Researcher Database

Koichiro Ishimori
Faculty of Science Chemistry Physical Chemistry
Professor

Researcher Profile and Settings

Affiliation

  • Faculty of Science Chemistry Physical Chemistry

Job Title

  • Professor

Degree

  • Ph. D.(Kyoto University)

URL

J-Global ID

Research Interests

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

Research Areas

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

Academic & Professional Experience

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

Education

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

Association Memberships

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

Research Activities

Published Papers

  • Takeshi Uchida, Nobuhiko Dojun, Kazuki Ota, Yukari Sekine, Yuina Nakamura, Sayaka Umetsu, Koichiro Ishimori
    Archives of biochemistry and biophysics 677 108165 - 108165 0003-9861 2019/11/30 [Refereed][Not invited]
     
    HutZ from Vibrio cholerae is a dimeric enzyme that catalyzes degradation of heme. The highly conserved Arg92 residue in the HutZ family is proposed to interact with an iron-bound water molecule in the distal heme pocket. To clarify the specific role of Arg92 in the heme degradation reaction, the residue was substituted with alanine, leucine, histidine or lysine to modulate electrostatic interactions with iron-bound ligand. All four Arg92 mutants reacted with hydrogen peroxide to form verdoheme, a prominent intermediate in the heme degradation process. However, when ascorbic acid was used as an electron source, iron was not released even at pH 6.0 despite a decrease in the Soret band, indicating that non-enzymatic heme degradation occurred. Comparison of the rates of heme reduction, ligand binding and verdoheme formation suggested that proton transfer to the reduced oxyferrous heme, a potential rate-limiting step of heme degradation in HutZ, is hampered by mutation. In our previous study, we found that the increase in the distance between heme and Trp109 from 16 to 18 Å upon lowering the pH from 8.0 to 6.0 leads to activation of ascorbic acid-assisted heme degradation by HutZ. The distance in Arg92 mutants was >19 Å at pH 6.0, suggesting that subunit-subunit interactions at this pH are not suitable for heme degradation, similar to Asp132 and His63 mutants. These results suggest that interactions of Arg92 with heme-bound ligand induce alterations in the distance between subunits, which plays a key role in controlling the heme degradation activity of HutZ.
  • Norifumi Muraki, Chihiro Kitatsuji, Yasunori Okamoto, Takeshi Uchida, Koichiro Ishimori, Shigetoshi Aono
    Chemical communications (Cambridge, England) 55 (92) 13864 - 13867 1359-7345 2019/11/14 [Refereed][Not invited]
     
    The crystal structures of the conserved region domains of HtaA and HtaB, which act as heme binding/transport proteins in the heme uptake machinery in Corynebacterium glutamicum, are determined for the first time. The molecular mechanism of heme transfer among these proteins is proposed based on the spectroscopic and structural analyses.
  • Yu H, Taniguchi M, Uesaka K, Wiseschart A, Pootanakit K, Nishitani Y, Murakami Y, Ishimori K, Miyazaki K, Kitahara K
    Microbiology resource announcements 8 (45) 2019/11 [Refereed][Not invited]
  • Konno S, Namiki T, Ishimori K
    Scientific reports 9 (1) 16654  2019/11 [Refereed][Not invited]
  • Dojun N, Muranishi K, Ishimori K, Uchida T
    Journal of inorganic biochemistry 203 110916  0162-0134 2019/11 [Refereed][Not invited]
  • 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.
  • 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.
  • Nishitani Y, Okutani H, Takeda Y, Uchida T, Iwai K, Ishimori K
    Journal of Inorganic Biochemistry 198 2019 [Refereed][Not invited]
  • Saio T, Ishimori K
    Biochimica et Biophysica Acta - General Subjects 2019 [Refereed][Not invited]
  • Shohei Konno, Kentaro Doi, Koichiro Ishimori
    Biophysics and Physicobiology Biophysical Society of Japan 16 (0) 18  2189-4779 2019 [Refereed][Not invited]
  • Kawagoe S, Nakagawa H, Kumeta H, Ishimori K, Saio T
    The Journal of Biological Chemistry 2018/08 [Refereed][Not invited]
  • 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.
  • Tomohide Saio, Soichiro Kawagoe, Koichiro Ishimori, Charalampos G Kalodimos
    eLife {eLife} Sciences Organisation, Ltd. 7 2050-084X 2018/05 [Refereed][Not invited]
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Misaki Kinoshita, Ju Yaen Kim, Satoshi Kume, Yuxi Lin, K. Hun Mok, Yosky Kataoka, Koichiro Ishimori, Natalia Markova, Genji Kurisu, Toshiharu Hase, Young-Ho Lee
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 482 (4) 909 - 915 0006-291X 2017/01 [Refereed][Not invited]
     
    In spite of a number of studies to characterize ferredoxin (Fd):ferredoxin NADP(+) reductase (FNR) interactions at limited conditions, detailed energetic investigation on how these proteins interact under near physiological conditions and its linkage to FNR activity are still lacking. We herein performed systematic Fd:FNR binding thermodynamics using isothermal titration calorimetry (ITC) at distinct pH (6.0 and 8.0), NaCl concentrations (0-200 mM), and temperatures (19-28 degrees C) for mimicking physiological conditions in chloroplasts. Energetically unfavorable endothermic enthalpy changes were accompanied by Fd:FNR complexation at all conditions. This energetic cost was compensated by favorable entropy changes, balanced by conformational and hydrational entropy. Increases in the NaCl concentration and pH weakened inter protein affinity due to the less contribution of favorable entropy change regardless of energetic gains from enthalpy changes, suggesting that entropy drove complexation and modulated affinity. Effects of temperature on binding thermodynamics were much smaller than those of pH and NaCl. NaCl concentration and pH-dependeht enthalpy and heat capacity changes provided clues for distinct binding modes. Moreover, decreases in the enthalpy level in the Hammond's postulate-based energy landscape implicated kinetic advantages for FNR activity. All these energetic interplays were comprehensively demonstrated by the driving force plot with the enthalpy-entropy compensation which may serve as an energetic buffer against outer stresses. We propose that high affinity at pH 6.0 may be beneficial for protection from proteolysis of Fd and FNR in rest states, and moderate affinity at pH 8.0 and proper NaCl concentrations with smaller endothermic enthalpy changes may contribute to increase FNR activity. (C) 2016 Elsevier Inc. All rights reserved.
  • Kazuo Kobayashi, Megumi Nakagaki, Haruto Ishikawa, Kazuhiro Iwai, Mark R. O'Brian, Koichiro Ishimori
    BIOCHEMISTRY 55 (29) 4047 - 4054 0006-2960 2016/07 [Refereed][Not invited]
     
    The iron response regulator (Irr) protein from Bradyrhizobium japonicum mediates iron-dependent regulation of heme biosynthesis. In degrades in response to heme availability through a process that involves the binding of heme to Cys-29 in the heme regulatory motif (HRM) in the presence of molecular oxygen. In this work, we assessed the dynamics of one-electron reduction of heme-bound Irr by monitoring the formation of transient intermediates by pulse radiolysis. Hydrated electrons generated by pulse radiolysis reduced heme iron-bound Irr, facilitating the binding of molecular oxygen to the heme iron in Irr through an initial intermediate with an absorption maximum at 420 nm. This initial intermediate was converted to a secondary intermediate with an absorption maximum at 425 nm, with a first-order rate constant of 1.0 x 10(4) s(-1). The Cys-29 -> Ala (C29A) mutant of Irr, on the other hand, did not undergo the secondary phase, implying that ligand exchange of Cys-29 for another ligand takes place during the process. Spectral changes during the reduction of the heme-bound Irr revealed that binding of CO to ferrous heme consisted of two phases with k(on) values of 1.3 x 10(5) and 2.5 x 10(4) M-1 s(-1), a finding consistent with the presence of two distinct hemes in Irr. In aerobic solutions, by contrast, oxidation of the ferrous heme to the ferric form was found to be a two-phase process. The C29A mutant was similarly oxidized, but this occurred as a single-phase process. We speculate that a reactive oxygen species essential for degradation of the protein is generated during the oxidation process.
  • 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.
  • Norifumi Muraki, Chihiro Kitatsuji, Mariko Ogura, Takeshi Uchida, Koichiro Ishimori, Shigetoshi Aono
    International journal of molecular sciences 17 (6) 2016/05/27 [Refereed][Not invited]
     
    Corynebacteria contain a heme uptake system encoded in hmuTUV genes, in which HmuT protein acts as a heme binding protein to transport heme to the cognate transporter HmuUV. The crystal structure of HmuT from Corynebacterium glutamicum (CgHmuT) reveals that heme is accommodated in the central cleft with His141 and Tyr240 as the axial ligands and that Tyr240 forms a hydrogen bond with Arg242. In this work, the crystal structures of H141A, Y240A, and R242A mutants were determined to understand the role of these residues for the heme binding of CgHmuT. Overall and heme environmental structures of these mutants were similar to those of the wild type, suggesting that there is little conformational change in the heme-binding cleft during heme transport reaction with binding and the dissociation of heme. A loss of one axial ligand or the hydrogen bonding interaction with Tyr240 resulted in an increase in the redox potential of the heme for CgHmuT to be reduced by dithionite, though the wild type was not reduced under physiological conditions. These results suggest that the heme environmental structure stabilizes the ferric heme binding in CgHmuT, which will be responsible for efficient heme uptake under aerobic conditions where Corynebacteria grow.
  • Norifumi Muraki, Chihiro Kitatsuji, Mariko Ogura, Takeshi Uchida, Koichiro Ishimori, Shigetoshi Aono
    International journal of molecular sciences 17 (6) 2016/05/27 [Refereed][Not invited]
     
    Corynebacteria contain a heme uptake system encoded in hmuTUV genes, in which HmuT protein acts as a heme binding protein to transport heme to the cognate transporter HmuUV. The crystal structure of HmuT from Corynebacterium glutamicum (CgHmuT) reveals that heme is accommodated in the central cleft with His141 and Tyr240 as the axial ligands and that Tyr240 forms a hydrogen bond with Arg242. In this work, the crystal structures of H141A, Y240A, and R242A mutants were determined to understand the role of these residues for the heme binding of CgHmuT. Overall and heme environmental structures of these mutants were similar to those of the wild type, suggesting that there is little conformational change in the heme-binding cleft during heme transport reaction with binding and the dissociation of heme. A loss of one axial ligand or the hydrogen bonding interaction with Tyr240 resulted in an increase in the redox potential of the heme for CgHmuT to be reduced by dithionite, though the wild type was not reduced under physiological conditions. These results suggest that the heme environmental structure stabilizes the ferric heme binding in CgHmuT, which will be responsible for efficient heme uptake under aerobic conditions where Corynebacteria grow.
  • Yasuaki Kabe, Takanori Nakane, Ikko Koike, Tatsuya Yamamoto, Yuki Sugiura, Erisa Harada, Kenji Sugase, Tatsuro Shimamura, Mitsuyo Ohmura, Kazumi Muraoka, Ayumi Yamamoto, Takeshi Uchida, So Iwata, Yuki Yamaguchi, Elena Krayukhina, Masanori Noda, Hiroshi Handa, Koichiro Ishimori, Susumu Uchiyama, Takuya Kobayashi, Makoto Suematsu
    NATURE COMMUNICATIONS 7 2041-1723 2016/03 [Refereed][Not invited]
     
    Progesterone-receptor membrane component 1 (PGRMC1/Sigma-2 receptor) is a haem-containing protein that interacts with epidermal growth factor receptor (EGFR) and cytochromes P450 to regulate cancer proliferation and chemoresistance; its structural basis remains unknown. Here crystallographic analyses of the PGRMC1 cytosolic domain at 1.95 angstrom resolution reveal that it forms a stable dimer through stacking interactions of two protruding haem molecules. The haem iron is five-coordinated by Tyr113, and the open surface of the haem mediates dimerization. Carbon monoxide (CO) interferes with PGRMC1 dimerization by binding to the sixth coordination site of the haem. Haem-mediated PGRMC1 dimerization is required for interactions with EGFR and cytochromes P450, cancer proliferation and chemoresistance against anti-cancer drugs; these events are attenuated by either CO or haem deprivation in cancer cells. This study demonstrates protein dimerization via haem-haem stacking, which has not been seen in eukaryotes, and provides insights into its functional significance in cancer.
  • Yuxi Lin, Jozsef Kardos, Mizue Imai, Tatsuya Ikenoue, Misaki Kinoshita, Toshihiko Sugiki, Koichiro Ishimori, Yuji Goto, Young-Ho Lee
    LANGMUIR 32 (8) 2010 - 2022 0743-7463 2016/03 [Refereed][Not invited]
     
    Despite extensive studies on the folding and function of cytochrome c, the mechanisms underlying its aggregation remain largely unknown. We herein examined the aggregation behavior of the physiologically relevant two types of cytochrome c, metal-bound cytochrome c, and its fragment with high amyloidogenicity as predicted in alcohol/water mixtures. Although the aggregation propensity of holo cytochrome c was low due to high solubility, markedly unfolded apo cytochrome c, lacking the heme prosthetic group, strongly promoted the propensity for amorphous aggregation with increases in hydrophobicity. Silver-bound apo cytochrome c increased the capacity of fibrillar aggregation (i.e., protofibrils or immature fibrils) due to subtle structural changes of apo cytochrome c by strong binding of silver. However, mature amyloid fibrils were not detected for any of the cytochrome c variants or its fragment, even with extensive ultrasonication, which is a powerful amyloid inducer. These results revealed the intrinsically low amyloidogenicity of cytochrome c, which is beneficial for its homeostasis and function by facilitating the folding and minimizing irreversible amyloid formation. We propose that intrinsically low amyloidogenicity of cytochrome c is attributed to the low metastability of supersaturation. The phase diagram constructed based on solubility and aggregate type is useful for a comprehensive understanding of protein aggregation. Furthermore, amorphous aggregation, which is also viewed as a generic property of proteins, and amyloid fibrillation can be distinguished from each other by the metastability of supersaturation.
  • 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.
  • Yoshiaki Furukawa, Itsuki Anzai, Shuji Akiyama, Mizue Imai, Fatima Joy C. Cruz, Tomohide Saio, Kenichi Nagasawa, Takao Nomura, Koichiro Ishimori
    JOURNAL OF BIOLOGICAL CHEMISTRY 291 (8) 4144 - 4155 0021-9258 2016/02 [Refereed][Not invited]
     
    Misfolding of Cu,Zn-superoxide dismutase (SOD1) is a pathological change in the familial form of amyotrophic lateral sclerosis caused by mutations in the SOD1 gene. SOD1 is an enzyme that matures through the binding of copper and zinc ions and the formation of an intramolecular disulfide bond. Pathogenic mutations are proposed to retard the post-translational maturation, decrease the structural stability, and hence trigger the misfolding of SOD1 proteins. Despite this, a misfolded and potentially pathogenic conformation of immature SOD1 remains obscure. Here, we show significant and distinct conformational changes of apoSOD1 that occur only upon reduction of the intramolecular disulfide bond in solution. In particular, loop regions in SOD1 lose their restraint and become significantly disordered upon dissociation of metal ions and reduction of the disulfide bond. Such drastic changes in the solution structure of SOD1 may trigger misfolding and fibrillar aggregation observed as pathological changes in the familial form of amyotrophic lateral sclerosis.
  • 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.
  • Mizue Imai, Tomohide Saio, Hiroyuki Kumeta, Takeshi Uchida, Fuyuhiko Inagaki, Koichiro Ishimori
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 469 (4) 978 - 984 0006-291X 2016/01 [Refereed][Not invited]
     
    Redox-dependent changes in the structure and dynamics of human cytochrome c (Cyt c) were investigated by solution NMR. We found significant structural changes in several regions, including residues 23-28 (loop 3), which were further corroborated by chemical shift differences between the reduced and oxidized states of Cyt c. These differences are essential for discriminating redox states in Cyt c by cytochrome c oxidase (CcO) during electron transfer reactions. Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments identified that the region around His33 undergoes conformational exchanges on the mu s-ms timescale, indicating significant redox-dependent structural changes. Because His33 is not part of the interaction site for CcO, our data suggest that the dynamic properties of the region, which is far from the interaction site for CcO, contribute to conformational changes during electron transfer to CcO. (C) 2015 Elsevier Inc. All rights reserved.
  • 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.
  • Koichiro Ishimori, Yuta Watanabe
    CHEMISTRY LETTERS 43 (11) 1680 - 1689 0366-7022 2014/11 [Refereed][Not invited]
     
    Heme is a typical and common prosthetic group for various types of proteins and is utilized for active centers in many biologically important processes in vivo. However, heme has also been shown to function as a signaling molecule that regulates the functions of proteins, suggesting that novel signaling cascades are mediated by heme. We focused on the spectroscopic characterization of such "heme-regulated" proteins, showing unique heme environmental structures.
  • 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.
  • 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.
  • Shin-Ichi Ozaki, Takehiro Sato, Yukari Sekine, Catharina T Migita, Takeshi Uchida, Koichiro Ishimori
    Journal of inorganic biochemistry 138 31 - 38 2014/09 [Refereed][Not invited]
     
    Heme acquisition system A (HasA) is known as a hemophore in Gram-negative pathogens. The ferric heme iron is coordinated by Tyr-75 and His-32 in holo-HasA from Pseudomonas aeruginosa (HasApa). In contrast, in holo-HasA from Yersinia pseudotuberculosis (HasAyp), our spectroscopic studies suggest that only Tyr-75 coordinates to the ferric heme iron. The substitution of Gln-32 with alanine in HasAyp does not alter the spectroscopic properties, indicating that Gln-32 is not an axial ligand for the heme iron. Somewhat surprisingly, the Y75A mutant of HasAyp can capture a free hemin molecule but the rate of hemin uptake is slower than that of wild type, suggesting that the hydrophobic interaction in the heme pocket may also play a role in heme acquisition. Unlike in wild type apoprotein, ferric heme transfer from Hb to Y75A apo-HasAyp has not been observed. These results imply that coordination (bonding/interaction) between Tyr-75 and the heme iron is important for heme transfer from Hb. Interestingly, HasAyp differs from HasApa in its ability to bind the ferrous heme iron. Apo-HasAyp can capture ferrous heme and resonance Raman spectra of ferrous-carbon monoxide holo-HasAyp suggest that Tyr-75 is protonated when the heme iron is in the ferrous state. The ability of HasAyp to acquire the ferrous heme iron might be beneficial to Y. pseudotuberculosis, a facultative anaerobe in the Enterobacteriaceae family.
  • Takanori Uzawa, Takashi Isoshima, Yoshihiro Ito, Koichiro Ishimori, Dmitrii E. Makarov, Kevin W. Plaxco
    Biophysical Journal 104 (11) 2485 - 2492 0006-3495 2013/06/04 [Refereed][Not invited]
     
    Intramolecular collision dynamics play an essential role in biomolecular folding and function and, increasingly, in the performance of biomimetic technologies. To date, however, the quantitative studies of dynamics of single-stranded nucleic acids have been limited. Thus motivated, here we investigate the sequence composition, chain-length, viscosity, and temperature dependencies of the end-to-end collision dynamics of single-stranded DNAs. We find that both the absolute collision rate and the temperature dependencies of these dynamics are base-composition dependent, suggesting that base stacking interactions are a significant contributor. For example, whereas the end-to-end collision dynamics of poly-thymine exhibit simple, linear Arrhenius behavior, the behavior of longer poly-adenine constructs is more complicated. Specifically, 20- and 25-adenine constructs exhibit biphasic temperature dependencies, with their temperature dependences becoming effectively indistinguishable from that of poly-thymine above 335 K for 20-adenines and 328 K for 25-adenines. The differing Arrhenius behaviors of poly-thymine and poly-adenine and the chain-length dependence of the temperature at which poly-adenine crosses over to behave like poly-thymine can be explained by a barrier friction mechanism in which, at low temperatures, the energy barrier for the local rearrangement of poly-adenine becomes the dominant contributor to its end-to-end collision dynamics. © 2013 Biophysical Society.
  • Takeshi Uchida, Yukari Sekine, Toshitaka Matsui, Masao Ikeda-Saito, Koichiro Ishimori
    Chemical communications (Cambridge, England) 48 (53) 6741 - 3 1359-7345 2012/07/07 [Refereed][Invited]
     
