non-covalently bound heme provide evidence for the modification of one of the heme vinyl side chains by the protein, most likely the 2-vinyl group.
• SOUL in mouse eyes is a new hexameric heme-binding protein with characteristic optical absorption, resonance Raman spectral, and heme-binding properties

  E Sato, Sagami, I, T Uchida, A Sato, T Kitagawa, J Igarashi, T Shimizu

  BIOCHEMISTRY 43 (44) 14189 - 14198 0006-2960 2004/11 [Refereed][Not invited]
   

  SOUL is specifically expressed in the retina and pineal gland and displays more than 40% sequence homology with p22HBP, a heme protein ubiquitously expressed in numerous tissues. SOUL was purified as a dimer in the absence of heme from the Escherichia coli expression system but displayed a hexameric structure upon heme binding. Heme-bound SOUL displayed optical absorption and resonance Raman spectra typical of 6-coordinate low-spin heme protein, with one heme per monomeric unit for both the Fe(III) and Fe(II) complexes. Spectral data additionally suggest that one of the axial ligands of the Fe(III) heme complex is His. Mutation of His42 (the only His of SOUL) to Ala resulted in loss of heme binding, confirming that this residue is an axial ligand of SOUL. The K-d value of heme for SOUL was estimated as 4.8 x 10(-9) M from the association and dissociation rate constants, suggesting high binding affinity. On the other hand, p22HBP was obtained as a monomer containing one heme per subunit, with a K-d value of 2.1 x 10(-11) M. Spectra of heme-bound p22HBP were different from those of SOUL but similar to those of heme-bound bovine serum albumin in which heme bound to a hydrophobic cavity with no specific axial ligand coordination. Therefore, the heme-binding properties and coordination structure of SOUL are distinct from those of p22HBP, despite high sequence homology. The physiological role of the new heme-binding protein, SOUL, is further discussed in this report.
• [UV/Vis absorption, infrared and Raman spectroscopy].

  Uchida T, Kitagawa T

  Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 49 (11 Suppl) 1693 - 1699 0039-9450 2004/08 [Refereed][Not invited]
  
• Structural characterization of a binuclear center of a Cu-containing NO reductase homologue from Roseobacter denitrificans: EPR and resonance Raman studies

  Y Matsuda, T Uchida, H Hori, T Kitagawa, H Arata

  BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS 1656 (1) 37 - 45 0005-2728 2004/05 [Refereed][Not invited]
   

  Aerobic phototrophic bacterium Roseobacter denitrificans has a nitric oxide reductase (NOR) homologue with cytochrome e oxidase (CcO) activity. It is composed of two subunits that are homologous with NorC and NorB, and contains heme c, heme b, and copper in a 1:21 stoichiometry. This enzyme has virtually no NOR activity. Electron paramagnetic resonance (EPR) spectra of the air-oxidized enzyme showed signals of two low-spin hemes at 15 K. The high-spin heme species having relatively low signal intensity indicated that major part of heme b(3) is EPR-silent due to an antiferromagnetic coupling to an adjacent Cu-B forming a Fe-Cu binuclear center. Resonance Raman (RR) spectrum of the oxidized enzyme suggested that heme b(3) is six-coordinate high-spin species and the other hemes are six-coordinate low-spin species. The RR spectrum of the reduced enzyme showed that all the ferrous hemes are six-coordinate low-spin species. nu(Fe-CO) and nu(C-O) stretching modes were observed at 523 and 1969 cm(-1), respectively, for CO-bound enzyme. In spite of the similarity to NOR in the primary structure, the frequency of nu(Fe-CO) mode is close to those of aa(3)- and bo(3)-type oxidases rather than that of NOR. (C) 2004 Elsevier B.V. All rights reserved.
• Structural characterization of a binuclear center of a Cu-containing NO reductase homologue from Roseobacter denitrificans: EPR and resonance Raman studies.

  Matsuda Y, Uchida T, Hori H, Kitagawa T, Arata H

  Biochimica et biophysica acta 1656 (1) 37 - 45 0006-3002 2004/05 [Refereed][Not invited]
  
• 1P267 Resonance Raman Investigation of (6-4)Photolyase : FAD Cofactor Is in a Strong Hydrogen-bonding Environment

  Li Jiang, Uchida T., Todo T., Kitagawa T.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 44 S96  2004
• 2P078 Interactions between Coral Allene Oxide Synthase and Substrate : Resonance Raman Investigation

  Tosha T., Uchida T., Brash Alan R., Kitagawa T.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 44 S129  2004
• Multiple monovalent ion-dependent pathways for the folding of the L-21 Tetrahymena thermophila ribozyme

  T Uchida, K Takamoto, Q He, MR Chance, M Brenowitz

  JOURNAL OF MOLECULAR BIOLOGY 328 (2) 463 - 478 0022-2836 2003/04 [Refereed][Not invited]
   

  Synchrotron hydroxyl radical (OH) footprinting is a technique that monitors the local changes in solvent accessibility of the RNA backbone on milliseconds to minutes time-scales. The Mg2+-dependent folding of the L-21 Sca I Tetrahymena thermophila ribozyme has been followed using this technique at an elevated concentration of monovalent ion (200 mM NaCl) and as a function of the initial annealing conditions and substrate. Previous studies conducted at low concentrations of monovalent ion displayed sequential folding of the P4-P6 domain, the peripheral helices and the catalytic core, with each protection displaying monophasic kinetics. For ribozyme annealed in buffer containing 200 mM NaCl and folded by the addition of 10 mM MgCl2, multiple kinetic phases are observed for .OH protections throughout the ribozyme. The independently folding P4-P6 domain is the first to fold with its protections displaying 50-90% burst phase amplitudes. That the folding of P4-P6 within the ribozyme does not display the 100% burst phase of isolated P4-P6 at 200 mM NaCl shows that interactions with the remainder of the ribozyme impede this domain's folding. In addition, .OH protections constituting each side of a tertiary contact are not coincident in some cases, consistent with the formation of transient non-native interactions. While the peripheral contacts and triple helical scaffold exhibit substantial burst phases, the slowest protection to appear is J8/7 in the catalytic core, which displays a minimal burst amplitude and whose formation is coincident with the recovery of catalytic activity. The number of kinetic phases as well as their amplitudes and rates are different when the ribozyme is annealed in low-salt buffer and folded by the concomitant addition of monovalent and divalent cations. Annealed substrate changes the partitioning of the ribozyme among the multiple folding populations. These results provide a map of the early steps in the ribozyme's folding landscape and the degree to which the preferred pathways are dependent upon the initial reaction conditions. (C) 2003 Elsevier Science Ltd. All rights reserved.
• Linkage of monovalent and divalent ion binding in the folding of the P4-P6 domain of the Tetrahymena ribozyme

  T Uchida, Q He, CY Ralston, M Brenowitz, MR Chance

  BIOCHEMISTRY 41 (18) 5799 - 5806 0006-2960 2002/05 [Refereed][Not invited]
   

  We have explored the linkage of monovalent and divalent ion binding in the folding of the P4-P6 domain of Tetrahymena thermophila ribozyme by examining the Mg2+-induced folding and the urea-induced denaturation of the folded state as a function of Na+ under equilibrium folding conditions using hydroxyl radical footprinting. These studies allowed a thermodynamic examination of eight discrete protection sites within P4-P6 that are involved in several tertiary structure contacts. Monovalent ions compete with Mg2+ ions in mediating P4-P6 folding. The urea denaturation isotherms demonstrated DeltaDeltaG values of > 2 kcal mol(-1) in experiments conducted in 10 versus 200 mM NaCl at a constant 10 mM MgCl2. However, the individual-site isotherms reported by footprinting revealed that larger than average changes in DeltaG values were localized to specific sites within the Mg2+-rich A-bulge. The competitive effects of monovalent ions were less when K+ rather than Na+ was the monovalent cation present. This result indicates the importance of the specific K+ binding sites that are associated with AA-platform structures to P4-P6 folding and stability. These site-specific footprinting data provide quantitative and site-specific measurements of the ion-linked stability for P4-P6 that are interpreted with respect to crystallographic data.
• Resonance Raman and ligand binding studies of the oxygen-sensing signal transducer protein HemAT from Bacillus subtilis