    HutZ, one of the crucial proteins of the iron uptake system in Vibrio cholerae, was purified, which binds to heme at a stoichiometry of 1 : 1. In the presence of ascorbic acid, the HutZ-bound heme degrades via the same intermediates observed in heme oxygenase, suggesting that HutZ works as a heme degradation enzyme.
  • Takeshi Uchida, Ikuko Sagami, Toru Shimizu, Koichiro Ishimori, Teizo Kitagawa
    JOURNAL OF INORGANIC BIOCHEMISTRY 108 188 - 195 0162-0134 2012/03 [Refereed][Invited]
     
    Neuronal PAS domain protein 2 (NPAS2), which is a CO-dependent transcription factor, consists of a basic helix-loop-helix domain (bHLH), and two heme-containing PAS domains (PAS-A and PAS-B). In our previous study on the isolated PAS-A domain, we concluded that His119 and Cys170 are the axial ligands of the ferric heme, while Cys170 is replaced by His171 upon reduction of heme (Uchida et al., J. Biol. Chem. 270, (2005) 21358-21368.). Recently, we characterized the PAS-A domain combined with the N-terminal bHLH domain, and found that some spectroscopic features were different from those of the isolated PAS-A domain (Mukaiyama et al., FEBS J. 273, (2006) 2528-2539.). Therefore, we reinvestigated the coordination structure of heme in the bHLH-PAS-A domain and prepared four histidine and one cysteine mutants. Resonance Raman spectrum of the Cys170Ala mutant is the same as that of wild type with a dominant 6-coordinate heme in the ferric form. In contrast, His119Ala and His171Ala mutants significantly increase amounts of the 5-coordinate species, indicating that His119 and His171, not Cys170, are axial ligands of the ferric heme in the bHLH-PAS-A domain. We had confirmed that the coordination structure of the isolated PAS-A domain is in equilibrium between Cys-Fe-His and His-Fe-His coordinated species but newly found that interaction of the PAS-A domain with the bHLH domain shifts the equilibrium toward the latter structure. Such flexibility in the heme coordination structure seems to be in favor of signal transduction in NPAS2. (C). 2011 Elsevier Inc. All rights reserved.
  • 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]
  • Kenichi Kitanishi, Kazuo Kobayashi, Takeshi Uchida, Koichiro Ishimori, Jotaro Igarashi, Toru Shimizu
    JOURNAL OF BIOLOGICAL CHEMISTRY 286 (41) 35522 - 35534 0021-9258 2011/10 [Refereed][Not invited]
     
    Two-component signal transduction systems regulate numerous important physiological functions in bacteria. In this study we have identified, cloned, overexpressed, and characterized a dimeric full-length heme-bound (heme: protein, 1: 1 stoichiometry) globin-coupled histidine kinase (AfGcHK) from Anaeromyxobacter sp. strain Fw109-5 for the first time. The Fe(III), Fe(II)-O(2), and Fe(II)-CO complexes of the protein displayed autophosphorylation activity, whereas the Fe(II) complex had no significant activity. A H99A mutant lost heme binding ability, suggesting that this residue is the heme proximal ligand. Moreover, His-183 was proposed as the autophosphorylation site based on the finding that the H183A mutant protein was not phosphorylated. The phosphate group of autophosphorylated AfGcHK was transferred to Asp-52 and Asp-169 of a response regulator, as confirmed from site-directed mutagenesis experiments. Based on the amino acid sequences and crystal structures of other globin-coupled oxygen sensor enzymes, Tyr-45 was assumed to be the O(2) binding site at the heme distal side. The O(2) dissociation rate constant, 0.10 s(-1), was substantially increased up to 8.0 s(-1) upon Y45L mutation. The resonance Raman frequencies representing nu(Fe-O2) (559 cm(-1)) and nu(O-O) (1149 cm(-1)) of the Fe(II)-O(2) complex of Y45F mutant AfGcHK were distinct from those of the wildtype protein (nu(Fe-O2), 557 cm(-1); nu(O-O), 1141 cm(-1)), supporting the proposal that Tyr-45 is located at the distal side and forms hydrogen bonds with the oxygen molecule bound to the Fe(II) complex. Thus, we have successfully identified and characterized a novel heme-based globin-coupled oxygen sensor histidine kinase, AfGcHK, in this study.
  • 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.
  • Takao Nomura, Rui Kamada, Issaku Ito, Koichi Sakamoto, Yoshiro Chuman, Koichiro Ishimori, Yasuyuki Shimohigashi, Kazuyasu Sakaguchi
    BIOPOLYMERS 95 (6) 410 - 419 0006-3525 2011/06 [Refereed][Not invited]
     
    Stabilization of protein structures and protein protein interactions are critical in the engineering of industrially useful enzymes and in the design of pharmaceutically valuable ligands. Hydrophobic interactions involving phenylalanine residues play crucial roles in protein stability and protein-protein/peptide interactions. To establish an effective method to explore the hydrophobic environments of phenylalanine residues, we present a strategy that uses pentafluorophenylalanine (F(5)Phe) and cyclohexylalanine (Cha). In this study, substitution of F(5)Phe or Cha for three Phe residues at positions 328, 338, and 341 in the tetramerization domain of the tumor suppressor protein p53 was performed. These residues are located at the interfaces of p53 p53 interactions and are important in the stabilization of the tetrameric structure. The stability of the p53 tetrameric structure did not change significantly when F(5)Phe-containing peptides at positions Phe328 or Phe338 were used. In contrast, the substitution of Cha for Phe341 in the hydrophobic core enhanced the stability of the tetrameric structure with a T(m) value of similar to 100 degrees C. Phe328 and Phe338 interact with each other through pi-interactions, whereas Phe341 is buried in the surrounding alkyl side-chains of the hydrophobic core of the p53 tetramerization domain. Furthermore, high pressure-assisted denaturation analysis indicated improvement in the occupancy of the hydrophobic core. Considerable stabilization of the p53 tetramer was achieved by filling the identified cavity in the hydrophobic core of the p.5.3 tetramer. The results indicate the status of the Phe residues, indicating that the "pair substitution" of Cha and F(5)Phe is highly suitable for probing the environments of Phe residues. (C) 2011 Wiley Periodicals, Inc. Biopolymers 95: 410-419, 2011.
  • 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.
  • 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.
  • 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.
  • Yasuyuki Tsuboi, Tatsuya Shoji, Masayuki Nishino, Seiji Masuda, Koichiro Ishimori, Noboru Kitamura
    APPLIED SURFACE SCIENCE 255 (24) 9906 - 9908 0169-4332 2009/09 [Refereed][Not invited]
     
    Optical trapping of lysozyme, cytochrome c, or myoglobin based on photon pressure generated by focusing 1064 nm laser beam in an aqueous solution was explored. For all the proteins, microparticle formation was observed at the focal point under an optical microscope. Furthermore, the microparticles were identified to the molecular assemblies of the corresponding protein by means of confocal Raman microspectroscopy. For lysozyme, molecular clusters in solution were optically trapped to form the microparticle and it took more than 1 h to produce the microparticle. By contrast, molecular assembling proceeded within 1 min for cytochrome c and myoglobin. Since heme in cytochrome c or myoglobin would have a high polarizability, that would contribute to rapid assembling of the protein. Thus we demonstrated that a focused laser beam was a powerful tool to manipulate protein molecules in solution. (C) 2009 Elsevier B.V. All rights reserved.
  • Takanori Uzawa, Chiaki Nishimura, Shuji Akiyama, Koichiro Ishimori, Satoshi Takahashi, H. Jane Dyson, Peter E. Wright
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 105 (37) 13859 - 13864 0027-8424 2008/09 [Refereed][Not invited]
     
    The earliest steps in the folding of proteins are complete on an extremely rapid time scale that is difficult to access experimentally. We have used rapid-mixing quench-flow methods to extend the time resolution of folding studies on apomyoglobin and elucidate the structural and dynamic features of members of the ensemble of intermediate states that are populated on a submillisecond time scale during this process. The picture that emerges is of a continuum of rapidly interconverting states. Even after only 0.4 ms of refolding time a compact state is formed that contains major parts of the A, G, and H helices, which are sufficiently well folded to protect amides from exchange. The B, C, and E helix regions fold more slowly and fluctuate rapidly between open and closed states as they search docking sites on this core; the secondary structure in these regions becomes stabilized as the refolding time is increased from 0.4 to 6 ms. No further stabilization occurs in the A, G, H core at 6 ms of folding time. These studies begin to time-resolve a progression of compact states between the fully unfolded and native folded states and confirm the presence an ensemble of intermediates that interconvert in a hierarchical sequence as the protein searches conformational space on its folding trajectory.
  • Tetsunari Kimura, Akio Maeda, Shingo Nishiguchi, Koichiro Ishimori, Isao Morishima, Takashi Konno, Yuji Goto, Satoshi Takahashi
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 105 (36) 13391 - 13396 0027-8424 2008/09 [Refereed][Not invited]
     
    Kinetic IR spectroscopy was used to reveal beta-sheet formation and water expulsion in the folding of single-chain monellin (SMN) composed of a five-stranded beta-sheet and an alpha-helix. The time-resolved IR spectra between 100 mu s and 10 s were analyzed based on two consecutive intermediates, I(1) and I(2), appearing within 100 mu s and with a time constant of approximate to 100 ms, respectively. The initial unfolded state showed broad amide I' corresponded to a fluctuating conformation. In contrast, I(1) possessed a feature at 1,636 cm(-1) for solvated helix and weak features assignable to turns, demonstrating the rapid formation of helix and turns. I(2) possessed a line for solvated helix at 1,637 cm(-1) and major and minor lines for beta-sheet at 1,625 and 1,680 cm(-1), respectively. The splitting of the major and minor lines is smaller than that of the native state, implying an incomplete formation of the beta-sheet. Furthermore, both major and minor lines demonstrated a low-frequency shift compared to those of the native state, which was interpreted to be caused by hydration of the C=O group in the beta-sheet. Together with the identification of solvated helix, the core domain of I(2) was interpreted as being hydrated. Finally, slow conversion of the water-penetrated core of I(2) to the dehydrated core of the native state was observed. We propose that both the expulsion of water, hydrogen-bonded to main-chain amides, and the completion of the secondary structure formation contribute to the energetic barrier of the rate-limiting step in SMN folding.
  • 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.
  • 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.
  • Chihiro Kitatsuji, Masaki Kurogochi, Shin-Ichiro Nishimura, Koichiro Ishimori, Keisuke Wakasugi
    JOURNAL OF MOLECULAR BIOLOGY 368 (1) 150 - 160 0022-2836 2007/04 [Refereed][Not invited]
     
    Oxidized human neuroglobin (Ngb), a heme protein expressed in the brain, has been proposed to act as a guanine nucleotide dissociation inhibitor (GDI) for the GDP-bound form of the heterotrimeric G protein alpha-subunit (G(xi). Here, to elucidate the molecular mechanism underlying the GD1 activity of Ngb, we used an glutathione-S-transferase pun-down assay to confirm that Ngb competes with G-protein beta gamma-subunits (G beta gamma) for binding to G alpha(i), and identified the G alpha(i)-binding site in Ngb by chemical cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and sulfo-N-hydroxysuccinimide, coupled with mass spectrometry (MS). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis for tryptic peptides derived from the cross-linked Ngb-G alpha(i) complex revealed several binding regions in Ngb. Furthermore, MALDI-TOF/TOF MS analysis of the cross-linked Ngb and Gai peptides, together with the MS/MS scoring method, predicted cross-linking between Glu60 (Ngb) and Ser206 (G alpha(i)), and between Glu53 (Ngb) and Ser44 (G alpha(i)). Because Ser206 of G alpha(i) is located in the region that contacts G beta gamma, binding of Ngb could facilitate the release of G beta gamma from G alpha(i). Binding of Ngb to Gai would also inhibit the exchange of GDP for GTP, because Ser44 (G alpha(i)) is adjacent to the GDP-binding site and Glu53 (Ngb), which is cross-linked to Ser44 (G alpha(i)), could be located close to GDP. Thus, we have identified, for the first time, the sites of interaction between Ngb and Gai, enabling us to discuss the functional significance of this binding on the GDI activity of Ngb. (c) 2007 Elsevier Ltd. All rights reserved.
  • Koichiro Ishimori
    Review of High Pressure Science and Technology/Koatsuryoku No Kagaku To Gijutsu 17 (1) 13 - 22 1348-1940 2007 [Refereed][Not invited]
     
    By using spectroscopies under high pressure, we determined the volume changes associated with protein folding of reduced cytochrome c from the unfolded state to the native state. The pressure dependence of the equilibrium constant for the denaturation and the folding rate revealed that the volume change for the protein folding and the activation volume for the native state are negative. Such negative volumes can be accounted for by a decrease in volume resulting from the dehydration of hydrophobic groups, primarily the heme group, and the dehydration is mainly induced in the formation of the transition for the native state. We, therefore, propose that dehydration can compensate for the decreased entropy in the formation of protein structures, entropically promoting the protein folding reactions.
  • T Uzawa, T Kimura, K Ishimori, Morishima, I, T Matsui, M Ikeda-Saito, S Takahashi, S Akiyama, T Fujisawa
    JOURNAL OF MOLECULAR BIOLOGY 357 (3) 997 - 1008 0022-2836 2006/03 [Refereed][Not invited]
     
    Polypeptide collapse is generally observed as the initial folding dynamics of proteins with more than 100 residues, and is suggested to be caused by the coil-globule transition explained by Flory's theory of polymers. To support the suggestion by establishing a scaling behavior between radius of gyration (R-g) and chain length for the initial folding intermediates, the folding dynamics of heme oxygenase (HO) was characterized by time-resolved, small-angle X-ray scattering. HO is a highly helical protein without disulfide bridges, and is the largest protein (263 residues) characterized by the method. The folding process of HO was found to contain a transient oligomerization; however, the conformation within 10 ms was demonstrated to be monomeric and to possess R-g of 26.1(+/- 1.1) angstrom. Together with the corresponding data for proteins with different chain lengths, the seven R-g values demonstrated the scaling relationship to chain length with a scaling exponent of 0.35 +/- 0.11, which is close to the theoretical value of 1/3 predicted for globules in solutions where monomer-monomer interactions are favored over monomer-solvent interactions (poor solvent). The finding indicated that the initial folding dynamics of proteins bears the signature of the coil-globule transition, and offers a clue to explain the folding mechanisms of proteins with different chain lengths. (c) 2006 Elsevier Ltd. All rights reserved.
  • T Kimura, K Sakamoto, Morishima, I, K Ishimori
    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 128 (3) 670 - 671 0002-7863 2006/01 [Refereed][Not invited]
  • M Shintaku, K Matsuura, S Yoshioka, S Takahashi, K Ishimori, Morishima, I
    JOURNAL OF BIOLOGICAL CHEMISTRY 280 (49) 40934 - 40938 0021-9258 2005/12 [Refereed][Not invited]
     
    A microsecond-resolved absorption spectrometer was developed to investigate the elementary steps in hydrogen peroxide (H2O2) activation reaction of horseradish peroxidase (HRP) at ambient temperature. The kinetic absorption spectra of HRP upon the mixing with various concentrations of H2O2 (0.5-3 mM) were monitored in the time range from 50 to 300 mu s. The time-resolved spectra in the Soret region possessed isosbestic points that were close to those between the resting state and compound I. The kinetic changes in the Soret absorbance could be well fitted by a single exponential function. Accordingly, no distinct spectrum of the putative intermediate between the resting state and compound I was identified. These results were consistent with the proposal that the O-O bond activation in heme peroxidases is promoted by the imidazolium form of the distal histidine that exists only transiently. It was estimated that the rate constant for the breakage of the O-O bond in H2O2 by HRP is significantly faster than 1 x 10(4) s(-1).
  • H Ishikawa, M Kato, H Hori, K Ishimori, T Kirisako, F Tokunaga, K Iwai
    MOLECULAR CELL 19 (2) 171 - 181 1097-2765 2005/07 [Refereed][Not invited]
     
    yIron regulatory protein 2 (IRP2), a regulator of iron metabolism, is modulated by ubiquitination and degradation. We have shown that IRP2 degradation is triggered by heme-mediated oxidation. We report here that not only Cys201, an invariant residue in the heme regulatory motif (HRM), but also His204 is critical for IRP2 degradation. Spectroscopic studies revealed that Cys201 binds ferric heme, whereas His204 is a ferrous heme binding site, indicating the involvement of these residues in sensing the redox state of the heme iron and in generating the oxidative modification. Moreover, the HRM in IRP2 has been suggested to play a critical role in its recognition by the HOIL-1 ubiquitin ligase. Although HRMs are known to sense heme concentration by simply binding to heme, the HRM in IRP2 specifically contributes to its oxidative modification, its recognition by the ligase, and its sensing of iron concentration after iron is integrated into heme.
  • T Kimura, S Akiyama, T Uzawa, K Ishimori, Morishima, I, T Fujisawa, S Takahashi
    JOURNAL OF MOLECULAR BIOLOGY 350 (2) 349 - 362 0022-2836 2005/07 [Refereed][Not invited]
     
    Nature of the burst-phase signals of protein folding has been the subject of much debate as to whether the signals represent the formation of early intermediates or the non-specific collapse of unfolded polypeptides. To distinguish the two possibilities, the submillisecond folding dynamics of ribonuclease A (RNase A) was examined, and compared with those of the disulfide bond-ruptured analog of RNase A (r-RNase A). The circular dichroism measurements on RNase A showed the burst-phase signal within 320 mu s after the initiation of the folding reaction, which was identical to that observed for r-RNase A. In contrast, the burst phase increase in the extrinsic fluorescence from 1-anilino-8-naphthalene sulfonate (ANS) was observed for RNase A but not for r-RNase A. The kinetic titration experiment of the ANS fluorescence intensity showed the presence of a specific binding site for ANS in the fast-refolding component of RNase A. The small-angle X-ray scattering measurements at similar to 22 ms after initiating the folding reaction demonstrated that the burst phase conformations of the medium and slow-refolding components of RNase A were distinctly smaller than that of r-RNase A. These results indicated the difference in the burst phase conformations of RNase A and r-RNase A. Since r-RNase A is denatured in the physiological solution condition, the burst-phase signal of RNase A was interpreted as the formation of the folding intermediate with specific conformations. (c) 2005 Elsevier Ltd. All rights reserved.
  • JH Yang, K Ishimori, MR O'Brian
    JOURNAL OF BIOLOGICAL CHEMISTRY 280 (9) 7671 - 7676 0021-9258 2005/03 [Refereed][Not invited]
     
    The iron response regulator (Irr) protein from Bradyrhizobium japonicum is a conditionally stable protein that degrades in response to cellular iron availability. This turnover is heme-dependent, and rapid degradation involves heme binding to a heme regulatory motif (HRM) of Irr. Here, we show that Irr confers iron-dependent instability on glutathione S-transferase (GST) when fused to it. Analysis of Irr-GST derivatives with C-terminal truncations of Irr implicated a second region necessary for degradation, other than the HRM, and showed that the HRM was not sufficient to confer instability on GST. The HRM-defective mutant IrrC29A degraded in the presence of iron but much more slowly than the wild-type protein. This slow turnover was heme-dependent, as discerned by the stability of Irr in a heme-defective mutant strain. Whereas the HRM of purified recombinant Irr binds ferric ( oxidized) heme, a second site that binds ferrous ( reduced) heme was identified based on spectral analysis of truncation and substitution mutants. A mutant in which histidines 117 - 119 were changed to alanines severely diminished ferrous, but not ferric, heme binding. Introduction of these substitutions in an Irr-GST fusion stabilized the protein in vivo in the presence of iron. We conclude that normal iron-dependent Irr degradation involves two heme binding sites and that both redox states of heme are required for rapid turnover.
  • K Matsuura, S Yoshioka, T Tosha, H Hori, K Ishimori, T Kitagawa, Morishima, I, N Kagawa, MR Waterman
    JOURNAL OF BIOLOGICAL CHEMISTRY 280 (10) 9088 - 9096 0021-9258 2005/03 [Refereed][Not invited]
     