  S Aono, T Kato, M Matsuki, H Nakajima, T Ohta, T Uchida, T Kitagawa

  JOURNAL OF BIOLOGICAL CHEMISTRY 277 (16) 13528 - 13538 0021-9258 2002/04 [Refereed][Not invited]
   

  HemAT-Bs is a heme-containing signal transducer protein responsible for aerotaxis of Bacillus subtilis. The recombinant HemAT-Bs expressed in Escherichia coli was purified as the oxy form in which oxygen was bound to the ferrous heme. Oxygen binding and dissociation rate constants were determined to be k(on) = 32 muM(-1) s(-1) and k(off) = 23 s(-1), respectively, revealing that HemAT-Bs has a moderate oxygen affinity similar to that of sperm whale myoglobin (Mb). The rate constant for autoxidation at 37 degreesC was 0.06 h(-1), which is also close to that of Mb. Although the electronic absorption spectra of HemAT-Bs were similar to those of Mb, HemAT-Bs showed some unique characteristics in its resonance Raman spectra. Oxygen-bound HemAT-Bs gave the nu(Fe-O2) band at a noticeably low frequency (560 cm(-1)), which suggests a unique hydrogen bonding between a distal amino acid residue and the proximal atom of the bound oxygen molecule. Deoxy HemAT-Bs gave the nu(Fe-His) band at a higher frequency (225 cm(-1)) than those of ordinary His-coordinated deoxy heme proteins. CO-bound HemAT-Bs gave the nu(Fe-CO) and nu(C-O) bands at 494 and 1964 cm(-1), respectively, which fall on the same nu(C-O) versus nu(Fe-CO) correlation line as that of Mb. Based on these results, the structural and functional properties of HemAT-Bs are discussed.
• 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.
• Role of Tyr288 at the dioxygen reduction site of cytochrome bo studied by stable isotope labeling and site-directed mutagesis

  Uchida T., Mogi T., Nakamura H., Kitagawa T.

  Seibutsu Butsuri 一般社団法人 日本生物物理学会 41 S116  2001
• First synthetic NO-heme-thiolate complex relevant to nitric oxide synthase and cytochrome P450nor [33]

  N. Suzuki, T. Higuchi, Y. Urano, K. Kikuchi, T. Uchida, M. Mukai, T. Kitagawa, T. Nagano

  Journal of the American Chemical Society 122 (48) 12059 - 12060 0002-7863 2000/12/06 [Refereed][Not invited]
  
• Active site structure of SoxB-type cytochrome bo(3) oxidase from thermophilic Bacillus

  T Uchida, M Tsubaki, T Kurokawa, H Hori, J Sakamoto, T Kitagawa, N Sone

  JOURNAL OF INORGANIC BIOCHEMISTRY 82 (1-4) 65 - 72 0162-0134 2000/11 [Refereed][Not invited]
   

  Two-subunit SoxB-type cytochrome c oxidase in Bacillus stearothermophilus was over-produced, purified, and examined for its active site structures by electron paramagnetic resonance (EPR) and resonance Raman (RR) spectroscopies. This is cytochrome bo(3) oxidase containing heme B at the low-spin heme site and heme O at the high-spin heme site of the binuclear center. EPR spectra of the enzyme in the oxidized form indicated that structures of the high-spin heme O and the low-spin heme B were similar to those of SoxM-type oxidases based on the signals at g=6.1, and g=3.04. However, the EPR signals from the Cu-A center and the integer spin system at the binuclear center showed slight differences. RR spectra of the oxidized form showed that heme O was in a 6-coordinated high-spin (nu (3) = 1472 cm(-1)), and heme B was in a 6-coordinated low-spin (nu (3) = 1500 cm(-1)) state. The Fe2+-His stretching mode was observed at 211 cm(-1), indicating that the Fe2+-His bond strength is not so much different from those of SoxM-type oxidases. On the contrary, both the Fe2+-CO stretching and Fe2+-C-O bending modes differed distinctly from those of SoxM-type enzymes, suggesting some differences in the coordination geometry and the protein structure in the proximity of bound CO in cytochrome bo, from those of SoxM-type enzymes. (C) 2000 Elsevier Science B.V. All rights reserved.
• Identification of histidine 77 as the axial heme ligand of carbonmonoxy CooA by picosecond time-resolved resonance Raman spectroscopy