    To gain insights into the molecular basis of the design for the selective azole anti-fungals, we compared the binding properties of azole-based inhibitors for cytochrome P450 sterol 14alpha-demethylase (CYP51) from human (HuCYP51) and Mycobacterium tuberculosis (MtCYP51). Spectroscopic titration of azoles to the CYP51s revealed that HuCYP51 has higher affinity for ketoconazole (KET), an azole derivative that has long lipophilic groups, than MtCYP51, but the affinity for fluconazole (FLU), which is a member of the anti-fungal armamentarium, was lower in HuCYP51. The affinity for 4-phenylimidazole (4-PhIm) to MtCYP51 was quite low compared with that to HuCYP51. In the resonance Raman spectra for HuCYP51, the FLU binding induced only minor spectral changes, whereas the prominent high frequency shift of the bending mode of the heme vinyl group was detected in the KET- or 4-PhIm-bound forms. On the other hand, the bending mode of the heme propionate group for the FLU-bound form of MtCYP51 was shifted to high frequency as found for the KET- bound form, but that for 4-PhIm was shifted to low frequency. The EPR spectra for 4-PhIm-bound MtCYP51 and FLU-bound HuCYP51 gave multiple g values, showing heterogeneous binding of the azoles, whereas the single g(x) and g(z) values were observed for other azole-bound forms. Together with the alignment of the amino acid sequence, these spectroscopic differences suggest that the region between the B' and C helices, particularly the hydrophobicity of the C helix, in CYP51s plays primary roles in determining strength of interactions with azoles; this differentiates the binding specificity of azoles to CYP51s.
  • Y Furukawa, T Ban, D Hamada, K Ishimori, Y Goto, Morishima, I
    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 127 (7) 2098 - 2103 0002-7863 2005/02 [Refereed][Not invited]
     
    To observe an electron transfer (ET) process in a single protein molecule, we constructed a model system, Alexa-HCytb(5), in which cytochrome b(5) (Cytb(5)) is modified with a fluorescent probe, Alexa. Fluor 647 dye. In this model system, intramolecular transfer of an electron from the Alexa dye to heme in Cytb(5) is supposed to oxidize the probe and quench its fluorescence, and the ET reaction at the single-molecule level can be monitored as the intermittent change in the fluorescence intensity. Alexa-HCytb(5) was fixed on the glass surface, and illumination of laser light by the total internal reflection resulted in blinking of the fluorescence from the single Alexa-HCytb(5) molecule in the time scale of several hundred milliseconds. Each Alexa-HCytb(5) molecule is characterized by its own rate constant of the blinking, corresponding to the ET rate constant at the single-molecule level, and its variation ranges between 1 and 10 s(-1). The current system thus enables us to visualize the ET reaction in the single protein molecule, and the protein ET reaction was found to be explained by the distribution of the rate constants. On the basis of the Marcus theory, we suggest that the origin of this rate distribution is the distance change associated with the structural fluctuation in the protein molecule.
  • T Kimura, T Uzawa, K Ishimori, Morishima, I, S Takahashi, T Konno, S Akiyama, T Fujisawa
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 102 (8) 2748 - 2753 0027-8424 2005/02 [Refereed][Not invited]
     
    Characterization of the conformational landscapes for proteins with different secondary structures is important in elucidating the mechanism of protein folding. The folding trajectory of single-chain monellin composed of a five-stranded beta-sheet and a helix was investigated by using a pH-jump from the alkaline unfolded to native state. The kinetic changes in the secondary structures and in the overall size and shape were measured by circular dichroism spectroscopy and small-angle x-ray scattering, respectively. The formation of the tertiary structure was monitored by intrinsic and extrinsic fluorescence. A significant collapse was observed within 300 mus after the pH-jump, leading to the intermediate with a small amount of secondary and tertiary structures but with an overall oblate shape. Subsequently, the stepwise formation of secondary and tertiary structures was detected. The current observation was consistent with the theoretical prediction that a more significant collapse precedes the formation of secondary structures in the folding of beta-sheet proteins than that of helical proteins [Shea, J. E., Onuchic, J. N. & Brooks, C. L., III (2002) Proc. Nati. Acad. Sci. USA 99, 16064-16068]. Furthermore, it was implied that the initial collapse was promoted by the formation of some specific structural elements, such as tight turns, to form the oblate shape.
  • Takehiko TOSHA, Koichiro ISHIMORI, Isao MORISHIMA
    Seibutsu Butsuri Biophysical Society of Japan 45 (2) 78  2005 [Refereed][Not invited]
  • H Ishikawa, S Takahashi, K Ishimori, Morishima, I
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 324 (3) 1095 - 1100 0006-291X 2004/11 [Refereed][Not invited]
     
    Structural factors to regulate the heme reorientation reaction in myoglobin were examined and we found that the side chain at position 107 (Ile107), which is located between the 2-vinyl and 3-methyl groups of heme, forms a kinetic barrier for the heme rotation about the alpha-gamma axis. The phenylalanine-substituted mutant showed an extremely slow heme reorientation rate, compared to that of the wild-type protein, while replacement by the decreased side chain, valine, at position 107 accelerated the reorientation reaction. Considering that the spectroscopic data show only minor structural changes in the heme environments of the Ile107 mutants, the side chain at position 107 sterically interacts with the heme peripheral groups in the activation state for the heme reorientation, which supports the intramolecular mechanism that the heme rotates about the alpha-gamma axis without leaving the "protein cage." (C) 2004 Elsevier Inc. All rights reserved.
  • K Matsuura, T Tosha, S Yoshioka, S Takahashi, K Ishimori, Morishima, I
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 323 (4) 1209 - 1215 0006-291X 2004/10 [Refereed][Not invited]
     
    To elucidate molecular mechanisms for the enhanced oxygenation activity in the three mutants of cytochrome P450cam screened by 'laboratory evolution' [Nature 399 (1999) 670], we purified the mutants and characterized their functional and structural properties. The electronic absorption and resonance Raman spectra revealed that the structures of heme binding site of all purified mutants were quite similar to that of the wild-type enzyme, although the fraction of the inactivated form, called "P420," was increased. In the reaction with H2O2, only trace amounts of the naphthalene hydroxylation product were detected by gas chromatography. We, therefore, conclude that the three mutants do not exhibit significant changes in the structural and functional properties from those of wild-type P450cam except for the stability of the axial ligand in the reduced form. The enhanced fluorescence in the whole-cell assay would reflect enhancement in the oxygenation activity below the detectable limit of the gas chromatography and/or contributions of other reactions catalyzed by the heme iron. (C) 2004 Elsevier Inc. All rights reserved.
  • T Tosha, S Yoshioka, K Ishimori, Morishima, I
    JOURNAL OF BIOLOGICAL CHEMISTRY 279 (41) 42836 - 42843 0021-9258 2004/10 [Refereed][Not invited]
     
    To investigate the functional and structural characterization of a crucial cytochrome P450cam ( P450cam)putidaredoxin (Pdx) complex, we utilized a mutant whose spectroscopic property corresponds to the properties of the wild type P450cam in the presence of Pdx. The H-1 NMR spectrum of the carbonmonoxy adduct of the mutant, the Leu- 358 --> Pro mutant (L358P), in the absence of Pdx showed that the ring current-shifted signals arising from D-camphor were upfield-shifted and observed as resolved signals, which are typical for the wild type enzyme in the presence of Pdx. Signals from the beta-proton of the axial cysteine and the gamma-methyl group of Thr-252 were also shifted upfield and downfield, respectively, in the L358P mutant as observed for Pdx-bound wild type P450cam. The close similarity in the NMR spectra suggests that the heme environment of the L358P mutant mimics that of the Pdx-bound enzyme. The functional analysis of the L358P mutant has revealed that the oxygen adduct of the L358P mutant can promote the oxygenation reaction for D-camphor with nonphysiological electron donors such as dithionite and ascorbic acid, showing that oxygenated L358P is "activated" to receive electron from the donor. Based on the structural and functional characterization of the L358P mutant, we conclude that the Pdx-induced structural changes in P450cam would facilitate the electron transfer from the electron donor, and the Pdx binding to P450cam would be a trigger for the electron transfer to oxygenated P450cam.
  • S Nagano, T Tosha, K Ishimori, Morishima, I, TL Poulos
    JOURNAL OF BIOLOGICAL CHEMISTRY 279 (41) 42844 - 42849 0021-9258 2004/10 [Refereed][Not invited]
     
    The cytochrome P450cam active site is known to be perturbed by binding to its redox partner, putidaredoxin (Pdx). Pdx binding also enhances the camphor monooxygenation reaction (Nagano, S., Shimada, H., Tarumi, A., Hishiki, T., Kimata- Ariga, Y., Egawa, T., Suematsu, M., Park, S.- Y., Adachi, S., Shiro, Y., and Ishimura, Y. ( 2003) Biochemistry 42, 14507 - 14514). These effects are unique to Pdx because nonphysiological electron donors are unable to support camphor monooxygenation. The accompanying H-1 NMR paper ( Tosha, T., Yoshioka, S., Ishimori, K., and Morishima, I. ( 2004) J. Biol. Chem. 279, 42836 - 42843) shows that the conformation of active site residues, Thr-252 and Cys-357, and the substrate in the ferrous ( Fe(II)) CO complex of the L358P mutant mimics that of the wild-type enzyme complexed to Pdx. To explore how these changes are transmitted from the Pdx-binding site to the active site, we have solved the crystal structures of the ferrous and ferrous-CO complex of wild-type and the L358P mutant. Comparison of these structures shows that the L358P mutation results in the movement of Arg-112, a residue known to be important for putidaredoxin binding, toward the heme. This change could optimize the Pdx-binding site leading to a higher affinity for Pdx. The mutation also pushes the heme toward the substrate and ligand binding pocket, which relocates the substrate to a position favorable for regio-selective hydroxylation. The camphor is held more firmly in place as indicated by a lower average temperature factor. Residues involved in the catalytically important proton shuttle system in the I helix are also altered by the mutation. Such conformational alterations and the enhanced reactivity of the mutant oxy complex with nonphysiological electron donors suggest that Pdx binding optimizes the distal pocket for monooxygenation of camphor.
  • S Akiyama, T Fujisawa, K Ishimori, Morishima, I, S Aon
    JOURNAL OF MOLECULAR BIOLOGY 341 (3) 651 - 668 0022-2836 2004/08 [Refereed][Not invited]
     
    CooA, a heme-containing transcriptional activator, binds CO to the heme moiety and then undergoes a structural change that promotes the specific binding to the target DNA. To elucidate the activation mechanism coupled to CO binding, we investigated the CO-dependent structural transition of CooA with small-angle X-ray scattering (SAXS). In the absence of CO, the radius of gyration (Rg) and the second virial coefficient (A(2)) were 25.3(+/-0.5) A and - 0.39(+/-0.25) x 10(-4) ml mol g(-2), respectively. CO binding caused a slight increase in R-g (by 0.5 Angstrom) and a marked decrease in A(2) (by 5.09 x 10(-4) ml mol g(-2)). The observed decrease in A(2) points to higher attractive interactions between CO-bound CooA molecules in solution compared with CO-free CooA. Although the minor alternation of R-g rules out changes in the overall structure, the marked change in the surface properties points to a CO-induced conformational transition. The experimental Rg and SAXS curves of the two states did not agree with the crystal structure of CO-free CooA. We thus simulated the solution structures of CooA based on the experimental data using rigid-body refinements as well as low-resolution model reconstructions. Both results demonstrate that the hinge region connecting the N-terminal heme domain and C-terminal DNA-binding domain is kinked in CO-free CooA, so that the two domains are positioned close to each other. The CO-dependent structural change observed by SAXS corresponds to a slight swing of the DNA-binding domains away from the heme domains coupled with their rotation by about 8degrees around the axis of 2-fold symmetry. (C) 2004 Elsevier Ltd. All rights reserved.
  • K Matsuura, S Yoshioka, S Takahashi, K Ishimori, T Mogi, H Hori, Morishima, I
    BIOCHEMISTRY 43 (8) 2288 - 2296 0006-2960 2004/03 [Refereed][Not invited]
     
    The mechanism of the dioxygen (O-2) reduction conducted by cytochrome bo-type quinol oxidase was investigated using submillisecond-resolved freeze-quench EPR spectroscopy. The fully reduced form of the wild-type enzyme (WT) with the bound ubiquinone-8 at the high-affinity quinone-binding site was mixed with an O-2-saturated solution, and the subsequent reaction was quenched at different time intervals from 0.2 to 50 ms. The EPR signals derived from the binuclear center and heme b were weak in the time domain from 0.2 to 0.5 ms. The signals derived from the ferric heme b and hydroxide-bound ferric heme o increased simultaneously after I ms, indicating that the oxidation of heme b is coupled to the formation of hydroxy heme o. In contrast, the enzyme without the bound ubiquinone-8 (DeltaUbiA) showed the faster oxidation of heme b and the slower formation of hydroxy heme o than WT. It is interpreted that the F-I intermediate possessing ferryl-oxo heme o. cupric Cu-B, and ferric heme b is converted to the F-II intermediate within 0.2 ms by an electron transfer from the bound ubiquinonol-8 to ferric heme b. The conversion of the F-II intermediate to the hydroxy intermediate occurred after 1 ms and was accompanied by the one-electron transfer from heme b to the binuclear center. Finally, it is suggested that the hydroxy intermediate possesses no bridging ligand between heme o and Cu-B and is the final intermediate in the turnover cycle of cytochrome bo under steady-state conditions.
  • T Inuzuka, BG Yun, H Ishikawa, S Takahashi, H Hori, RL Matts, K Ishimori, Morishima, I
    JOURNAL OF BIOLOGICAL CHEMISTRY 279 (8) 6778 - 6782 0021-9258 2004/02 [Refereed][Not invited]
     
    The heme-regulated eukaryotic initiation factor-2alpha (eIF2alpha) kinase (HRI) regulates the initiation of protein synthesis in reticulocytes. The binding of NO to the N-terminal heme-binding domain (NTD) of HRI positively modulates its kinase activity. By utilizing UV-visible absorption, resonance Raman, EPR and CD spectroscopies, two histidine residues have been identified that are crucial for the binding of heme to the NTD. The UV-visible absorption and resonance Raman spectra of all the histidine to alanine mutants constructed were similar to those of the unmutated NTD. However, the change in the CD spectra of the NTD construct containing mutation of His(78) to Ala (H78A) indicated loss of the specific binding of heme. The EPR spectrum for the ferric H78A mutant was also substantially perturbed. Thus, His(78) is one of the axial ligands for the NTD of HRL Significant changes in the EPR spectrum of the H123A mutant were also observed, and heme readily dissociated from both the H123A and the H78A NTD mutants, suggesting that His(123) was also an axial heme ligand. However, the CD spectrum for the Soret region of the H123A mutant indicated that this mutant still bound heme specifically. Thus, while both His(78) and His(123) are crucial for stable heme binding, the effects of their mutations on the structure of the NTD differed. His(78) appears to play the primary role in the specific binding of heme to the NTD, acting analogously to the "proximal histidine" ligand of globins, while His(123) appears to act as the "distal" heme ligand.
  • T Uzawa, S Akiyama, T Kimura, S Takahashi, K Ishimori, Morishima, I, T Fujisawa
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 101 (5) 1171 - 1176 0027-8424 2004/02 [Refereed][Not invited]
     
    The characterization of protein folding dynamics in terms of secondary and tertiary structures is important in elucidating the features of intraprotein interactions that lead to specific folded structures. Apomyoglobin (apoMb), possessing seven helices termed A-E, G, and H in the native state, has a folding intermediate composed of the A, G, and H helices, whose formation in the submillisecond time domain has not been clearly characterized. In this study, we used a rapid-mixing device combined with circular dichroism and small-angle x-ray scattering to observe the submillisecond folding dynamics of apoMb in terms of helical content (f(H)) and radius of gyration (R-g), respectively. The folding of apoMb from the acid-unfolded state at pH 2.2 was initiated by a pH jump to 6.0. A significant collapse, corresponding to approximate to50% of the overall change in R-g from the unfolded to native conformation, was observed within 300 Its after the pH jump. The collapsed intermediate has a f(H) of 33% and a globular shape that involves >80% of all its atoms. Subsequently, a stepwise helix formation was detected, which was interpreted to be associated with a conformational search for the correct tertiary contacts. The characterized folding dynamics of apoMb indicates the importance of the initial collapse event, which is suggested to facilitate the subsequent conformational search and the helix formation leading to the native structure.
  • T Tosha, S Yoshioka, S Takahashi, K Ishimori, H Shimada, Morishima, I
    JOURNAL OF BIOLOGICAL CHEMISTRY 278 (41) 39809 - 39821 0021-9258 2003/10 [Refereed][Not invited]
     
    We investigated putidaredoxin-induced structural changes in carbonmonoxy P450cam by using NMR spectroscopy. The resonance from the beta-proton of the axial cysteine was upfield shifted by 0.12 ppm upon the putidaredoxin binding, indicating that the axial cysteine approaches to the heme-iron by about 0.1 Angstrom. The approach of the axial cysteine to the heme-iron would enhance the electronic donation from the axial thiolate to the heme-iron, resulting in the enhanced heterolysis of the dioxygen bond. In addition to the structural perturbation on the axial ligand, the structural changes in the substrate and ligand binding site were observed. The resonances from the 5-exo- and 9-methyl-protons of d-camphor, which were newly identified in this study, were upfield shifted by 1.28 and 0.20 ppm, respectively, implying that d-camphor moves to the heme-iron by 0.15-0.7 Angstrom. Based on the radical rebound mechanism, the approach of d-camphor to the heme-iron could promote the oxygen transfer reaction. On the other hand, the downfield shift of the resonance from the gamma-methyl group of Thr-252 reflects the movement of the side chain away from the heme-iron by similar to0.25 Angstrom. Because Thr-252 regulates the heterolysis of the dioxygen bond, the positional rearrangement of Thr-252 might assist the scission of the dioxygen bond. We, therefore, conclude that putidaredoxin induces the specific heme environmental changes of P450cam, which would facilitate the oxygen activation and the oxygen transfer reaction.
  • T Egawa, S Yoshioka, S Takahashi, H Hori, S Nagano, H Shimada, K Ishimori, Morishima, I, M Suematsu, Y Ishimura
    JOURNAL OF BIOLOGICAL CHEMISTRY 278 (43) 41597 - 41606 0021-9258 2003/10 [Refereed][Not invited]
     
    The reaction of metmyoglobin with H2O2 was investigated in a pH range between 8.5 and 6.0 with the aid of stopped flow-rapid scan and rapid freezing-EPR techniques. Singular value decomposition analyses of the stopped flow data at pH 8.5 revealed that a spectral species previously unknown accumulated during the reaction and exhibited a Soret absorption maximum at greater than or equal to423 nm. In the EPR experiments, the new species exhibited a set of g values at 2.32, 2.19, and 1.94, indicating that the species was assignable to a ferric hydroperoxy (Fe(III)[O-O-H](-)) compound. In contrast, the hydroperoxy compound scarcely accumulated in the reaction at pH 6.0, and the dominant intermediate species accumulated was compound I, which was derived from the oxygen-oxygen bond cleavage of the hydroperoxy compound. The accumulated amount of the hydroperoxy compound relative to compound I showed a pH dependence with an apparent pK(a) (pK(a)(app)) from 6.95 to 7.27 depending on the metmyoglobins examined. This variation in pK(a)(app) paralleled that in pK(a) of the acid-alkaline transition (pK(a)(AB)) of metmyoglobins, suggesting that the accumulation of hydroperoxy compound is controlled by the distal histidine. We propose that the H2O2 activation by metmyoglobin is promoted at the acidic condition due to the imidazolium form of the distal histidine, and we further propose that the controlled protonation state of the distal histidine is important for the facile O-O bond cleavage in heme peroxidases.
  • Koji Matsuura, Shiro Yoshioka, Satoshi Takahashi, Koichiro Ishimori, Tatsushi Mogi, Hiroshi Hori, Isao Morishima
    Journal of Inorganic Biochemistry Elsevier {BV} 96 (1) 188  2003/07 [Refereed][Not invited]
  • K Yamanaka, H Ishikawa, Y Megumi, F Tokunaga, M Kanie, TA Rouault, Morishima, I, N Minato, K Ishimori, K Iwai
    NATURE CELL BIOLOGY 5 (4) 336 - 340 1465-7392 2003/04 [Refereed][Not invited]
     
    The ubiquitin system is involved in several basic cellular functions(1-3). Ubiquitination is carried out by a cascade of three reactions catalysed by the El, E2 and E3 enzymes. Among these, the E3 ubiquitin-protein ligases have a pivotal role in determining the specificity of the system by recognizing the target substrates through defined targeting motifs(1-3). Although RING finger proteins constitute an important family of E3 ligases(4), only a few post-transcriptional modifications, including phosphorylation(1), proline hydroxylation(5,6) and glycosylation(7), are known to function as recognition signals for E3. Iron regulatory protein 2 (IRP2), a modulator of iron metabolism, is regulated by iron-induced ubiquitination and degradation(8). Here we show that the RING finger protein HOIL-1 functions as an E3 ligase for oxidized IRP2, suggesting that oxidation is a specific recognition signal for ubiquitination. The oxidation of IRP2 is generated by haem, which binds to IRP2 in iron-rich cells, and by oxygen, indicating that the iron sensing of IRP2 depends on the synthesis and availability of haem.
  • S Neya, K Imai, H Hori, H Ishikawa, K Ishimori, D Okuno, S Nagatomo, T Hoshino, M Hata, N Funasaki
    INORGANIC CHEMISTRY 42 (5) 1456 - 1461 0020-1669 2003/03 [Refereed][Not invited]
     
    The iron complex of hemiporphycene, a molecular hybrid of porphyrin with porphycene, was incorporated into the apomyoglobin pocket to examine ligand binding ability of the iron atom in the novel porphyrinoid. Apomyoglobin was successfully coupled with a stoichiometric amount of ferric hemiporphycene to afford the reconstituted myoglobin equipped with the iron coordination structure of native protein. Cyanide, imidazole, and fluoride coordinated to the ferric protein with affinities comparable with those for native myoglobin. The ferrous myoglobin was functionally active to bind O-2 and CO reversibly at pH 7.4 and 20 degreesC. The O-2 affinity is 12-fold higher than that of native myoglobin while the CO affinity is slightly lower, suggesting decreased discrimination between O-2 and CO in the heme pocket. The functional anomaly was interpreted to reflect increased sigma-bonding character in the Fe(II)-O-2 bond. In contrast with 6-coordinate native NO protein, the NO myoglobin containing ferrous hemiporphycene is in a mixed 5- and 6-coordinate state. This observation suggests that the in-plane configuration of the iron atom in hemiporphycene is destabilized by NO. Influence of the core deformation was also detected with both the infrared absorption for the ferrous CO derivative and electron paramagnetic resonance for ferric imidazole complex. Anomalies in the ferric and ferrous derivatives were ascribed to the modified iron-N(pyrrole) interactions in the asymmetric metallo core of hemiporphycene.
  • M Tanaka, K Matsuura, S Yoshioka, S Takahashi, K Ishimori, H Hori, Morishima, I
    BIOPHYSICAL JOURNAL 84 (3) 1998 - 2004 0006-3495 2003/03 [Refereed][Not invited]
     
    To observe the formation process of compound I in horseradish peroxidase (HRP), we developed anew freeze-quench device with similar to200 mus of the mixing-to-freezing time interval and observed the reaction between HRP and hydrogen peroxide (H2O2). The developed device consists of a submillisecond solution mixer and rotating copper or silver plates cooled at 77 K; it freezes the small droplets of mixed solution on the surface of the rotating plates. The ultraviolet-visible spectra of the sample quenched at similar to1 ms after the mixing of HRP and H2O2 suggest the formation of compound 1. The electron paramagnetic resonance spectra of the same reaction quenched at similar to200 mus show a convex peak at g = 2.00, which is identified as compound I due to its microwave power and temperature dependencies. The absence of ferric signals in the electron paramagnetic resonance spectra of the quenched sample indicates that compound I is formed within similar to200 mus after mixing HRP and H2O2. We conclude that the activation of H2O2 in HRP at ambient temperature completes within similar to200 mus. The developed device can be generally applied to investigate the electronic structures of short-lived intermediates of metalloenzymes.
  • Motomasa Tanaka, Koji Matsuura, Shiro Yoshioka, Satoshi Takahashi, Koichiro Ishimori, Hiroshi Hori, Isao Morishima
    Biophysical journal 84 (3) 1998 - 2004 0006-3495 2003/03 [Refereed][Not invited]
     
    To observe the formation process of compound I in horseradish peroxidase (HRP), we developed a new freeze-quench device with approximately 200 micro s of the mixing-to-freezing time interval and observed the reaction between HRP and hydrogen peroxide (H(2)O(2)). The developed device consists of a submillisecond solution mixer and rotating copper or silver plates cooled at 77 K; it freezes the small droplets of mixed solution on the surface of the rotating plates. The ultraviolet-visible spectra of the sample quenched at approximately 1 ms after the mixing of HRP and H(2)O(2) suggest the formation of compound I. The electron paramagnetic resonance spectra of the same reaction quenched at approximately 200 micro s show a convex peak at g = 2.00, which is identified as compound I due to its microwave power and temperature dependencies. The absence of ferric signals in the electron paramagnetic resonance spectra of the quenched sample indicates that compound I is formed within approximately 200 micro s after mixing HRP and H(2)O(2). We conclude that the activation of H(2)O(2) in HRP at ambient temperature completes within approximately 200 micro s. The developed device can be generally applied to investigate the electronic structures of short-lived intermediates of metalloenzymes.
  • S Yoshioka, T Tosha, S Takahashi, K Ishimori, H Hori, Morishima, I
    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 124 (49) 14571 - 14579 0002-7863 2002/12 [Refereed][Not invited]
     
    Structural and functional roles of the hydrogen bonding network that surrounds the heme-thiolate coordination of P450(cam) from Pseudomonas putida were investigated. A hydrogen bond between the side chain amide of Gln360 and the carbonyl oxygen of the axial Cys357 was removed in Q360L. The side chain hydrogen bond and the electrostatic interaction between the polypeptide amide proton of Gln360 and the sulfur atom of Cys357 were simultaneously removed in Q360P. The increased electron donation of the axial thiolate in Q360L and Q360P was evidenced by negative shifts of their reduction potentials by 45 and 70 mV, respectively. Together with the results on L358P in which the amide proton at position 358 was removed (Yoshioka, S., Takahashi, S., Ishimori, K., Morishima, I.J. Inorg. Biochem. 2000, 81, 141-151), we propose that the side chain hydrogen bond and the electrostatic interaction of the amide proton with the thiolate ligand cause similar to45 and similar to35 mV of positive shifts, respectively, of the redox potential of the heme in P450(cam). The resonance Raman spectra of the ferrous-CO form of the Q360 mutants showed a downshifted Fe-CO stretching mode at 482similar to483 cm(-1) compared with that of wild-type P450(cam) at 484 cm(-1). The Q360 mutants also showed the upshift by 4similar to5 cm-1 of the Fe-NO stretching mode in the ferrous-NO form. These Raman results indicate the increase in the sigma-electron donation of the thiolate ligand in the reduced state of the 0360 mutants and were in contrast to the increased pi-back-do nation of the thiolate in L358P having an upshifted Fe-CO stretching mode at 489 cm(-1). The catalytic activities of the Q360 mutants for the unnatural substrates were similar to those of the wild-type enzyme, indicating that the increased sigma-electron donation does not promote the O-O bond heterolysis in the Q360 mutants, although the increased pi-electron donation in L358P promoted the heterolysis of the O-O bond. We conclude that the functions of the proximal hydrogen bonding network in P450(cam) are to stabilize the heme-thiolate coordination, and to regulate the redox potential of the heme iron. Furthermore, we propose that the pi-electron donation, not the sigma-electron donation, of the thiolate ligand promotes the heterolysis of the O-O bond of dioxygen.
  • H Ishikawa, BG Yun, S Takahashi, H Hori, RL Matts, K Ishimori, Morishima, I
    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 124 (46) 13696 - 13697 0002-7863 2002/11 [Refereed][Not invited]
  • T Tosha, S Yoshioka, H Hori, S Takahashi, K Ishimori, Morishima, I
    BIOCHEMISTRY 41 (47) 13883 - 13893 0006-2960 2002/11 [Refereed][Not invited]
     
    We characterized electron transfer (ET) from putidaredoxin (Pdx) to the mutants of cytochrome P450(cam) (P450(cam)), in which one of the residues located on the putative binding site to Pdx, Gln360, was replaced with Glu, Lys, and Len. The kinetic analysis of the ET reactions from reduced Pdx to ferric P450(cam) (the first ET) and to ferrous oxygenated P450(cam) (the second ET) showed the dissociation constants (K) that were moderately perturbed for the Lys and Len mutants and the distinctly increased for the Glu mutant. Although the alterations in K-m indicate that Gln360 is located at the Pdx binding site, the effects of the Gln360 mutations (0.66-20-fold of that of wild type) are smaller than those of the Arg112 mutants (25-2500-fold of that of wild type) [Unno, M., et al. (1996) J. Biol. Chem. 271, 17869-17874], allowing us to conclude that Gln360 much less contributes to the complexation with Pdx than Arg112. The first ET rate (35 s(-1) for wild-type P450(cam)) was substantially reduced in the Ght mutant (5.4 s(-1)), while less perturbation was observed for the Lys (53 s(-1)) and Leu (23 s(-1)) mutants. In the second ET reaction, the retarded ET rate was detected only in the Ght mutant but not in the Lys and Len mutants. These results showed the smaller mutational effects of Gln360 on the ET reactions than those of the Arg112 mutants. In contrast to the moderate perturbations in the kinetic parameters, the mutations at Gln360 significantly affected both the standard enthalpy and entropy of the redox reaction of P450(cam), which cause the negative shift of the redox potentials for the Fe3+/Fe2+ couple by 20-70 mV. Since the arnide group of Gln360 is located near the carbonyl oxygen of the amide group of the axial cysteine, it is plausible that the mutation at Gln360 perturbs the electronic interaction of the axial ligand with heme iron, resulting in the reduction of the redox potentials. We, therefore, conclude that Gln360 primarily regulates the ET reaction of P450cam by modulating the redox potential of the heme iron and not by the specific interaction with Pdx or the formation of the ET pathway that are proposed as the regulation mechanism of Arg 112.
  • T Kimura, S Takahashi, S Akiyama, T Uzawa, K Ishimori, Morishima, I
    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 124 (39) 11596 - 11597 0002-7863 2002/10 [Refereed][Not invited]
  • Y Furukawa, K Ishimori, Morishima, I
    BIOCHEMISTRY 41 (31) 9824 - 9832 0006-2960 2002/08 [Refereed][Not invited]
     
    To characterize the protein-protein interaction during electron transfer, we used Zn-substituted cytochrome c (ZnCytc) as a model of ferrous Cytc and determined the volume change, DeltaV(d)(Zn), for the dissociation of its complex with ferric cytochrome b(5) (Cytb(5)) by the pressure dependence of its photoinduced electron-transfer kinetics. Under ambient pressure, the dissociation constant, K-d(Zn), of the ZnCytc/Cytb(5) complex was dependent on the buffer concentration, 1.5 and 12 muM in 2 and 10 mM Tris-HCl, pH 7.4, respectively, which was consistent with formation of salt bridges in its complexation. The dissociation of one salt bridge is usually associated with large volume changes of -10 to -30 cm(3) mol(-1), while pressure dependence of K-d(Zn) resulted in smaller value of DeltaV(d)(Zn), -8.5 cm(3) mol(-1). Therefore, the interaction between ZnCytc and Cytb5 cannot be explained only by salt bridge interaction, and the partial cancellation by the positive volume change due to the additional hydrophobic interaction is a plausible explanation for the observed DeltaV(d)(Zn). In addition, DeltaV(d)(Zn) of -8.5 cm(3) mol(-1) was considerably smaller than the previously reported volume change, DeltaV(d)(Fe), of -122 cm(3) mol(-1) in the ferric Cytc/Cytb(5) complex dissociation [Rodgers and Sligar (1991) J. Mol. Biol. 221, 1453-1460]. ZnCytc used here has been assumed to be a reliable model of ferrous Cytc, and thus the discrepancy between our present DeltaV(d)(Zn) and the previous DeltaV(d)(Fe) is discussed on the basis of the protein docking dependent on the oxidation states of heme iron in Cytc.
  • Y Furukawa, F Matsuda, K Ishimori, Morishima, I
    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 124 (15) 4008 - 4019 0002-7863 2002/04 [Refereed][Not invited]
     
    We have investigated the photoinduced electron transfer (ET) in the 1: 1 cross-linked complex (CL-ZnMb/b(5)) formed by a cross-linking reagent, EDC, between Zn-substituted myoglobin (ZnMb) and cytochrome b(5) (Cytb(5)) to reveal the mechanism of the inter-protein ET reactions under the condition of multiple encounter complexes. A variety of the ZnMb-Cytb(5) orientations was suggested because of failure to identify the single and specific cross-linking site on Cytb(5) by the peptide-mapping analysis using mass spectrometry. In CL-ZnMb/b(5), a laser pulse generates the triplet excited state of the ZnMb domain ((3)ZnMb(*)), which can transfer one electron to the Cytb(5) domain. The decay kinetics of (3)ZnMb(*) in CL-ZnMb/b(5) consists of a facile power-law ET phase to Cytb(5) domain (similar to30%) and a slower single-exponential phase (similar to70%). The application of the Marcus equation to this power-law phase indicates that CL-ZnMb/b(5) has a variety of ZnMb-Cytb(5) orientations for the facile ET in which the distance between the redox centers (D-A distance) is distributed over 13-20 Angstrom. The single-exponential phase in the (3)ZnMb(*) decay kinetics of CL-ZnMb/b(5) is similar to the intrinsic decay of (3)ZnMb(*) in its rate constant, 65 s(-1), This implies that the ET is impeded in about 70% of the total ZnMb-Cytb(5) orientations due to the D-A distance larger than 20 Angstrom. Combined with the results of the Brownian dynamics simulations for the encounter complexes, the overall bimolecular ET rate, k(app), can be reproduced by the sum of the ET rates for the minor encounter complexes of which D-A distance is less than 20 Angstrom. On the other hand, the encounter complexes with longer D-A distance, which are the majority of the encounter complexes between ZnMb and Cytb(5), have little contribution to the overall bimolecular ET rate. These observations experimentally demonstrate that ZnMb forms a variety of encounter complexes with Cytb(5), among which a minor set of the complexes with the shorter D-A distance < &SIM;20 &ANGS;) regulates the overall bimolecular ET between the proteins.
  • S Akiyama, S Takahashi, T Kimura, K Ishimori, Morishima, I, Y Nishikawa, T Fujisawa
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 99 (3) 1329 - 1334 0027-8424 2002/02 [Refereed][Not invited]
     
    To investigate protein folding dynamics in terms of compactness, we developed a continuous-flow mixing device to make small-angle x-ray scattering measurements with the time resolution of 160 mus and characterized the radius of gyration (R-g) of two folding intermediates of cytochrome c (cyt c). The early intermediate possesses approximate to20 Angstrom of R-g, which is smaller by approximate to4 Angstrom than that of the acid-unfolded state. The R-g of the later intermediate is approximate to18 Angstrom, which is close to that of the molten globule state. Considering the alpha-helix content (f(H)) of the intermediates, we clarified the folding pathway of cyt c on the conformational landscape defined by R-g and f(H). Cyt c folding proceeds with a collapse around a specific region of the protein followed by a cooperative acquisition of secondary structures and compactness.
  • 2002/02 [Refereed][Not invited]
  • Yoshiaki Furukawa, Yoichi Sugiyama, Satoshi Takahashi, Koichiro Ishimori, Isao Morishima
    Springer Nature 187  2002 [Refereed][Not invited]
  • K Yamamoto, H Ishikawa, S Takahashi, K Ishimori, Morishima, I, H Nakajima, S Aono
    JOURNAL OF BIOLOGICAL CHEMISTRY 276 (15) 11473 - 11476 0021-9258 2001/04 [Refereed][Not invited]
     
    CooA is a heme-containing transcriptional activator that anaerobically binds to DNA at CO atmosphere. To obtain information on the conformational transition of CooA induced by CO binding to the heme, we assigned ring current-shifted H-1 NMR signals of CooA using two mutants whose axial Ligands of the heme were replaced. In the absence of CO, the NMR spectral pattern of H77T CooA in which the axial histidine (His(77)) was replaced with tyrosine, was similar to that of wild-type CooA. In contrast, the spectra of CooA Delta N5, in which the NH2 termini including the other axial ligand (Pro(2)) were deleted, were drastically modulated. We assigned three signals of wild-type CooA at -4.5, -3.6, and -2.8 ppm to delta (1)-, alpha-, and delta (2)-protons of Pro(2), respectively. The Pro(2) signals were undetectable in the upfield region of the spectrum of the CO-bound state, which confirms that CO displaces Pro(2), Interestingly, the Pro(2) signals were observed for CO-bound H77Y CooA implying that CO binds to the trans position of Pro(2) in H77Y CooA. The abolished CO-dependent transcriptional activity of H77Y CooA is therefore the consequence of Pro(2) ligation. These observations are consistent with the view that the movement of the NH2 terminus triggers the conformational transition to the DNA binding form.
  • 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.
  • 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.
  • S Yoshioka, S Takahashi, H Hori, K Ishimori, Morishima, I
    EUROPEAN JOURNAL OF BIOCHEMISTRY 268 (2) 252 - 259 0014-2956 2001/01 [Refereed][Not invited]
     
    To investigate the functional and structural roles of the proximal thiolate ligand in cytochrome P450(cam), we prepared the C357H mutant of the enzyme in which the axial cysteine residue (Cys357) was replaced with a histidine residue. We obtained the unstable C357H mutant by developing a new preparation procedure involving in vitro folding of P450(cam) from the inclusion bodies. The C357H mutant in the ferrous-CO form exhibited the Soret peak at 420 nm and the Fe-CO stretching line at 498 cm(-1), indicating a neutral histidine residue as the axial ligand. However, another internal ligand is coordinated to the heme iron as the sixth ligand in the ferric and ferrous forms of the C357H mutant, suggesting the collapse of the substrate-binding site. The C357H mutant showed no catalytic activity for camphor hydroxylation and the reduced heterolytic/homolytic ratio of the O-O bond scission in the reaction with cumene hydroperoxide. The present observations indicate that the thiolate coordination in P450(cam) is important for the construction of the heme pocket and the heterolysis of the O-O bond.
  • S Yoshioka, S Takahashi, H Hori, K Ishimori, Morishima, I
    EUROPEAN JOURNAL OF BIOCHEMISTRY 268 (2) 252 - 259 0014-2956 2001/01 [Refereed][Not invited]
     