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

  Biochemistry 39 12747 - 12752 2000/09 [Refereed][Not invited]
  
• 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.
• Synthesis, structure, and H2O2-dependent catalytic functions of disulfide-bridged dicopper(I) and related thioether-copper(I) and thioether-copper(II) complexes

  T Ohta, T Tachiyama, K Yoshizawa, T Yamabe, T Uchida, T Kitagawa

  INORGANIC CHEMISTRY 39 (19) 4358 - 4369 0020-1669 2000/09 [Refereed][Not invited]
   

  A disulfide-bridged dicopper(I) complex, [Cu-2(Py2SSPy2)](ClO4)(2) (I) (Py2SSPy2 = bis{2-[N,N-bis(2-pyridylethyl)amino]-1,1-dimethylethyl}disulfide), a thioether-copper(I) complex, [Cu((PrSPy2)-Pr-i)](ClO4) (2) ((PrSPy2)-Pr-i = N-(2-isopropylthio-2-methyl)propyl-N,N-bis-2-(2-pyridyl)ethylamine), and a thioether-copper(II) complex, [Cu(PheSPy(2))(H2O)](ClO4)(2) (3) (PheSPy(2) = N-(2-methyl-2-phenethylthio)propyl-N,N-bis-2-(2-pyridyl)ethylamine), were newly synthesized by the reactions of Cu(ClO4)(2). 6H(2)O with a thiol ligand of Py2SH (N,N-bis[2-(2-pyridyl)ethyl]-1,1-dimethyl-2-mercaptoethylamine) and thioether ligands of (PrSPy2)-Pr-i and PheSPy(2), respectively, For complexes 1 and 2, X-ray analyses were performed. Complex 1 crystallizes in the triclinic space group P (1) over bar, and complex 2 crystallizes in the orthorhombic space group Pbca with the following unit cell parameters: for 1, a = 15.165 (3) Angstrom. b = 22.185 (4) Angstrom, c = 14.989 (3) Angstrom, alpha = 105.76 (1)degrees, beta = 90.82 (2)degrees, gamma = 75.23 (1)degrees, and Z = 2; for 2, a = 17.78 (2) Angstrom, b = 17.70.(1) Angstrom, c = 15.75 (1) Angstrom, and Z = 8. Complex 1 is the first structurally characterized example obtained by the redox reaction Cu(II) + RSH --> Cu(I) + RSSR and has two independent structures (la, Ib) which mainly differ in S-S bond distances, Cu(I) Cu(I) separations, and C-S-S-C dihedral angles of the disulfide units. The S-S bond distances of 2.088(7) Angstrom in 1a and 7.070(7) Angstrom in 1b are indicative of significant activation of the S-S bonds by the dicopper centers. Fragment molecular orbital (FMO) analyses and molecular orbital overlap population (MOOP) analyses based on the extended Huckel method clarify the preferable formation of the disulfide S-S bond in 1 rather than the formation of a thiolate-copper(II) complex within the Py2S- ligand framework. Catalytic functions of complexes 1-3 were investigated with peroxides (H2O2 and (BuOOH)-Bu-t) as oxidants. Complex 1 catalyzed the selective oxidation of cyclohexane to cyclohexanol and mediated the cyclohexene epoxidation in the presence of H2O2. A transient dark green intermediate observed in the reaction of 1 with H2O2 is characterized by UV-vis, EPR, and resonance Raman spectroscopies, identifying it as a Cu(II)-OOH species, 1(OOH). The resonance Raman features of the nu(O-O) bands at 822 and 836 cm(-1), which are red-shifted to 781 and 791 cm(-1), respectively, upon introduction of (H2O2)-O-18, are indicative of formation of two kinds of Cu-OOH species rather than the Fermi doublet and the significant weakening of the O-O bonds. These mechanistic studies demonstrate that by virtue of the electron-donating ability of the disulfide unit the Cu-OOH species can be actually activated for one-electron oxidation. which has been reported so far unfavorable for other vibrationally characterized Cu-OOH species.
• Resonance raman studies of oxo intermediates in the reaction of pulsed cytochrome bo with hydrogen peroxide