    To investigate the functional and structural roles of the proximal thiolate ligand in cytochrome P450(cam), we prepared the C357H mutant of the enzyme in which the axial cysteine residue (Cys357) was replaced with a histidine residue. We obtained the unstable C357H mutant by developing a new preparation procedure involving in vitro folding of P450(cam) from the inclusion bodies. The C357H mutant in the ferrous-CO form exhibited the Soret peak at 420 nm and the Fe-CO stretching line at 498 cm(-1), indicating a neutral histidine residue as the axial ligand. However, another internal ligand is coordinated to the heme iron as the sixth ligand in the ferric and ferrous forms of the C357H mutant, suggesting the collapse of the substrate-binding site. The C357H mutant showed no catalytic activity for camphor hydroxylation and the reduced heterolytic/homolytic ratio of the O-O bond scission in the reaction with cumene hydroperoxide. The present observations indicate that the thiolate coordination in P450(cam) is important for the construction of the heme pocket and the heterolysis of the O-O bond.
  • M Ihara, M Shintaku, S Takahashi, K Ishimori, Morishima, I
    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 122 (46) 11535 - 11536 0002-7863 2000/11 [Refereed][Not invited]
  • T Uchida, H Ishikawa, K Ishimori, Morishima, I, H Nakajima, S Aono, Y Mizutani, T Kitagawa
    BIOCHEMISTRY 39 (42) 12747 - 12752 0006-2960 2000/10 [Refereed][Not invited]
     
    The heme proximal ligand of carbonmonoxy CooA, a CO-sensing transcriptional activator, in the CO-bound form was identified to be His77 by using picosecond time-resolved resonance Raman spectroscopy. On the basis of the inverse correlation between Fe-CO and C-O stretching frequencies, we proposed previously that His77 is the axial ligand trans to CO [Uchida et al. (1998) J. Biol. Chem. 273, 19988-19992], whereas later a possibility of displacement of His77 by CO with retention of another unidentified axial ligand was reported [Vogel et al. (1999) Biochemistry 38, 2629-2682]. Although our previous resonance Raman study failed to detect the Fe-His stretching [v(Fe-His)] mode of CO-photodissociated CooA of the carbonmonoxy adduct due to the rapid recombination, application of the picosecond time-resolved resonance Raman technique enabled us to observe a new intense line assignable to v(Fe-His) at 211 cm(-1) immediately after photolysis, while it became nondiscernible after 100-ps delay. The low v(Fe-His) frequency of photodissociated CooA indicates the presence of some strain in the Fe-His bond in CO-bound CooA. This and the rapid recombination of CO characterize the heme pocket of CooA. The 211 cm(-1) band was completely absent in the spectrum of the CO-photodissociated form of the His77-substituted mutant but the Fe-Im stretching band was observed in the presence of exogenous imidazole (Im). Thus, we conclude that His77 is the axial ligand of CO-bound CooA and CO displaces the axial ligand trans to His77 with retention of ligated His77 to activate CooA as the transcriptional activator.
  • Y Furukawa, K Ishimori, Morishima, I
    BIOCHEMISTRY 39 (36) 10996 - 11004 0006-2960 2000/09 [Refereed][Not invited]
     
    We have investigated photoinduced electron transfer (ET) reactions between zinc-substituted cytochrome P450cam (ZnP450) and several inorganic reagents by using the laser flash photolysis method, to reveal roles of the electrostatic interactions in the regulation of the ET reactions. The laser pulse irradiation to ZnP450 yielded a strong reductant, the triplet excited state of ZnP450, (3)ZnP450*, which was able to transfer one electron to anionic redox partners, OsCl62- and Fe(CN)(6)(3-), with formation of the porphyrin pi-cation radical, ZnP450(+). In contrast, the ET reactions from (3)ZnP450* to cationic redox partners, such as Ru(NH3)(6)(3+) and Co(phen)(3)(3+), were not observed even in the presence of 100-fold excess of the oxidant. One of the possible interpretations for the preferential ET to the anionic redox partner is that the cationic patch on the P450cam surface, a putative interaction site for the anionic reagents, is located near the heme (less than 10 Angstrom from the heme edge), while the anionic surface is far from the heme moiety (more than 16 Angstrom from the heme edge), which would yield 8000-fold faster ET rates through the cationic patch. The ET rate through the anionic patch to the cationic partner would be substantially slower than that of the phosphorescence process in (3)ZnP450*, resulting in no ET reactions to the cationic reagents. These results demonstrate that the asymmetrical charge distribution on the protein surface is critical for the ET reaction in P450cam.
  • 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.
  • S Yoshioka, S Takahashi, K Ishimori, Morishima, I
    JOURNAL OF INORGANIC BIOCHEMISTRY 81 (3) 141 - 151 0162-0134 2000/08 [Refereed][Not invited]
     
    To examine the roles of the axial thiolate in cytochrome P450-catalyzed reactions, a mutant of cytochrome P450cam, L358P, was prepared to remove one of the conserved amide protons that are proposed to neutralize the negative charge of the thiolate sulfur. The increased push effect of the thiolate in L358P was evidenced by the reduced reduction potential of the heme. The N-15-NMR and resonance Raman spectra of the mutant in the ferric-CN- and in the ferrous-CO forms, respectively, also supported the increased push effect. The maintenance of stereo- and regioselectivities for d-camphor hydroxylation by the mutant suggests the minimum structural change at the distal site. The heterolysis/homolysis ratios of cumene hydroperoxide were the same for wild-type and L358P. However, we observed the enhanced monooxygenations of the unnatural substrates using dioxygen and electrons supplied from the reconstituted system, which indicate the significant role of the push effect in dioxygen activation. We interpret that the enhanced push effect inhibits the protonation of the inner oxygen atom and/or promotes the protonation of the outer oxygen atom in the putative iron-hydroperoxo intermediate (Fe3+-O-OH) of P450cam. This work is the first experimental indication of the significance of the axial cysteine for the P450 reactivity. (C) 2000 Elsevier Science S.A. All rights reserved.
  • S Akiyama, S Takahashi, K Ishimori, Morishima, I
    NATURE STRUCTURAL BIOLOGY 7 (6) 514 - 520 1072-8368 2000/06 [Refereed][Not invited]
     
    Two models have been proposed to describe the folding pathways of proteins. The framework model assumes the initial formation of the secondary structures whereas the hydrophobic collapse model supposes their formation after the collapse of backbone structures. To differentiate between these models for real proteins, we have developed a novel CD spectrometer that enables us to observe the submillisecond time frame of protein folding and have characterized the timing of secondary structure formation in the folding process of cytochrome c (cyt c). We found that similar to 20% of the native helical content was organized in the first phase of folding, which is completed within milliseconds. Furthermore, we suggest the presence of a second intermediate, which has alpha-helical content resembling that of the molten globule state. Our results indicate that many of the alpha-helices are organized after collapse in the folding mechanism of cyt c.
  • M Ihara, S Takahashi, K Ishimori, Morishima, I
    BIOCHEMISTRY 39 (20) 5961 - 5970 0006-2960 2000/05 [Refereed][Not invited]
     
    Cytochrome b(5) (cyt b(5)) holds heme using two axial histidines, His63 and His39, that are located in the centers of the two heme-binding loops. The previous NMR study on the apo form of cyt b(5) (apocyt b(5)) revealed that the loop including His63 exhibits a larger fluctuation compared to the other loop including His39 [Falzone, C. J., Mayer, M. R., Whiteman, E. L., Moore, C. D., and Lecomte, J. T. (1996) Biochemistry 35, 6519-6526]. To understand the significance of the fluctuation, the heme association and dissociation rates of the two loops were compared using two mutants of cyt b(5) in which one of the axial histidines was replaced with leucine. It was demonstrated that the fluctuating loop possesses a significantly slower heme dissociation rate and a faster heme association rate than the other loop. To further verify the importance of the fluctuating loop, the heme association process of wild-type apocyt b(5) was investigated using optical absorption and CD spectroscopies. It was indicated that the process proceeds through the two pathways, and that the dominant pathway involves the initial coordination of His63 located in the fluctuating loop. The urea concentration dependency of the rate constants revealed that the folding of the fluctuating loop is associated with the coordination of His63. It was suggested that the fluctuation enables the loop to have a larger heme-loop contact in the heme-bound conformation. The fluctuating heme-binding loops might be useful for the artificial design of heme-binding proteins.
  • K Inaba, K Ishimori, K Imai, Morishima, I
    JOURNAL OF BIOLOGICAL CHEMISTRY 275 (17) 12438 - 12445 0021-9258 2000/04 [Refereed][Not invited]
     
    In our previous work, we demonstrated that the replacement of the "heme binding module," a segment from F1 to G5 site, in myoglobin with that of hemoglobin alpha-subunit converted the heme proximal structure of myoglobin into the alpha-subunit type (Inaba, K., Ishimori, K. and Morishima, I. (1998) J. Mel. Biol. 283, 311-327). To further examine the structural regulation by the heme binding module in hemoglobin, we synthesized the beta alpha(HBM)-subunit, in which the heme binding module (HBM) of hemoglobin beta-subunit was replaced by that of hemoglobin a-subunit. Based on the gel chromatography, the beta alpha(HBM)-subunit was preferentially associated with the alpha-subunit to form a heterotetramer, alpha(2)[beta alpha(HBM)(2)], just as is native beta-subunit. Deoxy-alpha(2)[beta alpha(HBM)(2)] tetramer exhibited the hyperfine-shifted NMR resonance from the proximal histidyl NdeltaH proton and the resonance Raman band from the Fe-His vibrational mode at the same positions as native hemoglobin. Also, NMR spectra of carbonmonoxy and cyanomet alpha(2)[beta alpha(HBM)(2)] tetramer were quite similar to those of native hemoglobin. Consequently, the heme environmental structure of the beta alpha(HBM)-subunit in tetrameric alpha(2)[beta alpha(HBM)(2)] was similar to that of the beta-subunit in native tetrameric Hb A, and the structural conversion by the module substitution was not clear in the hemoglobin subunits, The contrastive structural effects of the module substitution on myoglobin and hemoglobin subunits strongly suggest different regulation mechanisms of the heme proximal structure between these two globins, Whereas the heme proximal structure of monomeric myoglobin is simply determined by the amino acid sequence of the heme binding module, that of tetrameric hemoglobin appears to be closely coupled to the subunit interactions.
  • Y Furukawa, K Ishimori, Morishima, I
    JOURNAL OF PHYSICAL CHEMISTRY B 104 (8) 1817 - 1825 1089-5647 2000/03 [Refereed][Not invited]
     
    The activation volumes (Delta V-not equal) for intramolecular electron transfer (ET) reactions in Ru-modified Zn-porphyrin (ZnP) substituted myoglobins (Ru-ZnMb) have been determined to investigate the pressure effects on the redox potentials and donor-acceptor distance (D-A distance) for the ET reaction. Three Ru-ZnMbs, in which D-A distances for the ET reactions are 12.7 Angstrom (His48Mb), 15.5 Angstrom (His83Mb), and 19.3 Angstrom (His81Mb), were constructed. The activation volumes for the forward ET reactions (Delta V-f(not equal)) were -1.6 (His83Mb), +3.7 (His81Mb), and +6.5 cm(3) mol(-1) (His48Mb). We also measured the pressure dependence of the back ET reactions (from Ru2+ complex to ZnP+), showing that the back ET reactions exhibited negative activation volumes (Delta V-b(not equal)) for all of the Ru--ZnMbs: -11, -5.3, and -6.2 cm(3) mol(-1) for His83Mb, His81Mb, and His48Mb, respectively. On the basis of these activation volumes, the pressure dependence of the redox potentials, (partial derivative Delta G degrees/partial derivative P)(T) was estimated as about 2.94 x 10(-4) eV MPa-1, regardless of the position of the Ru complex. Since (partial derivative Delta G degrees/partial derivative P)T in the present study is close to that of RU(NH3)(6)(2+/3+) (2.97 x 10(-4) eV MPa-1), the pressure-induced redox changes of the Ru complex were primarily responsible for that of the ET reaction and the contribution of ZnP to the pressure dependence of the redox potential on the ET reactions would be small. In sharp contrast to (partial derivative Delta G degrees/partial derivative P)(T), the pressure dependence of the D-A distance, (partial derivative d/partial derivative P)(T), highly depends on the ET pathway and microenvironments of the redox centers. The linear compressibility, (-1/d(0))(partial derivative d/partial derivative P)(T), was (2.2 +/- 0.1) x 10(-10), (5.1 +/- 0.5) x 10(-11), and (-2.6 +/- 3.2) x 10(-11) m(2) N-1 for His83Mb, His81Mb, and His48Mb, respectively. The different linear compressibility for the three ET reaction systems suggests that the structural fluctuation in proteins is not unique in protein structure and site specific local fluctuations would be one of the factors regulating the protein ET reactions.
  • Yoshiaki Furukawa, Koichiro Ishimori, Isao Morishima
    The Journal of Physical Chemistry B American Chemical Society ({ACS}) 104 (8) 1817  2000/03 [Refereed][Not invited]
  • M Tanaka, K Ishimori, Morishima, I
    BIOCHEMISTRY 38 (32) 10463 - 10473 0006-2960 1999/08 [Refereed][Not invited]
     
    To enhance the oxidation activity for luminol in horseradish peroxidase (HRP), we have prepared three HRP mutants by mimicking a possible binding site for luminol in Arthromyces ramosus peroxidase (ARP) which shows 500-fold higher oxidation activity for luminol than native HRP. Spectroscopic studies by H-1 NMR revealed that the chemical shifts of 7-propionate and 8-methyl protons of the heme in cyanide-ligated ARP were deviated upon addition of luminol (4 mM), suggesting that the charged residues, Lys49 and Glu190, which are located near the 7-propionate and 8-methyl groups of the heme. are involved in the specific binding to luminol. The positively charged Lys and negatively charged Glu were introduced into the corresponding positions of Ser35 (S35K) and Gln176 (Q176E) in HRP, respectively, to build the putative binding site for luminol. A double mutant, S35K/Q176E, in which both Ser35 and Gln176 were replaced, was also prepared. Addition of luminol to the HRP mutants induced more pronounced effects on the resonances from the heme substituents and heme environmental residues in the H-1 NMR spectra than that to the wild-type enzyme, indicating that the mutations in this study induced interactions with luminol in the vicinity of the heme. The catalytic efficiencies (V-max/K-m) for luminol oxidation of the S35K and S35K/Q176E mutants were 1.5- and 2-fold improved, whereas that of the Q176E mutant was slightly depressed. The increase in luminol activity of the S35K and S35K/Q176E mutants was rather small but significant, suggesting that the electrostatic interactions between the positive charge of Lys35 and the negative charge of luminol can contribute to the effective binding for the luminol oxidation. On the other hand, the negatively charged residue would not be so crucial for the luminol oxidation. The absence of drastic improvement in the luminol activity suggests that introduction of the charged residues into the heme vicinity is not enough to enhance the oxidation activity for luminol as observed for ARP.
  • Annika Lindgren, Motomasa Tanaka, Tautgirdas Ruzgas, Lo Gorton, Irina Gazaryan, Koichiro Ishimori, Isao Morishima
    Electrochemistry Communications Elsevier {BV} 1 (5) 171  1999/05 [Refereed][Not invited]
  • Morimoto A, Tanaka M, Takahashi S, Ishimori K, Hori H, Morishima I, Pond A.E, Dawson J.H
    Chemtracts 12 (2) 87 - 95 1999 [Refereed][Not invited]
  • Shirai T, Fujikake M, Yamane T, Inaba K, Ishimori K, Morishima I
    Journal of Molecular Biology 287 (2) 369 - 382 1999 [Refereed][Not invited]
  • T Nakatsukasa, N Nomura, G Miyazaki, K Imai, Y Wada, K Ishimori, Morishima, I, H Morimoto
    FEBS LETTERS 441 (1) 93 - 96 0014-5793 1998/12 [Refereed][Not invited]
     
    It was previously reported that Hb Philly with a mutation of Phe for Tyr at 35(C1)beta showed non-cooperative oxygen binding with a very high affinity and instability leading to hemolysis, Further, it lacked the H-1-NMR signal at 13.1 ppm from 2,2-dimethyl-2-silapentane-5-sulfonate in normal hemoglobin (Hb A), so that this signal was assigned to a hydrogen bond formed by Tyr-35(C1)beta. Surprisingly, our artificial mutant hemoglobin with the same mutation as hb Philly showed slightly lowered oxygen affinity, almost normal cooperativity, the H-1-NMR signal at 13.1 ppm and no sign of instability. Our results indicate that the mutation reported for Hb Philly and the assignment of the 13.1 ppm signal need reexamination. (C) 1998 Federation of European Biochemical Societies.
  • K Inaba, K Ishimori, Morishima, I
    JOURNAL OF MOLECULAR BIOLOGY 283 (1) 311 - 327 0022-2836 1998/10 [Refereed][Not invited]
     
    To investigate structural and functional significance of a newly proposed structural unit in globins, the "heme binding module", we synthesized a "heme binding module"-substituted chimeric globin and characterized its function and structure. In our previous study we proposed that the heme binding module, corresponding to the segment from Leu(F1) to Phe(G5) in hemoglobin alpha-subunit, plays a key role in constructing the heme proximal structure in globins. The replacement of the heme binding module in myoglobin with that of hemoglobin alpha-subunit converted the absorption spectra into that of the alpha-subunit, and, in the resonance Raman spectra, the vibration mode characteristic of myoglobin completely disappeared after the module replacement. The hyperfine-shifted NMR resonances for the cyanide-bound form of the module-substituted myoglobin also revealed that the orientation of the axial histidine is close to that of the alpha-subunit rather than that of myoglobin, while the deviations of the resonance positions of the NMR signals from the amino acid residues located in the distal site were subtle, supporting the preferential structural alterations in the heme proximal site. The present finding for the structural alterations in the module-substituted myoglobin confirms that the heme binding module can be a segment regulating the heme proximal structure in globin proteins. (C) 1998 Academic Press.
  • T Shirai, M Fujikake, T Yamane, K Inaba, K Ishimori, Morishima, I
    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 32 (3) 263 - 267 0887-3585 1998/08 [Refereed][Not invited]
     
    A chimera beta alpha-subunit of human hemoglobin was crystallized into a carbon-monoxy form, The protein was assembled by substituting the structural portion of a beta-subunit of hemoglobin (M4 module of the subunit) for its counterpart in the alpha-subunit. In order to overcome the inherent instability in the crystallization of the chimera subunit, a site-directed mutagenesis (F133V) technique was employed based on a computer model. The crystal was used for an X-ray diffraction study yielding a data set with a resolution of 2.5 Angstrom. The crystal belongs to the monoclinic space group P2(1), with cell dimensions of a = 62.9, b = 81.3, c = 55.1 Angstrom, and beta = 91.0 degrees. These dimensions are similar to the crystallographic parameters of the native beta-subunit tetramers in three different ligand states, one of which is a cyanide form that was also crystallized in this study. Proteins 32:263-267, 1998. (C) 1998 Wiley-Liss, Inc.
  • T Shirai, M Fujikake, T Yamane, K Inaba, K Ishimori, Morishima, I
    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 32 (3) 263 - 267 0887-3585 1998/08 [Refereed][Not invited]
     
    A chimera beta alpha-subunit of human hemoglobin was crystallized into a carbon-monoxy form, The protein was assembled by substituting the structural portion of a beta-subunit of hemoglobin (M4 module of the subunit) for its counterpart in the alpha-subunit. In order to overcome the inherent instability in the crystallization of the chimera subunit, a site-directed mutagenesis (F133V) technique was employed based on a computer model. The crystal was used for an X-ray diffraction study yielding a data set with a resolution of 2.5 Angstrom. The crystal belongs to the monoclinic space group P2(1), with cell dimensions of a = 62.9, b = 81.3, c = 55.1 Angstrom, and beta = 91.0 degrees. These dimensions are similar to the crystallographic parameters of the native beta-subunit tetramers in three different ligand states, one of which is a cyanide form that was also crystallized in this study. Proteins 32:263-267, 1998. (C) 1998 Wiley-Liss, Inc.
  • M Aoki, K Ishimori, Morishima, I
    BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY 1386 (1) 157 - 167 0167-4838 1998/07 [Refereed][Not invited]
     
    To investigate the interaction of putidaredoxin (Pdx) with its redox partners in the cytochrome P450cam system, we focused on the role of negatively charged surface amino acid residues. The amino acid residues we examined in this mutational study are Asp-58, Glu-65, Glu-72, and Glu-77, which are located on the alpha-helical segment to form a negatively charged region on the surface of Pdx and have been supposed to play key roles in the association with the redox partners, NADH-putidaredoxin reductase (PdR) and P450cam. The neutralization of the single negative charge on these amino acid residues did not significantly inhibit the electron-transfer reaction with the redox partners, except for the mutation at Glu-72. Together with the previous results, we can conclude that the negatively charged cluster on the alpha-helical segment is not so crucial for the electron transfer of the Pdx/PdR complex, and, instead of the negative charges, the steric hindrance is essential for the binding of Pdx with PdR. In the electron transfer from Pdx to P450cam, the alpha-helical region would not be included in the binding site with P450cam and some specific hydrogen bonds on the surface loop near the Fe-S center contribute to the electron transfer to P450cam. Such different binding sites and interactions for Pdx will shed light on the electron-transfer mechanism mediated by Pdx, the shuttle mechanism. (C) 1998 Elsevier Science B.V. All rights reserved.
  • M Aoki, K Ishimori, Morishima, I
    BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY 1386 (1) 168 - 178 0167-4838 1998/07 [Refereed][Not invited]
     
    To characterize the electron-transfer reaction in the P450cam monooxygenation system, the binding regions of putidaredoxin (Pdx) to NADH-putidaredoxin reductase (PdR) and P450cam were investigated using isotope-filtered NMR experiments in which uniformly N-15-labeled Pdx ([U-N-15]Pdx) is mixed with unlabeled PdR and P450cam. By addition of PdR to Pdx, site specific signal broadening was observed for the N-H correlation peaks from Val-28, Glu-72, Ile-88, and Gln-105. Although previous studies have suggested the contribution from acidic amino acid residues on the G-helix of Pdx to the binding with PdR, no site specific broadening was observed for the resonances from these residues except for Glu-72. The lesser contribution of electrostatic interactions to the Pdx/PdR complex formation was also suggested by our previous study (M. Aoki, K. Ishimori, H. Fukada, K. Takahashi, I. Morishima, Biochim. Biophys. Acta 1384 (1998) 180-188), which is in sharp contrast to the complex formation between adrenodoxin and adrenodoxin reductase. Upon the complex formation between Pdx and P450cam, the site specific NMR line broadening was observed for several amino acid residues distributed near the iron-sulfur cluster, corresponding to the large binding site in the complex formation with P450cam. Since some of the amino acid residues included in the binding site are not conserved for the electron-transfer iron-sulfur proteins such as ferredoxin and adrenodoxin, the interactions formed by these amino acid residues would be highly specific to the binding with P450cam, consistent with very low cross-reactivity to other iron-sulfur proteins in the P450cam monooxygenation system. (C) 1998 Elsevier Science B.V. All rights reserved.
  • Masaaki Aoki, Koichiro Ishimori, Isao Morishima
    Biochimica et Biophysica Acta ({BBA}) - Protein Structure and Molecular Enzymology Elsevier {BV} 1386 (1) 157  1998/07 [Refereed][Not invited]
  • Masaaki Aoki, Koichiro Ishimori, Isao Morishima
    Biochimica et Biophysica Acta ({BBA}) - Protein Structure and Molecular Enzymology Elsevier {BV} 1386 (1) 168  1998/07 [Refereed][Not invited]
  • A Morimoto, M Tanaka, S Takahashi, K Ishimori, H Hori, Morishima, I
    JOURNAL OF BIOLOGICAL CHEMISTRY 273 (24) 14753 - 14760 0021-9258 1998/06 [Refereed][Not invited]
     
    The crucial reaction intermediate in the reaction of peroxidase with hydrogen peroxide (H2O2), compound I, contains a porphyrin pi-cation radical in horseradish peroxidase (HRP), which catalyzes oxidation of small organic and inorganic compounds, whereas cytochrome c peroxidase (CcP) has a radical center on the tryptophan residue (Trp-191) and oxidizes the redox partner, cytochrome c. To investigate the roles of the amino acid residue near the heme active center in discriminating the function of the peroxidases in these two enzymes, we prepared a CcP-like HRP mutant, F221W (Phe-221 --> Trp). Although the rapid spectral scanning and stopped-flow experiments confirmed that the F221W mutant reacts with H2O2 to form the porphyrin pi-cation radical at the same rate as for the wild-type enzyme, the characteristic spectral features of the porphyrin pi-cation radical disappeared rapidly, and were converted to the compound II-type spectrum. The EPR spectrum of the resultant species produced by reduction of the porphyrin pi-cation radical, however, was quite different from that of compound II in HRP, showing typical signals from a Trp radical as found for CcP. The sequential radical formation from the porphyrin ring to the Trp residue implies that the proximal Trp is a key residue in the process of the radical transfer from the porphyrin ring, which differentiates the function of peroxidases.
  • M Aoki, K Ishimori, Morishima, I, Y Wada
    INORGANICA CHIMICA ACTA 272 (1-2) 80 - 88 0020-1693 1998/05 [Refereed][Not invited]
     
    In the P450cam reaction cycle, the interactions between putidaredoxin (Pdx) and P450cam or NADH-putidaredoxin reductase (PdR) have been considered to be essential in the electron-transfer process. In this study, three mutant putidaredoxins were prepared to examine the hydrophobic and electrostatic interactions in the reaction complexes. Val-98, which is exposed to solvent and has been previously suggested to play a key role in hydrophobic interactions, was substituted by alanine (V98A) or threonine (V98T) to perturb the hydrophobicity at the 98 position. Another mutation site was Glu-72 which is located in the middle of the negative charge-rich region. This glutamate was altered to glutamine (E72Q) to neutralize one of the negative charges on the surface of Pdx. The electronic absorption and H-1 NMR spectra of oxidized and reduced forms of these mutants, and their redox potentials were similar to those of wild type Pdx, indicating that the environment of the Fe-2-S-2 cluster was not very seriously affected by these mutations. In cytochrome c reduction activity, however, the ionic strength dependence of E72Q mutant differs slightly from that of the wild type protein. The mutation at glutamine at the 72 position weakened the association to PAR, indicating that Glu-72 is one of the amino acid residues contributing to form the reaction complex for the electron-transfer between Pdx and PdR. Based on the NADH consumption activity of these mutants, the hydrophobic interactions at Val-98 are involved in binding to P450cam, while electrostatic interactions at Glu-72 are rather small to affect the reaction between Pdx and P450cam. These different effects of the mutations suggest that the interaction sites to P450cam on Pdx are not completely superimposed on that to PUR. (C) 1998 Elsevier Science S.A. All rights reserved.
  • Masaaki Aoki, Koichiro Ishimori, Isao Morishima, Yoshinao Wada
    Inorganica Chimica Acta Elsevier {BV} 272 (1-2) 80  1998/05 [Refereed][Not invited]
  • M Tanaka, A Morimoto, K Ishimori, Morishima, I
    PURE AND APPLIED CHEMISTRY 70 (4) 911 - 916 0033-4545 1998/04 [Refereed][Not invited]
     
    The distal His-Asn-Glu hydrogen bond network in the heme distal site is highly conserved among various fungal and plant peroxidases. To gain an insight into the functional roles of this hydrogen network in peroxidase activity, we have mutated the Asn70 to Val (N70V) or Asp (N70D), the Glu64 to Pro (E64P), or GIy (E64G) or Ser (E64S) in horseradish peroxidase. All the mutants disrupted the distal His-Asn hydrogen bond with maintaining the heme electronic structures as revealed by H-1 NMR spectra and exhibited substantial depression of the peroxidase activity. The depression of the activity was ascribed to the decreased basicity and dislocation of the distal His induced by these mutations. The replacement of the distal His by Glu (H42E) to mimic the heme distal site of chloroperoxidase impaired the peroxidase activity. We have also studied F221W HRP mutant in which the proximal Phe is replaced with Trp to mimic the heme proximal structure of cytochrome c peroxidase and shown that the HRP-type compound I was formed, followed by transformation to the CcP-type Trp radical.
  • K Inaba, K Ishimori, K Imai, Morishima, I
    JOURNAL OF BIOLOGICAL CHEMISTRY 273 (14) 8080 - 8087 0021-9258 1998/04 [Refereed][Not invited]
     
    Functional and structural significance of the "module" in proteins has been investigated for globin proteins. Our previous studies have revealed that some modules in globins are responsible for regulating the subunit association and heme environmental structures, whereas the module substitution often induces fatal structural destabilization, resulting in failure of functional regulation. In this paper, to gain further insight into functional and structural significance of the modular structure in globins, we focused upon the "pseudo-module" in globin structure where boundaries are located at the center of modules. Although the pseudo-module has been supposed not to retain a compactness, the beta alpha(PM3)-subunit, in which one of the pseudo-modules, the F1-H6 regoin, of the alpha-subunit is implanted into the beta-subunit, conserved stable globin structure, and its association property was converted into that of the alpha-subunit, as the case for the module substituted globin, the beta alpha(M4)-subunit. These results suggest that modules are not unique structural and functional units for globins. Interestingly, however, the recent reconsideration of the module boundary indicates that the modules in globins can be further divided into two small modules, and one of the boundaries for the new small modules coincides with that of the pseudo-module we substituted in this study. Although it would be premature to conclude the significance of the modular structure in globins, it can be safely said that we have found new structural units in globin structure, probably new modules.
  • M Aoki, K Ishimori, H Fukada, K Takahashi, Morishima, I
    BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY 1384 (1) 180 - 188 0167-4838 1998/04 [Refereed][Not invited]
     
    Putidaredoxin (Pdx), an iron-sulfur protein containing a 2Fe-2S cluster, serves as a physiological electron mediator from NADH-putidaredoxin reductase (PdR) to P450cam in the P450cam monooxygenation reaction cycle, Previous studies have revealed that the associations of Pdx with P450cam and PdR are not strongly dominated by electrostatic interactions, although such interactions stabilize most electron-transfer complexes [A.R. De Pascalis, I. Jelesarov, F. Ackermann, W.H. Koppenol, M. Hiroasawa, D.B. Knaff, H.R. Bosshard, Protein Sci. 2 (1993) 1126-1135]. In the present study, to elucidate the interactions dominating the specific associations in the electron-transfer reaction mediated by Pdx, the thermodynamic properties-entropy (Delta S), enthalpy (Delta H), and heat capacity changes (Delta Cp)-for PdR/Pdx and P450cam/Pdx association reactions have been examined by isothermal titration calorimetry (ITC). Although the binding enthalpy change, Delta H-bind, for the PdR/Pdx association is positive at 10 degrees C, it declines linearly with temperature in the range 10-22 degrees C and becomes negative above 11 degrees C. On the other hand, the binding entropy changer Delta S-bind, is positive at all temperatures examined in this study, indicating that the association of Pdx to PdR is entropically driven. On the basis of the temperature dependence of Delta H-bind, Delta Cp-bind for the association of Pdx to PdR was estimated as -1.24 kJ mol(-1) K-1. This value is larger than those reported for other electron-transfer protein systems (e.g., -0.68 kJ mol(-1) K-1 for ferredoxin/ferredoxin:NADP(+) reductase), suggesting that the PdR/Pdx association may be dominated by hydrophobic rather than electrostatic components. For the P450cam/Pdx association. the negative Delta S-bind and highly favorable Delta H-bind were observed, behavior that stands in sharp contrast to the association reactions in other electron-transfer proteins. The energetics of the P450cam/Pdx association are similar to those of binding reaction of antibody to antigen in which van der Waals and hydrogen bonding interactions are dominant, resulting in high specificity in the association of Pdx with P450cam. (C) 1998 Elsevier Science B.V.
  • M Tanaka, K Ishimori, Morishima, I
    BIOCHEMISTRY 37 (8) 2629 - 2638 0006-2960 1998/02 [Refereed][Not invited]
     
    One of the highly conserved amino acid residues in the heme distal site of various fungal and plant peroxidases, glutamic acid 64 (Glu64) in horseradish peroxidase (HRP), interacts with a distal calcium ion through a hydrogen bond with a water molecule and its peptide carbonyl oxygen on the main-chain forms the hydrogen bond network to the distal His via the adjacent Asn residue, suggesting that the Glu residue is related to the stabilization of the calcium ion and catalytic activity of peroxidase [Nagano, S., Tanaka, M., Ishimori, K,, Watanabe, Y., and Morishima, I, (1996) Biochemistry 35, 14251-14258]. To perturb the hydrogen bond with the adjacent Asn, we replaced the Glu with Pro (E64G) or Gly (E64G), which would alter the configuration of the main chain at position 64. Both of the mutants exhibited substantially depressed oxidation activities for hydroquinone and elementary reaction rates in the catalytic cycle. However, the E64S (Glu64 --> Ser) mutant, in which the configuration of the main chain and the hydrogen bond with Asn70 would not be affected but the interactions with the calcium ion are seriously perturbed by removal of the carboxylate, also showed quite low catalytic activity as observed for the E64P and E64G mutants. Spectral features for the E64S mutant are similar to those of the other mutants: the reorientation of the distal His, disruption of the hydrogen bond between the distal His and Asn70, and loss of the calcium ion. Thus, we can conclude that, in addition to forming the hydrogen bond network in the distal site, the Glu residue is a key residue for stable binding of the calcium ion, which maintains the structural integrity of the distal cavity, resulting in high peroxidase activity.
  • Uchida T, Ishikawa H, Takahashi S, Ishimori K, Morishima I, Ohkubo K, Nakajima H, Aono S
    Journal of Biological Chemistry 273 (32) 19988 - 19992 1998 [Refereed][Not invited]
  • Shingo Nagano, Motomasa Tanaka, Koichiro Ishimori, Isao Morishima, Yoshihito Watanabe, Masahiro Mukai, Takashi Ogura, Teizo Kitagawa
    Springer Nature 354  1998 [Refereed][Not invited]
  • M. Tanaka, A. Morimoto, Koichiro Ishimori, I. Morishima
    Pure and Applied Chemistry Walter de Gruyter {GmbH} 70 (4) 1998/01 [Refereed][Not invited]
  • 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.
  • K Inaba, K Wakasugi, K Ishimori, T Konno, M Kataoka, Morishima, I
    JOURNAL OF BIOLOGICAL CHEMISTRY 272 (48) 30054 - 30060 0021-9258 1997/11 [Refereed][Not invited]
     
    The alpha- and beta-subunits of human hemoglobin consist of the modules M1, M2 + M3, and M4, which correspond to the exons 1, 2, and 3, respectively (Go, M. (1981) Nature 291, 90-92). To gain further insight into functional and structural significance of the modules, we designed two kinds of chimeric hemoglobin subunits (chimeric alpha alpha beta- and beta beta alpha-subunits), in which the module M4 was replaced by the partner subunits. CD spectra in the far-UV region showed that the secondary structure of the chimeric alpha alpha beta-subunit drastically collapsed, while the chimeric beta beta alpha-subunit conserved the native globin structure (Wakasugi, K., Ishimori, It., Imai, IT., Wada, Y., and Morishima, I. (1994) J. Biol. Chem. 269, 18750-18756). SAXS data also suggested a partially disordered structure of the chimeric alpha alpha beta-subunit. Based on tryptophan-fluorescence spectra and computer modeling from x-ray structures of native globins, steric constraint between Trp(14) and Tyr(125) would be induced in the chimeric alpha alpha beta-subunit, which would perturb the packing of the A- and H-helices and destabilize the globule structure, On the other hand, such a steric constraint was not found for the counterpart chimeric subunit, the beta beta alpha-subunit The different stabilities of these module-substituted globins imply that nodules would not always be stable ''structural'' units, and interactions between modules are crucial to construct stable globin subunits.
  • Y Sugiyama, S Takahashi, K Ishimori, Morishima, I
    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 119 (40) 9582 - 9583 0002-7863 1997/10 [Refereed][Not invited]
  • K Wakasugi, K Ishimori, Morishima, I
    BIOPHYSICAL CHEMISTRY 68 (1-3) 265 - 273 0301-4622 1997/10 [Refereed][Not invited]
     
    Based on the detailed structural analysis of proteins, Go [M. Go, Nature 291 (1981) 90-92] found that protein structures can be divided into some structural units, 'modules,' which correspond to peptides coded by exons. In the present study, to investigate functional and structural roles of modular structures in proteins, we have engineered eight chimera globins, in which the exons are shuffled among human myoglobin, human hemoglobin alpha- and beta-subunits, in addition to the chimera beta beta alpha-globin described previously [K. Wakasugi, K. Ishimori, K. Imai, Y. Wada, I. Morishima, J. Biol. Chem. 269 (1994) 18750-18756]. Although all of the chimera globins stoichiometrically bound the heme and their alpha-helical contents increased by heme incorporation as found for native globins, the alpha-helical contents of the chimera globins were significantly lower than those of native globins, suggesting that 'module' substitutions seriously affect the protein folding and stability in globins. The comparisons among several chimera globins demonstrated that such structural alterations are mainly attributed to loss of some key intermodular interactions for protein folding. By simultaneous substitution of the modules M1 and M4 from the same globin, the protein structure was stabilized, which indicates that the module packing between modules M1 and M4 would be one of the crucial interaction to stabilize the globin fold. Present results allow us to conclude that module substitutions would be available for designing and producing novel functional proteins if we can reproduce the stable modular packing in the 'module'-substituted proteins. (C) 1997 Elsevier Science B.V.
  • Keisuke Wakasugi, Koichiro Ishimori, Isao Morishima
    Biophysical Chemistry Elsevier {BV} 68 (1-3) 265  1997/10 [Refereed][Not invited]
  • M Tanaka, S Nagano, K Ishimori, Morishima, I
    BIOCHEMISTRY 36 (32) 9791 - 9798 0006-2960 1997/08 [Refereed][Not invited]
     
    The distal His in peroxidases forms a hydrogen bond with the adjacent Asn, which is highly conserved among many plant and fungal peroxidases. Our previous work [Nagano, S., Tanaka, M., Ishimori, K., Watanabe, Y., & Morishima, I. (1996) Biochemistry 35, 14251-14258] has revealed that the replacement of Asn70 in horseradish peroxidase C (HRP) by Val (N70V) and Asp (N70D) discourages the oxidation activity for guaiacol, and the elementary reaction rate constants for the mutants was decreased by 10-15-fold. In order to delineate the structure-function relationship of the His-Asn couple in peroxidase activity, heme environmental structures of the HRP mutant, N70D, were investigated by CD, H-1 NMR, and IR spectroscopies as well as Fe2+/Fe3+ redox potential measurements. While N70D mutant exhibited quite similar CD spectra and redox potential to those of native enzyme, the paramagnetic NMR spectrum clearly showed that the hydrogen bond between the distal His and Asp70 is not formed in the mutant. The disappearance of the splitting in the H-1 NMR signal. of heme peripheral g-methyl group observed in 50% H2O/50% D2O solution of N70D-CN suggests that the hydrogen bond between the distal His and heme-bound cyanide is also disrupted by the mutation, which was supported by the low C-N vibration frequency and large dissociation constant of the heme-bound cyanide in the mutant. Together with the results from various spectroscopies and redox potentials, we can conclude that the improper positioning of the distal His induced the cleavages of the hydrogen bonds around the distal His, resulting in the substantial decrease of the catalytic activity without large structural alterations of the enzyme. The His-Asn hydrogen bond in the distal site of peroxidases, therefore, is essential for the catalytic activity by controlling the precise location of the distal His.
  • M Tanaka, K Ishimori, M Mukai, T Kitagawa, Morishima, I
    BIOCHEMISTRY 36 (32) 9889 - 9898 0006-2960 1997/08 [Refereed][Not invited]
     