  Takeshi Uchida, Tatsushi Mogi, Teizo Kitagawa

  Biochemistry 39 (22) 6669 - 6678 0006-2960 2000/06/06 [Refereed][Not invited]
   

  Cytochrome bo from Escherichia coli, a member of the heme-copper terminal oxidase superfamily, physiologically catalyzes reduction of O2 by quinols and simultaneously translocates protons across the cytoplasmic membrane. The reaction of its ferric pulsed form with hydrogen peroxide was investigated with steady-state resonance Raman spectroscopy using a homemade microcirculating system. Three oxygen-isotope-sensitive Raman bands were observed at 805/X, 783/753, and (767)/730 cm-1 for intermediates derived from H216O2/H218O2. The experiments using H216O18O yielded no new bands, indicating that all the bands arose from the Fe=O stretching (v(Fe=O)) mode. Among them, the intensity of the 805/X cm-1 pair increased at higher pH, and the species giving rise to this band seemed to correspond to the P intermediate of bovine cytochrome c oxidase (CcO) on the basis of the reported fact that the P intermediate of cytochrome bo appeared prior to the formation of the F species at higher pH. For this intermediate, a Raman band assignable to the CO stretching mode of a tyrosyl radical was deduced at 1489 cm-1 from difference spectra. This suggests that the P intermediate of cytochrome bo contains an Fe(IV)=O heme and a tyrosyl radical like compound I of prostaglandin H synthase. The 783/753 cm-1 pair, which was dominant at neutral pH and close to the v(Fe=O) frequency of the oxoferryl intermediate of CcO, presumably arises from the F intermediate. On the contrary, the (767)/730 cm-1 species has no counterpart in CcO. Its presence may support the branched reaction scheme proposed previously for O2 reduction by cytochrome bo.
• An approach to the O-2 activating mononuclear non-heme Fe enzymes: structural characterization of Fe(II)-acetato complex and formation of alkylperoxoiron(III) species with the highly hindered hydrotris(3-tert-butyl-5-isopropyl-1-pyrazolyl)borate

  T Ogihara, S Hikichi, M Akita, T Uchida, T Kitagawa, Y Moro-oka

  INORGANICA CHIMICA ACTA 297 (1-2) 162 - 170 0020-1693 2000/01 [Refereed][Not invited]
   

  Structural characterization of an Fe(II)-acetato complex and attempts to synthesize mononuclear Fe(III) dioxygen complexes bearing the highly sterically demanding Tp(tBu,iPr) (= hydrotris(3-tert-butyl-5-isopropyl-1-pyrazolyl)borate) ligand have been investigated. X-ray crystallography reveals that the acetato complex consists of the distorted square pyramidal Fe(II) center as found for the previously reported O-2-reactive Tp(iPr2) derivative. In contrast to the less hindered Tp(iPr2), (= hydrotris(3,5-diisopropyl-1-pyrazolyl)borate) complexes, oxidative addition of O-2 to the coordinatively unsaturated Fe(II) centers of the acetato and a hydroxo complexes with Tp(tBu,iPr) has never been observed in any conditions. Reaction of the ferrous hydroxo complex with ROOH (R = H, alkyl) results in the formation of the thermally unstable intermediates. Especially, the Fe(III)-alkylperoxo complex is characterized by UV-Vis, ESR and resonance Raman spectroscopy. The extremely bulky Tp(tBu,iPr) ligand hinders the approach of the exogenous O-2 molecule to the Fe(II) centers but stabilizes the unstable Fe(III)alkylperoxo intermediate enough to be detected. (C) 2000 Elsevier Science S.A. All rights reserved.
• Novel iron porphyrin-alkanethiolate complex with intramolecular NH···S hydrogen bond: Synthesis, spectroscopy, and reactivity [3]

  Noriyuki Suzuki, Tsunehiko Higuchi, Yasuteru Urano, Kazuya Kikuchi, Hidehiro Uekusa, Yuji Ohashi, Takeshi Uchida, Teizo Kitagawa, Tetsuo Nagano

  Journal of the American Chemical Society 121 (49) 11571 - 11572 0002-7863 1999/12/15 [Refereed][Not invited]
  
• 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.
• 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.
• 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.