    The distal histidine (His) is highly conserved in peroxidases and has been considered to play a major role as a general acid-base catalyst for peroxidase reaction cycle. Recently, however, the X-ray structure of chloroperoxidase from the marine fungus Caldariomyces fumago has revealed that a glutamic acid is located at the position where most of the peroxidase has a histidine residue, suggesting that the carboxyl group in the glutamic acid (Glu) can also assist cleavage of an O-O bond in peroxides [Sundaramoorthy, M., Terner, J., & Poulos, T. L. (1995) Structure 3, 1367-1377]; In order to investigate catalytic roles of the glutamic acid at the distal cavity, two horseradish peroxidase mutants were prepared, in which the distal His42 has been replaced by Glu (H42E) or Gln (H42Q). The formation rate of compound I in the H42E mutant was significantly greater than that for the H42Q mutant, indicating that the distal Glu can play a role as a general acid-base catalyst. However, the peroxidase activity of the H42E mutant was still lower, compared to that for native enzyme. On the basis of the CD, resonance Raman, and EPR spectra, it was suggested that the basicity of the distal Glu is lower than that of the distal His and the position of the distal Glu is not fixed at the optimal position as a catalytic amino acid residue, although no prominent structural changes around heme environment were detected. The less basicity and improper positioning of the distal Glu would destabilize the heme-H2O2-distal Glu ternary intermediate for the peroxidase reaction. Another characteristic feature in the mutants was the enhancement of the peroxygenase activity. Since the peroxygenase activity was remarkably enhanced in the H42E mutant, the distal Glu is also crucial to facilitate the peroxygenase activity as well as the enlarged distal cavity caused by the amino acid substitution. These observations indicate that the distal amino acid residue is essential for function of peroxidases and subtle conformational changes around the distal cavity would control the catalytic reactions in peroxidase.
  • Motomasa Tanaka, Atsushi Morimoto, Koichiro Ishimori, Isao Morishima
    Journal of Inorganic Biochemistry Elsevier {BV} 67 (1-4) 80  1997/07 [Refereed][Not invited]
  • M Mukai, S Nagano, M Tanaka, K Ishimori, Morishima, I, T Ogura, Y Watanabe, T Kitagawa
    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 119 (7) 1758 - 1766 0002-7863 1997/02 [Refereed][Not invited]
     
    Effects of a highly conserved hydrogen bond between the distal histidine (His) and a nearby asparagine residue (Asn) of peroxidases upon the active site structures were investigated using resonance Raman (RR) and EPR spectroscopy. Although there is no crystallographic data for horseradish peroxidase (HRP), Asn70 is deduced to be the hydrogen bond acceptor for HRP. Accordingly, site-directed mutagenesis of Asn70 to Val (N70V) and to Asp (N70D) was carried out with HRP, and their RR spectra were compared with those of native and wild type (WT) enzymes in the resting, reduced, CN-bound ferric and compound II states. In the resting state, the six-coordinate high-spin structure is the main component for N70V and N70D mutants, while the five-coordinate high-spin structure is dominant for the native and WT HRPs. This was confirmed with EPR spectra. The Fe-III-CN stretching (nu(Fe-CN)) and bending RR bands of the linear and bent forms were identified using (CN)-C-12-N-15 and (CN)-C-13-N-14 isotopes. The nu(Fe-CN) frequency of the linear form is lower for the mutants than for native enzyme, and the spectral patterns of the mutants at pH 7.0 resemble that of the basic form of native HRP. The Fe-histidine stretching bands of reduced HRPs exhibit pH dependent frequency shifts, and the midpoint pH values were 7.2, 5.9, and 5.5 for native, N70V, and N70D, respectively. This change is ascribed to the acid-base transition of the distal His. While the Fe-IV=0 stretching (nu(Fe=0)) frequency of compound LI at pD 7.0 is lower than that at pD 10.0 for native enzyme, the nu(Fe=0) band of the mutants show no pH dependent frequency shifts between pD 7.0 and pD 10.0. However, the H2O/D2O frequency change of nu(Fe=0) and the oxygen atom exchange with bulk water suggested the presence of the hydrogen bond between the oxygen ligand of the ferryloxo heme and distal His for these mutants at pD 10.0. On the basis of these observations, it is proposed that the hydrogen bond between the N-delta-proton of distal His and Asn70 regulates the pK(a) of the N-epsilon- proton (and thus the reactivity of compound LI at the distal side) and also affects the Fe-His bond at the proximal side via tertiary structure changes.
  • 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.
  • K Ishimori, S Sommer, A Bailone, M Takahashi, MM Cox, R Devoret
    JOURNAL OF MOLECULAR BIOLOGY 264 (4) 696 - 712 0022-2836 1996/12 [Refereed][Not invited]
     
    A recA mutant (recA423; Arg(169) --> His), with properties that should help clarify the relationship between the biochemical properties of RecA protein and its two major functions, homologous genetic recombination and recombinational DNA repair, has been isolated. The mutant has been characterized in vivo and the purified RecA423 protein has been studied in vitro. The recA423 cells are nearly as proficient in conjugational recombination, transductional recombination, and recombination of lambda red(-) gam(-) phage as wild-type cells. At the same time, the mutant cells are deficient for intra-chromosomal recombination and nearly as sensitive to UV irradiation as a recA deletion strain. The cells are proficient in SOS induction, and results indicate the defect involves the capacity of RecA protein to participate directly in recombinational DNA repair. In vitro, the RecA423 protein binds to single-stranded DNA slowly, with an associated decline in the ATP hydrolytic activity. The RecA423 protein promoted a limited DNA strand exchange reaction when the DNA substrates were homologous, but no bypass of a short heterologous insert in the duplex DNA substrate was observed. These results indicate that poor binding to DNA and low ATP hydrolysis activity can selectively compromise certain functions of RecA protein. The RecA423 protein can promote recombination between homologous DNAs during Hfr crosses, indicating that the biochemical requirements for such genetic exchanges are minimal. However, the deficiencies in recombinational DNA repair suggest that the biochemical requirements for this function are more exacting. (C) 1996 Academic Press Limited
  • S Nagano, M Tanaka, K Ishimori, Y Watanabe, Morishima, I
    BIOCHEMISTRY 35 (45) 14251 - 14258 0006-2960 1996/11 [Refereed][Not invited]
     
    There are highly conserved hydrogen bonds between the distal His and the adjacent Asn in many peroxidases. Although the crystal structure of horseradish peroxidase C (HRP) is not available, comparison of the amino acid sequence with cytochrome c peroxidase indicates that Asn70 is making the hydrogen bond with the distal His in the active site of HRP. To investigate the catalytic roles of the hydrogen bond, Asn70 in HRP was replaced with Val (N70V) or Asp (N70D), Though UV-vis, CD, and H-1-NMR spectra of native (plant enzyme), wild-type (recombinant enzyme), and mutant HRPs suggest that the active site and secondary structure are very similar even after the mutation, the mutants exhibit low V-max values for the hydroquinone oxidation (native, 281; wild-type, 283; N70V, 18; and N70D, 33 mu M . min(-1)). The rates of compound I formation were decreased to less than 10% of that of the native enzyme. The reduction rates of compounds I and II by guaiacol also were reduced to less than 10% of that of the native enzyme. Substituent effects of various phenol derivatives on the reduction of native, wild-type, and mutant compound I were examined. Large negative Hammett rho values (rho(N70V:fast) = -4.0, rho(N70V:slow) = -3.6, rho(N70D) = -3.8, rho(native) = -6.9, and rho(wild-type) = -6.8) are an indication of electron transfer being the rate-determining step in the phenol oxidation. However, these results also indicate the participation of the deprotonation step in the compound I reduction process. The proton abstraction from phenol must be harder for the mutants due to the decrease of basicity of the distal His upon mutation. Contrary to phenol oxidation, ABTS [2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] oxidation activity was substantially increased by the mutations (native, 73; wild-type, 71; N70V, 217; and N70D, 234 mu M . s(-1)). The redox potentials of N70V and N70D compounds II are 957 and 970 mV (vs NHE), which are 95 and 108 mV higher than that of native compound II (862 mV), respectively, Therefore, the high ABTS oxidation activities of mutants are attributed to these high redox potentials of compound II.
  • M Tanaka, K Ishimori, Morishima, I
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 227 (2) 393 - 399 0006-291X 1996/10 [Refereed][Not invited]
     
    The distal His is an essential amino acid residue as a general acid-base catalyst for peroxidase reaction cycle. However, the x-ray structure of chloroperoxidase revealed that Glu is located near the heme, suggesting that the carboxyl group also assists cleavage of O-O bond in peroxides. In this paper, we examined functional and structural properties of a horseradish peroxidase mutant having Glu instead of the distal His. Although this amino acid replacement depressed reaction rate with H2O2 and oxidation activity for guaiacol, the mutant still exhibited much higher activity than mutants in which the distal His was replaced by hydrophobic amino acid. Kinetic measurements suggest that the proton abstraction is decelerated in the mutant due to large fluctuation of the carboxyl group of the distal Glu. Therefore, we can conclude that Gla can be a potent acid-base catalyst for peroxidase reaction cycle, if the carboxyl group can be fixed at the optimum position. (C) 1996 Academic Press, Inc.
  • T Matsui, S Nagano, K Ishimori, Y Watanabe, Morishima, I
    BIOCHEMISTRY 35 (40) 13118 - 13124 0006-2960 1996/10 [Refereed][Not invited]
     
    We have reported that H93C human myoglobin (Mb), in which proximal histidine (His93, F8) was replaced by cysteine, gave nearly identical spectroscopic features of P-450 [Adachi, S., Nagano, S., Ishimori, K., Watanabe, Y., Morishima, I., Egawa, T., Kitagawa, T., & Makino R. (1993) Biochemistry, 32, 241-252]. More importantly, the thiolate ligand enhanced its oxygenation activities when supported by H2O2 due to the exclusive encouragement of heterolytic O-O bond cleavage of peroxides. While we have attributed the enhanced heterolysis to the electron donation from the thiolate ligand, possible participation of the distal histidine (H64, E7) in H93C Mb cannot be eliminated. In addition, the racemic product formation catalyzed by H93C Mb implied that its distal cavity could prevent substrates from accessing to the heme and the reactions may proceed other than by the P-450 type mechanism (ferryl oxygen transfer). In order to clarify whether the distal histidine is involved in the O-O bond cleavage step and to improve accessibility of substrates, the distal histidine of H93C Mb is replaced by smaller and nonpolar residues, glycine (H64G/H93C Mb) and valine (H64V/H93C Mb), by site-directed mutagenesis. Various spectroscopic studies on these double-mutated Mbs revealed the ligation of cysteine to the ferric heme as a thiolate form. In the reaction with cumene hydroperoxide, the anionic nature of the proximal cysteine in H64G/H93C and H64V/H93C Mbs was found to encourage the heterolytic O-O bond cleavage as observed for H93C Mb, The results clearly demonstrate that the distal histidine of H93C Mb is hardly involved in the O-O bond cleavage step and are in good agreement with the role of thiolate ligation for the formation of the reactive intermediate, equivalent to compound I, in the catalytic cycle of P-450 reactions. In the oxygenation of methyl p-tolyl sulfide, the ratios of ferryl oxygen transfer increased in H64G/H93C Mb (58%) and H64V/H93C Mb (78%) as compared to H93C Mb (53%). The increased ratios of ferryl oxygen transfer imply the active site of H64G/H93C and H64V/H93C Mbs being more accessible for substrates; however, the sulfoxidation by the ferric mutant Mbs/H2O2 system was much slower than that by H93C Mb. The poor activities of these mutant Mbs are attributed to the significantly discouraged binding of H2O2.
  • K Wakasugi, K Ishimori, Morishima, I
    BIOCHIMIE 78 (8-9) 763 - 770 0300-9084 1996 [Refereed][Not invited]
     
    In the active center of cytochrome P450cam, Thr-252 is one of the conserved amino acid residues in the cytochrome P450 superfamily and plays a key role in hydroxylation of camphor. T252A mutant, in which Thr-252 is replaced by alanine, consumed O-2 at a rate comparable to that of the wild-type enzyme, whereas the amount of exo-5-hydroxycamphor formed was less than 10% of that formed by the wild-type enzyme and H2O2 is the main product in the hydroxylation reaction. H2O2 was also yielded by the valine mutant and the consumption rate of O-2 was much lower than that for the wild-type enzyme (Imai et al (1989) Pi-oc Natl Acad Sci USA 86, 7823-7827). On the basis of the H-1- and N-15-NMR spectra. it was revealed that the anionic nature of the axial thiolate and the heme-environmental structures were substantially affected in the absence of d-camphor by the amino acid substitution at 252 Thr. In T252A mutant, however, the binding of camphor reduced these conformational alterations in the heme vicinity, probably due to the formation of interactions between camphor and enzyme. On the other hand, T252V mutant still exhibited large reduction of the anionic nature of the axial ligand in the presence of d-camphor and structural changes around heme were also enhanced, since the affinity of the valine mutant to d-camphor was low. These results imply that the hydrophobic and/or steric effects of the valine residue at 252 interfere with the interactions around heme and camphor binding sites, which correspond to the larger functional defects for T252V mutant.
  • Keisuke Wakasugi, Kenhi Inaba, Koichiro Ishimori, Isao Morishima
    Journal of Inorganic Biochemistry Elsevier {BV} 59 (2-3) 435  1995/08 [Refereed][Not invited]
  • M UNNO, K ISHIMORI, Y ISHIMURA, MORISHIMA, I
    BIOCHEMISTRY 33 (32) 9762 - 9768 0006-2960 1994/08 [Refereed][Not invited]
     
    The effects of camphor and camphor analogues on the CO recombination kinetics of ferrous cytochrome P450(CAM) (P450(CAM)) at 293 K have been studied as a function of hydrostatic pressure (0.1-200 MPa) by means of flash photolysis. At 0.1 MPa, the association rate constant (k(on)) for substrate-free P450(CAM) is 8.5 x 10(6) M(-1) s(-1). Measurements as a function of pressure lead to a determination of the activation volume (Delta V-double dagger) of +4 cm(3) mol(-1) for substrate-free protein. This positive Delta V-double dagger is interesting because the CO association reaction of various hemoproteins, such as myoglobin and hemoglobin, exhibit negative Delta V-double dagger values [Adachi, S., & Morishima, I. (1989) J. Biol. Chem. 264, 18896-18901; Unno, M., Ishimori, K., & Morishima, I. (1990) Biochemistry 29, 10199-10205]. The binding of d-camphor and some camphor analogues (d-fenchone, 3-endo-bromocamphor, and 3,3,5,5-tetramethylcyclohexanone) into the heme pocket strongly influences the kinetics, i.e., k(on) is reduced ((1-10) x 10(5) M(-1) s(-1)) and Delta V-double dagger is altered to a negative value (-14 to -32 cm(3) mol(-1)). The negative Delta V-double dagger suggests that the effects of camphor and these camphor analogues are due to an increase in the iron-ligand bond formation barrier. On the other hand, the binding of adamantane and norcamphor does not affect the kinetics. This result is particularly surprising because both substrate analogues are located in the immediate vicinity of the CO binding site. Since both adamantane and norcamphor show high mobility in the heme active site, we conclude that a substrate fluctuation at the heme active site is an important determinant of the rate of the bond formation process.
  • M UNNO, K ISHIMORI, Y ISHIMURA, MORISHIMA, I
    BIOCHEMISTRY 33 (32) 9762 - 9768 0006-2960 1994/08 [Refereed][Not invited]
     
    The effects of camphor and camphor analogues on the CO recombination kinetics of ferrous cytochrome P450(CAM) (P450(CAM)) at 293 K have been studied as a function of hydrostatic pressure (0.1-200 MPa) by means of flash photolysis. At 0.1 MPa, the association rate constant (k(on)) for substrate-free P450(CAM) is 8.5 x 10(6) M(-1) s(-1). Measurements as a function of pressure lead to a determination of the activation volume (Delta V-double dagger) of +4 cm(3) mol(-1) for substrate-free protein. This positive Delta V-double dagger is interesting because the CO association reaction of various hemoproteins, such as myoglobin and hemoglobin, exhibit negative Delta V-double dagger values [Adachi, S., & Morishima, I. (1989) J. Biol. Chem. 264, 18896-18901; Unno, M., Ishimori, K., & Morishima, I. (1990) Biochemistry 29, 10199-10205]. The binding of d-camphor and some camphor analogues (d-fenchone, 3-endo-bromocamphor, and 3,3,5,5-tetramethylcyclohexanone) into the heme pocket strongly influences the kinetics, i.e., k(on) is reduced ((1-10) x 10(5) M(-1) s(-1)) and Delta V-double dagger is altered to a negative value (-14 to -32 cm(3) mol(-1)). The negative Delta V-double dagger suggests that the effects of camphor and these camphor analogues are due to an increase in the iron-ligand bond formation barrier. On the other hand, the binding of adamantane and norcamphor does not affect the kinetics. This result is particularly surprising because both substrate analogues are located in the immediate vicinity of the CO binding site. Since both adamantane and norcamphor show high mobility in the heme active site, we conclude that a substrate fluctuation at the heme active site is an important determinant of the rate of the bond formation process.
  • K WAKASUGI, K ISHIMORI, K IMAI, Y WADA, MORISHIMA, I
    JOURNAL OF BIOLOGICAL CHEMISTRY 269 (29) 18750 - 18756 0021-9258 1994/07 [Refereed][Not invited]
     
    In the genes of alpha- and beta-subunits of hemoglobin, Go showed that modules F1, F2 + F3, and F4 correspond to exons 1, 2, and 3, respectively (Go, M. (1981) Nature 291, 90). The analysis of the correlation of function with its exon pattern showed that the residues associated with the defined function are concentrated in the specific exons encoding the ''module'' (Eaten, W.A. (1980) Nature 284, 183). To investigate the functional and structural significance of the ''modular structure,'' we engineered a ''chimera'' subunit, in which module F4 of the beta-subunit was replaced by that of the alpha-subunit by use of mutagenesis. The NMR and resonance Raman spectra of the isolated ''chimera beta alpha-subunit'' have revealed that it has a beta-subunit-like heme environmental structure. However, the gel chromatography and NMR spectra of mixtures of the chimera and native subunits clearly showed that the chimera beta alpha-subunit binds specifically to the beta-subunit to form a heterotetramer, not to the alpha-subunit. These results led us to conclude that the predominant role of the module F4 is the subunit association and suggest that the modules are structural and functional units that have advantages in producing stable functional proteins.
  • K ISHIMORI, M HASHIMOTO, K IMAI, K FUSHITANI, G MIYAZAKI, H MORIMOTO, Y WADA, MORISHIMA, I
    BIOCHEMISTRY 33 (9) 2546 - 2553 0006-2960 1994/03 [Refereed][Not invited]
     