MISC

• 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
• 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
• 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
• 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
• 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
• Structural and functional characterization of nanodisc-reconstituted cytochrome cbb3 oxidase from Vibrio cholerae

  INOUE Masanao, SHIBATA Akihiro, IMAI Mizue, UCHIDA Takeshi, MURAMOTO Kazumasa, SHIRO Noritsugu, FURUKAWA Shinya, ISHIMORI Koichiro  生物物理(Web)  56-  (Supplement 1-2)  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
• 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
• 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
• Resonance Raman studies of the heme chaperone protein, CcmE

  T Uchida, T Kitagawa, JM Stevens, O Daltrop, CW Higham, SJ Ferguson  JOURNAL OF INORGANIC BIOCHEMISTRY  96-  (1)  242  -242  2003/07  [Not refereed][Not invited]
  
• Monovalent cations mediate formation of native tertiary structure by the tetrahymena thermophila ribozyme: Implications for the folding kinetics.

  M Brenowitz, K Takamoto, S Morris, T Uchida, Shcherbakova, I, MR Chance  ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY  225-  U459  -U460  2003/03  [Not refereed][Not invited]
  
• Stability of RNA probed by chemical denaturation

  Q He, T Uchida, C Ralston, M Brenowitz, MR Chance  BIOPHYSICAL JOURNAL  80-  (1)  481A  -481A  2001/01  [Not refereed][Not invited]
  
• Investigating the effect of monovalent ion on folding mechanism of the Tetrahymena thermophila group I intron by synchrotron X-ray footprinting

  T Uchida, Q He, M Brenowitz, MR Chance  BIOPHYSICAL JOURNAL  80-  (1)  482A  -482A  2001/01  [Not refereed][Not invited]
  
• Resonance Raman studies of oxo intermediates in the reaction of cytochrome bo with hydrogen peroxide

  T Uchida, T Mogi, T Kitagawa  JOURNAL OF INORGANIC BIOCHEMISTRY  74-  (1-4)  321  -321  1999/04  [Not refereed][Not invited]
  
• 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
• 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
• 1a-G-6 Laser spectroscopic study of dynamics of myoglobin with a disulfide bond

  Kurita A., Matsuda K., Shibata Y., Kushida T., Uchida T., Morishima I.  Abstracts of the meeting of the Physical Society of Japan. Annual meeting  51-  (3)  752  -752  1996/03/15

Books etc

• Heme binding characteristics of mouse PER1, a transcriptional regulatory factor associated with circadian rhythms

  Nova Science Publishers 2011

Association Memberships

• 日本生物物理学会   日本化学会   日本生化学会   日本蛋白質科学会   

Research Projects

• Environmental pollutant degradation system using dye-decolorizing peroxidase

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

  Date (from‐to) : 2020/04 -2023/03 

  Author : 内田 毅

   

  DyP (Dye-decolorizing peroxidase) はヘムを含むペルオキシダーゼという酵素タンパク質の一種で、過酸化水素を利用し、アントラキノン系の色素を分解する酵素であることから、環境浄化酵素としての利用が期待されている。しかし、微生物やカビなどの一部に存在するタンパク質であるにもかかわらず、それらの生育条件とは離れたpH 4程度の酸性条件で活性が高く、中性付近ではほとんど活性がないという特徴が、環境浄化酵素としての実用化へのハードルとなっていた。そこで、反応機構を明らかにすることにより、pH依存性を決定している因子を明らかにし、アミノ酸置換を導入することにより、中性pHで活性を持たせることに成功した。試験管内の反応では中性で色素を分解可能になったため、これを大腸菌に発現させ、大腸菌を培養しながら、溶液内の色素を分解することを試みた。しかし、予想外に活性は著しく低く、環境浄化酵素として利用することでできなかった。原因を検討した結果、菌体内で発現させると活性中心であるヘムと結合していないことがわかった。ヘモグロビンなど多くのヘムタンパク質ではヘムはタンパク質と配位結合しているため、大腸菌内で利用しようとするとヘムを含まない可能性がある。そこで、タンパク質とヘムが共有結合しているシトクロムcにDyP活性を付与することを試みた。 はじめに天然型のシトクロムcのDyP活性を測定したところ、DyPの1/2程度の活性であることがわかった。次に、DyPで明らかにした反応機構をもとにシトクロムcに色素分解に必要なアミノ酸残基を導入することにより、天然型のシトクロムcの80倍の活性をもつ変異体シトクロムcを作成した。また、至適pHは8.0付近であったことから、試験管レベルではあるが、環境浄化酵素としての利用が期待できるものの作成に成功した。
• 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.
• 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.
• 病原菌に特徴的な鉄の取り込みタンパク質に着目した新規な抗菌剤の開発

  文部科学省：科学研究費補助金(基盤研究(C))

  Date (from‐to) : 2016/04 -2019/03 

  Author : 内田 毅
• Analysis of functional regulation of PGMRC1

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

  Date (from‐to) : 2015/04 -2018/03 

  Author : Kabe Yasuaki, UCHIDA Tsuyoshi

   

  Progesterone receptor membrane component 1 (PGRMC1) 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. Crystallographic analyses revealed that PGRMC1 forms a stable dimer through stacking interactions of two protruding haem molecules. The haem iron is five-coordinated by Tyr113. Haem-mediated PGRMC1 dimerization is required for interactions with EGFR and cytochromes P450, cancer proliferation and chemoresistance against anti-cancer drugs. 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.
• 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.
• 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.
• Characterization of the proteins involved in iron uptake system of pathogen and its application to development of anti-bacterial drugs

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

  Date (from‐to) : 2012 -2014 

  Author : UCHIDA Takeshi

   

  Iron is an essential element for bacteria survival. To obtain this element, bacterial pathogens utilize heme from hemoglobin in blood as an iron source, because the vast majority of iron in human body is present as heme. In this project, we found that VCA0907 (HutZ) from Vibrio cholerae is a heme-degrading enzyme. The activity of VCA0907 depends on the strength of the hydrogen bond between His170 that is a sole ligand of heme, and Asp132. We further found that VCA0908 (HutX) binds to heme with a slightly higher affinity than that of HutZ. Unexpectedly, heme, which is bound to HutX, irreversibly moves to HutZ. We concluded that heme that is transported into cytosol is captured by HutX, and then it is transferred to HutZ through the direct interaction with HutX. Finally, HutZ degrades heme to release iron, which is used for survival of V. cholerae. Because iron is an essential element, our findings lead to development of a new kind of medicine for pathogens.
• 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.
• Redox- and CO-dependent Transcription Mechanism by NPAS2

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(C))

  Date (from‐to) : 2008 -2010 

  Author : Takeshi UCHIDA

   

  The purpose of this project is to clarify the mechanism of the redoxand CO-dependent transcription by NPAS2. By using resonance Raman technique, we found that CO can bind to heme located inside NPAS2, which leads to a small conformational change around heme peripheral groups. Such a conformational change around heme is a trigger for the protein whole structural change to modulate the affinity of NPAS2 to the target DNA.
• 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。
• シトクロムcの翻訳後修飾を行うタンパク質システムの作用機構の解明

  文部科学省：科学研究費補助金(若手研究(B))

  Date (from‐to) : 2006 -2007 

  Author : 内田 毅

   