    The penultimate tyrosine in the hemoglobin subunit is considered to be one of the most important residues for the normal structure and function of hemoglobin. To elucidate the functional and structural role of the penultimate residue in the alpha-subunit, we prepared new artificial mutants; Hb Y140 alpha Q, in which Tyr-140 alpha is replaced by a nonaromatic residue, Gln, and Hb Y140 alpha F, which loses its hydrogen bond to Val-93 alpha by the substitution of Phe for Tyr. HB Y140 alpha Q exhibited a markedly increased oxygen affinity and almost completely diminished cooperativity, whereas Hb Y140 alpha F showed similar but less extensively impaired function, indicating that the aromatic residue at the penultimate position in the cr-subunit contributes to the stabilization of the T-quaternary structure as does the corresponding residue in the beta-subunit. However, the deoxygenated forms of these mutants bear significant T-state character in their spectroscopic properties observed at high protein concentrations. The tetramer-dimer equilibrium data of the mutants suggested that a significant part of the functional alterations observed for dilute solution appears to result from partial dissociation into alpha beta dimers rather than direct destabilization of the T-quaternary structure in the deoxygenated form. Therefore, we can conclude that the penultimate tyrosine in the alpha-chain plays a key role not only in the stabilization of the T-state but also in the subunit assembly. Such ease of dissociation from tetramer to dimers by amino acid substitution at the penultimate position did not occur in the beta-subunit mutant, implying different structural and functional roles of the penultimate tyrosine between the alpha- and P-subunits.
  • M HASHIMOTO, K ISHIMORI, K IMAI, G MIYAZAKI, H MORIMOTO, Y WADA, MORISHIMA, I
    BIOCHEMISTRY 32 (49) 13688 - 13695 0006-2960 1993/12 [Refereed][Not invited]
     
    To clarify the functional and structural roles of Thr-38alpha at the alpha1-beta2 interface, two artificial alpha-chain mutants, in which Thr-38alpha is replaced by Ser (Hb T38alphaS) or Val (Hb T38alphaV), were prepared. Thr-38alpha is one of the highly conserved amino acid residues in hemoglobins and forms a hydrogen bond to Asp-99beta, which is a crucial residue to stabilize the T state, via a water molecule in the deoxygenated form. We investigated their oxygen binding properties together with structural consequences of the mutations by using various spectroscopic probes. Their oxygen equilibrium curves showed small changes in the oxygen binding properties. Structural probes such as ultraviolet-region derivative and oxy-minus-deoxy difference spectra, rcsonance Raman scattering, and H-1-NMR spectra also indicated that the oxy and deoxy forms of these mutants show spectra characteristic of the R and T states, respectively, and the R-T transition is not very disturbed. The present structural and functional data of the mutants imply that the hydrogen bond between Thr-38alpha and Asp-99beta does not play a key role in stabilizing the deoxy T structure, which is in sharp contrast to the role of the hydrogen bond between Tyr-42alpha and Asp-99beta, and suggest that the interactions via the intersubunit hydrogen bonds are highly site-specific, depending on the amino acid residue which participates in them.
  • A TOGI, K ISHIMORI, M UNNO, T KONNO, MORISHIMA, I, G MIYAZAKI, K IMAI
    BIOCHEMISTRY 32 (38) 10165 - 10169 0006-2960 1993/09 [Refereed][Not invited]
     
    To clarify the effects of specific inter- and intrasubunit hydrogen bonds on the R-T transition in human hemoglobin (Hb A), the recombination reaction of carbon monoxide with artificial mutant Hbs was measured and analyzed. One of the hydrogen bonds we focused on is formed between Tyr-42alpha and Asp-99beta in the alpha1-beta2 interface of Hb A, which is one of the hydrogen bonds characteristic of the T state. Hb His-42alpha, in which Tyr-42alpha is replaced by His to perturb this hydrogen bond, showed that the ligand-free R to T transition rate was decreased by 20-fold compared with that for Hb A. This mutation caused the destabilization of the transition state in the R to T quaternary structure change by about 7 kJ mol-1, indicating that the hydrogen bond between Tyr-42alpha and Asp-99beta plays a definite role in the R-T transition as well as in stabilization of the equilibrium T state. Hb Phe-145beta, in which Tyr-145beta is replaced by Phe and the intrasubunit hydrogen bond between Tyr-145beta and Val-98beta is lacking, also showed a slow R-T transition rate as observed in Hb His-42alpha. The published crystallographic data suggest that this intrasubunit hydrogen bond stabilizes the transition state by reducing the freedom of motion of the C-terminus of the beta subunit and, thereby, facilitates the R-T transition.
  • S ADACHI, S NAGANO, K ISHIMORI, Y WATANABE, MORISHIMA, I, T EGAWA, T KITAGAWA, R MAKINO
    BIOCHEMISTRY 32 (1) 241 - 252 0006-2960 1993/01 [Refereed][Not invited]
     
    Histidine-93(F8) in human myoglobin (Mb), which is the proximal ligand of the heme iron, has been replaced with cysteine or tyrosine by site-directed mutagenesis. The resultant proximal cysteine and tyrosine mutant Mbs (H93C and H93Y Mbs, respectively) exhibit the altered axial ligation analogous to P-450, chloroperoxidase, and catalase. Coordination of cysteine or tyrosine to the ferric heme iron is confirmed by spectroscopic measurements including electronic absorption, hyperfine-shifted H-1-NMR, EPR, resonance Raman spectroscopies, and redox potential measurements of ferric/ferrous couple. H93C Mb is five-coordinate ferric high-spin with the proximal cysteine. H93Y Mb bearing the proximal tyrosine ligated to the iron is also in a ferric high-spin, five-coordinate state. The reactions of the mutants with cumene hydroperoxide show that the thiolate ligand enhances heterolytic O-O bond cleavage of the oxidant, while the phenolate ligand hardly affects the heterolysis/homolysis ratio for O-O bond scission in comparison with wild-type Mb. Monooxygenase activities such as epoxidation of styrene and N-demethylation of N,N-dimethylaniline, and catalase activity (dismutation of hydrogen peroxide) by wild-type Mb and the mutants, are examined by using H2O2. The increase of the catalytic activities by the mutation was, at most, 5-fold in the epoxidation reaction.
  • Ishimori K, Morishima I
    Magnetic Resonance in Chemistry 31 (13) S113 - S117 1993 [Refereed][Not invited]
  • Koichiro ISHIMORI, Isao MORISHIMA
    Seibutsu Butsuri Biophysical Society of Japan 33 (4) 212  1993 [Refereed][Not invited]
  • Shinichi Adachi, Shingo Nagano, Koichiro Ishimori, Yoshihito Watanabe, Isao Morishima, Tsuyoshi Egawa, Teizo Kitagawa, Ryu Makino
    Biochemistry American Chemical Society ({ACS}) 32 (1) 241  1993/01 [Refereed][Not invited]
  • S ADACHI, N SUNOHARA, K ISHIMORI, MORISHIMA, I
    JOURNAL OF BIOLOGICAL CHEMISTRY 267 (18) 12614 - 12621 0021-9258 1992/06 [Refereed][Not invited]
     
    Site-specific mutants of human myoglobin (Mb) have been prepared, in which Leu29(B10) is replaced by Ala(L29A) or Ile(L29I), in order to examine the influence of this highly conserved residue in the hydrophobic clusters of the heme distal site on the heme environmental structure and ligand binding properties of Mb. Structural characterizations of these recombinant Mbs are studied by electronic absorption, infrared (IR), one- and two-dimensional proton nuclear magnetic resonance spectroscopies, and ligand-binding kinetics by laser photolysis measurements under ambient and high pressures (up to 2000 bar). Multiple split carbon monoxide (CO) stretch bands in the IR spectra of mutant Mbs exhibit a relative decrease of the 1945 cm-1 band (approximately 50%) which is associated with an upright binding geometry of CO, accompanied by an increase of the tilted CO conformer at 1932 cm-1. On the basis of these results, replacement of Leu29(B10) by Ala or Ile appears to allow bound CO to rotate from a conformation pointing toward the beta-meso carbon of the heme group to the one pointing toward the alpha-meso carbon atom, presumably filling the space left by removal of the delta-2 carbon atom of Leu29(B10). These substitutions cause the rate constants for CO and O2 association to decrease almost 3-5-fold. Present results show that CO and O2 bindings to the heme iron of Mb are controlled by Leu29(B10) by influencing the structure of close vicinity of the heme and the geometry of iron-bound ligand. Further, mutant Mbs (Leu72(E15)-->Ala and Leu104 (G5)-->Ala) which have altered residues in another hydrophobic clusters around proximal and distal site are also examined.
  • S ADACHI, N SUNOHARA, K ISHIMORI, MORISHIMA, I
    JOURNAL OF BIOLOGICAL CHEMISTRY 267 (18) 12614 - 12621 0021-9258 1992/06 [Refereed][Not invited]
     
    Site-specific mutants of human myoglobin (Mb) have been prepared, in which Leu29(B10) is replaced by Ala(L29A) or Ile(L29I), in order to examine the influence of this highly conserved residue in the hydrophobic clusters of the heme distal site on the heme environmental structure and ligand binding properties of Mb. Structural characterizations of these recombinant Mbs are studied by electronic absorption, infrared (IR), one- and two-dimensional proton nuclear magnetic resonance spectroscopies, and ligand-binding kinetics by laser photolysis measurements under ambient and high pressures (up to 2000 bar). Multiple split carbon monoxide (CO) stretch bands in the IR spectra of mutant Mbs exhibit a relative decrease of the 1945 cm-1 band (approximately 50%) which is associated with an upright binding geometry of CO, accompanied by an increase of the tilted CO conformer at 1932 cm-1. On the basis of these results, replacement of Leu29(B10) by Ala or Ile appears to allow bound CO to rotate from a conformation pointing toward the beta-meso carbon of the heme group to the one pointing toward the alpha-meso carbon atom, presumably filling the space left by removal of the delta-2 carbon atom of Leu29(B10). These substitutions cause the rate constants for CO and O2 association to decrease almost 3-5-fold. Present results show that CO and O2 bindings to the heme iron of Mb are controlled by Leu29(B10) by influencing the structure of close vicinity of the heme and the geometry of iron-bound ligand. Further, mutant Mbs (Leu72(E15)-->Ala and Leu104 (G5)-->Ala) which have altered residues in another hydrophobic clusters around proximal and distal site are also examined.
  • K ISHIMORI, K IMAI, G MIYAZAKI, T KITAGAWA, Y WADA, H MORIMOTO, MORISHIMA, I
    BIOCHEMISTRY 31 (12) 3256 - 3264 0006-2960 1992/03 [Refereed][Not invited]
     
    In order to clarify the functional and structural role of intra- and intersubunit hydrogen bonds in human hemoglobin (Hb A), we prepared two artificial beta-chain mutant hemoglobins by site-directed mutagenesis. The mutant Hb Phe-37-beta, in which Trp-37-beta is replaced by Phe to remove the intersubunit hydrogen bond between Asp-94-alpha and Trp-37-beta at the alpha-1-beta-2 interface in deoxy Hb A, showed a markedly increased oxygen affinity and almost completely diminished Bohr effect and cooperativity. However, H-1-NMR data indicated that the structure of deoxy Hb Phe-37-beta is rather similar to that of deoxy Hb A. The enhanced tetramer-to-dimer dissociation previously observed in Hb Hirose (Trp-37-beta --> Ser) together with our observation of the effects of organic phosphate on the structure and function of Hb Phe-37-beta suggested that a large part of the abnormal properties of Hb Phe-37-beta observed for dilute solutions appears to result from partial dissociation into alpha-beta-dimers rather than direct destabilization of the T-quaternary structure in the deoxygenated state. Thus, the primary and direct role of the hydrogen bond between Asp-94-alpha and Trp-37-beta is to stabilize the tetrameric assembly, and thereby this hydrogen bond indirectly contributes to stabilization of the T-quaternary structure. The other mutant Hb Phe-145-beta has a Phe residue at the 145-beta site and lacks the intrasubunit hydrogen bond formed between Tyr-145-beta and the carbonyl group of Val-98-beta in deoxy Hb A. Although this hydrogen bond has been considered to fix the phenolic group of Tyr-145-beta in a pocket between the F and H helices, to strengthen the salt bridges formed by His-146-beta, and thereby to stabilize the T-quaternary structure, Hb Phe-145-beta exhibited only mild functional and structural alterations. This result led us to conclude that the van der Waals contacts between the benzene ring of Tyr-145-beta and the tyrosine pocket, rather than the hydrogen bond between Tyr-145-beta and Val-98-beta, make a major contribution to the stabilization of the T-quaternary structure. The present NMR spectra of deoxygenated Hb Phe-37-beta and Hb Phe-145-beta further showed that the exchangeable proton resonance observed at 6.4 ppm for deoxy Hb A originates from the intersubunit hydrogen bond between Asp-94-alpha and Trp-37-beta, although it has previously been assigned to the intrasubunit hydrogen bond between Val-98-beta and Try-145-beta.
  • M UNNO, K ISHIMORI, MORISHIMA, I, T NAKAYAMA, K HAMANOUE
    BIOCHEMISTRY 30 (44) 10679 - 10685 0006-2960 1991/11 [Refereed][Not invited]
     
    The effects of pressure on the recombination kinetics of carbon monoxide binding to the isolated alpha and beta-chains of human adult hemoglobin at pH 7, approximately 20-degrees-C, were studied by the use of millisecond and nanosecond laser photolyses. The kinetic data were analyzed on the basis of a simple three-species model, which assumes two elementary reaction processes of bond formation and ligand migration steps. The activation volume for each elementary step was obtained from the pressure dependence of the rate constants. A pressure-dependent activation volume change from negative to positive values in the bimolecular carbon monoxide association reaction was observed for both of the isolated chains. This finding is attributed to a change of the rate-limiting step from the bond formation step to the ligand migration step. For both of the isolated chains, the activation volumes for ligand migration into and from the protein were estimated as +12-16 and +7-11 cm3 mol-1, respectively. These positive activation volumes for the ligand migration process may be caused by conformational fluctuations of proteins, that is, the conformational changes from "closed" to "open" structure. In the iron-ligand bond formation process, the activation volumes are -15 to -22 cm3 mol-1, which are almost identical to that for the model heme complexes [Taube, D. J., Projahn, H.-D., van Eldik, R., Magde, D., & Traylor, T. G. (1990) J. Am. Chem. Soc. 112, 6880-68861. Accordingly, the surrounding protein contributions to the activation volumes for the bond formation process could be small. A slight difference in the activation volumes between the isolated chains was found for each elementary step. This is discussed in relation to characteristic features of the dynamic aspects of the isolated alpha and beta-chains.
  • S ADACHI, S NAGANO, Y WATANABE, K ISHIMORI, MORISHIMA, I
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 180 (1) 138 - 144 0006-291X 1991/10 [Refereed][Not invited]
  • K IMAI, K FUSHITANI, G MIYAZAKI, K ISHIMORI, T KITAGAWA, Y WADA, H MORIMOTO, MORISHIMA, I, DT SHIH, J TAME
    JOURNAL OF MOLECULAR BIOLOGY 218 (4) 769 - 778 0022-2836 1991/04 [Refereed][Not invited]
  • M UNNO, K ISHIMORI, MORISHIMA, I
    BIOCHEMISTRY 29 (44) 10199 - 10205 0006-2960 1990/11 [Refereed][Not invited]
  • T ARAI, SI NAKAO, K MORI, K ISHIMORI, MORISHIMA, I, T MIYAZAWA, B FRITZZIEROTH
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 169 (1) 153 - 158 0006-291X 1990/05 [Refereed][Not invited]
  • K ISHIMORI, A TSUNESHIGE, K IMAI, MORISHIMA, I
    BIOCHEMISTRY 28 (21) 8603 - 8609 0006-2960 1989/10 [Refereed][Not invited]
  • Koichiro Ishimori, Antonio Tsuneshige, Kiyohiro Imai, Isao Morishima
    Biochemistry American Chemical Society ({ACS}) 28 (21) 8603  1989/10 [Refereed][Not invited]
  • K ISHIMORI, MORISHIMA, I, K IMAI, K FUSHITANI, G MIYAZAKI, D SHIH, J TAME, J PEGNIER, K NIGAI
    JOURNAL OF BIOLOGICAL CHEMISTRY 264 (25) 14624 - 14626 0021-9258 1989/09 [Refereed][Not invited]
     
    Ishimori K, Morishima I, Imai K, Fushitani K, Miyazaki G, Shih D, Tame J, Pegnier J, Nigai K, The Journal of biological chemistry, 1989, vol. 264, no. 25, pp. 14624-14626, 1989
  • K ISHIMORI, MORISHIMA, I
    BIOCHEMISTRY 27 (13) 4747 - 4753 0006-2960 1988/06 [Refereed][Not invited]
  • K ISHIMORI, MORISHIMA, I
    BIOCHEMISTRY 27 (11) 4060 - 4066 0006-2960 1988/05 [Refereed][Not invited]
  • T MATUURA, M KOHNO, Y KANAYAMA, K YASUNARI, K MURAKAWA, T TAKEDA, K ISHIMORI, MORISHIMA, I, T YONEZAWA
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 143 (3) 1012 - 1017 0006-291X 1987/03 [Refereed][Not invited]
  • Isao Morishima, Mitsunobu Hara, Koichiro Ishimori
    Biochemistry American Chemical Society ({ACS}) 25 (22) 7243  1986/11 [Refereed][Not invited]
  • Koichiro Ishimori, Isao Morishima
    Biochemistry American Chemical Society ({ACS}) 25 (17) 4892  1986/08 [Refereed][Not invited]

Books etc

  • タンパク質科学
    化学同人 2005
  • Protein Science
    Kagaku Dojin 2005

MISC

Research Grants & Projects

  • Structural and Functional Characterization of Metalloproteins and Its Molecula Design
    Date (from‐to) : 1983

Educational Activities

Teaching Experience

  • Advanced Lecture of Physical Chemistry
    開講年度 : 2018
    課程区分 : 修士課程
    開講学部 : 総合化学院
    キーワード : 分子構造決定、高分子、分子集合体
  • Introductory of Physical Chemistry
    開講年度 : 2018
    課程区分 : 修士課程
    開講学部 : 総合化学院
    キーワード : 分子軌道法,対称,分光学,表面,平衡と速度論
  • Inter-Graduate School Classes(General Subject):Natural and Applied Sciences
    開講年度 : 2018
    課程区分 : 修士課程
    開講学部 : 大学院共通科目
    キーワード : 分子軌道法,対称,分光学,表面,平衡と速度論
  • Physical Chemistry Ⅲ
    開講年度 : 2018
    課程区分 : 学士課程
    開講学部 : 理学部
    キーワード : 相転移 混合の熱力学 相図 分子の対称 統計熱力学 磁気共鳴 分子間相互作用 回折

Campus Position History

  • 2013年10月1日 
    2014年3月31日 
    創成研究機構副機構長
  • 2013年4月1日 
    2014年3月31日 
    研究戦略室室員
  • 2013年4月1日 
    2014年3月31日 
    役員補佐
  • 2014年4月1日 
    2015年3月31日 
    教育研究評議会評議員
  • 2014年4月1日 
    2015年3月31日 
    大学院理学研究院副研究院長
  • 2015年4月1日 
    2017年3月31日 
    教育研究評議会評議員
  • 2015年4月1日 
    2017年3月31日 
    大学院理学研究院長
  • 2015年4月1日 
    2017年3月31日 
    理学部長
  • 2017年4月1日 
    2019年3月31日 
    教育研究評議会評議員
  • 2017年4月1日 
    2019年3月31日 
    大学院理学研究院長
  • 2017年4月1日 
    2019年3月31日 
    理学部長

Position History

  • 2013年10月1日 
    2014年3月31日 
    創成研究機構副機構長
  • 2013年4月1日 
    2014年3月31日 
    研究戦略室室員
  • 2013年4月1日 
    2014年3月31日 
    役員補佐
  • 2014年4月1日 
    2015年3月31日 
    教育研究評議会評議員
  • 2014年4月1日 
    2015年3月31日 
    大学院理学研究院副研究院長
  • 2015年4月1日 
    2017年3月31日 
    教育研究評議会評議員
  • 2015年4月1日 
    2017年3月31日 
    大学院理学研究院長
  • 2015年4月1日 
    2017年3月31日 
    理学部長
  • 2017年4月1日 
    2019年3月31日 
    教育研究評議会評議員
  • 2017年4月1日 
    2019年3月31日 
    大学院理学研究院長
  • 2017年4月1日 
    2019年3月31日 
    理学部長


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