  本研究は、シトクロムcという原核生物から真核生物までほぼすべての生物が有するタンパク質の成熟化を行うタンパク質群であるCcm(Cytochrome c maturation)の作用機構を明らかにすることを目的としている。シトクロムcはミトコンドリアの呼吸鎖における電子伝達タンパク質と機能する他、細胞死であるアポトーシスの引き金となるタンパク質であることから、生成及びその品質管理を理解することは重要であると共に、CcmはC型ヘムを有するタンパク質を大腸菌や無細胞系で発現させる時には必須である、その効率の点で未解明な部分が多く、Ccmの理解はタンパク質工学的にも重要な課題の一つである。本研究の共同研究者であるOxford大学のFerguson教授らの研究により、8個存在するCcmタンパク質の中でもCcmEがヘムをシトクロムcに引き渡し、チオエーテル結合の形成という翻訳後修飾を行うことが生化学的に報告されていた。我々はこの機構を詳細に検討することを試みた。Ferguson教授らは細菌(Hydrogenobacter thermophilus)由来のシトクロムcを用いていたが、今回、ホ乳類であるウマ、及び酵母であるSaccharomyces cerevisiae由来のシトクロムcを用い、翻訳後修飾過程を検討したが、翻訳後修飾は形成されなかった。これは、CcmEによるシトクロムcの選択性を表している。CcmEとこれらのシトクロムcの相互作用を表面プラズモン共鳴装置により解析したが、全く相互作用しなかった。以上のことから、CcmEはシトクロムcのある特定の部位を認識し、ヘムの受け渡しを行っていることがわかった。今後はH. thermophilus由来のシトクロムcの認識部位を明らかにすることを試みる。それにより、CcmEを利用したより広汎なC型ヘムをもつタンパク質の翻訳後修飾につながり、外来タンパク質の発現における有用な情報が得られると期待される。
• Development of Detection Methods of Dynamical Higher Order Structures of Proteins and Its Application to Elucidation of Structure-Function Relations of Proteins.

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

  Date (from‐to) : 2002 -2006 

  Author : KITAGAWA Teizo, MIZUTANI Yasuhisa, UCHIDA Takeshi

   

  Structural biology became a main subject in biophysical chemistry after the human genome problem, and 'Protein 3000' project has been successfully carried out. This project intended to uncover the protein structures with x-ray crystallography and NMR spectroscopy. Thus, the static structures of many proteins have been solved, but dynamical structures remained to be revealed to understand how proteins perform their functions. Our research aimed to provide more detailed structures of active sites of large proteins and also dynamical structures in their functioning state. For this purpose vibrational spectroscopy was adopted. New systems for obtaining time-resolved visible and ultraviolet resonance Raman spectra and microscope FTIR spectra were developed and applied to the following eights subjects; 1) Detection of ultrafast structural change of chromophores by using picosecond time-resolved visible resonance Raman spectroscopy, 2) Detection of fast changes of higher order structures of proteins by using subnanosecond time-resolved ultraviolet resonance Raman spectroscopy, 3) Elucidation of the primary process in protein folding/unfolding by using laser temperature jump technique, 4) Development of a novel spectroscopy for functionally-important protein fluctuation, 5) Elucidation of the primary process in the light-induced DNA repair by DNA photolyases, 6) FTIR microspectroscopic investigation of amyloid fibril structures and a trigger mechanism along its formation, 7) Mechanism of sensing of environment and information transduction to a functional domain by gas sensor heme proteins, 8) Elucidation of mechanism of oxygen activation and proton active transport by heme enzymes. In practice we have constructed several inventive time-resolved resonance Raman systems, which cover a wide ranges of excitation wavelengths (from ultraviolet to near infrared) and time-resolution (from picosecond to second). We applied these systems successfully to basic proteins like hemoglobin and myoglobin and some new proteins like gas sensor proteins. We succeeded in elucidating mechanisms of target discrimination and information transmission in the gas sensor proteins.
• 時間分解紫外共鳴ラマン法によるセンサー蛋白の情報感知・伝達及び機能発現機構の解明

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2002 -2002 

  Author : 北川 禎三, 内田 毅

   

  本研究の実施前に辞退する事になったため本格的な実施ではないが、特別推進研究においても関連課題を実施しているので成果の一部をここに記す。 バシラスという細菌の酵素分子センサーであるHemATという蛋白の共鳴ラマンスペクトルを測定し、酸素分子の特異的認識メカニズムを構造化学的に調べた。すなわち、HemATの鉄-酸素伸縮振動を同位体置換法で帰属し、この振動数が非常に低い事から、鉄に結合した酸素がまわりの蛋白と強く水素結合していると結論した。シグナル伝達部の長さを変えると、強く水素結合した酸素とそうでない酸素の存在比が変化したので、その水素結合が情報伝達に寄与していると考えられた。