Yoshitsugu Shiro

RIKEN, Вако, Saitama, Japan

Are you Yoshitsugu Shiro?

Claim your profile

Publications (228)917.91 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: UV–visible absorption spectroscopy is useful for probing the electronic and structural changes of protein active sites, and thus the on-line combination of X-ray diffraction and spectroscopic analysis is increasingly being applied. Herein, a novel absorption spectrometer was developed at SPring-8 BL26B2 with a nearly on-axis geometry between the X-ray and optical axes. A small prism mirror was placed near the X-ray beamstop to pass the light only 2° off the X-ray beam, enabling spectroscopic analysis of the X-ray-exposed volume of a crystal during X-ray diffraction data collection. The spectrometer was applied to NO reductase, a heme enzyme that catalyzes NO reduction to N 2 O. Radiation damage to the heme was monitored in real time during X-ray irradiation by evaluating the absorption spectral changes. Moreover, NO binding to the heme was probed via caged NO photolysis with UV light, demonstrating the extended capability of the spectrometer for intermediate analysis.
    No preview · Article · Jan 2016 · Journal of Synchrotron Radiation
  • Akihiro Doi · Hiro Nakamura · Yoshitsugu Shiro · Hiroshi Sugimoto
    [Show abstract] [Hide abstract]
    ABSTRACT: ChrA is a response regulator (RR) in the two-component system involved in regulating the degradation and transport of haem (Fe–porphyrin) in the pathogen Corynebacterium diphtheriae . Here, the crystal structure of full-length ChrA is described at a resolution of 1.8 Å. ChrA consists of an N-terminal regulatory domain, a long linker region and a C-terminal DNA-binding domain. A structural comparison of ChrA with other RRs revealed substantial differences in the relative orientation of the two domains and the conformation of the linker region. The structural flexibility of the linker could be an important feature in rearrangement of the domain orientation to create a dimerization interface to bind DNA during haem-sensing signal transduction.
    No preview · Article · Aug 2015 · Acta Crystallographica Section F: Structural Biology Communications
  • [Show abstract] [Hide abstract]
    ABSTRACT: L-Tryptophan 2,3-dioxygenase (TDO) is a protoheme-containing enzyme catalyzing the production of N-formylkynurenine by inserting O2 into the pyrrole ring of L-tryptophan. Although a ferrous-oxy form (Fe2+-O2) has been established to be an obligate intermediate in the reaction, details of the ring opening reaction remain still elusive. In the present study, the O2 inserting reaction catalyzed by Pseudomonas TDO (PaTDO) was examined using heme-modification approach, which enabled to draw quantitative correlation between the inductive electronic effects of the heme-substituents and the substituent-induced changes in the functional behaviors of the ferrous-oxy form. We succeeded in the preparation of reconstituted PaTDO with synthetic hemes, which were different in the inductive electron-withdrawing nature of the heme-substituents at the 2- and 4-positions. An increase in the electron-withdrawing power of the heme-substituents elevated the redox potential of reconstituted PaTDO, showing that the stronger the electron-withdrawing ability of the heme-substituents, the lesser the electron density on the heme-iron. The decrease in the electron density of the heme-iron resulted in the higher frequency shift of the C-O stretch of the heme-bound CO and enhanced the O2 dissociation from the ferrous-oxy intermediate. This result was interpreted due to a lesser  back-donation from the heme-iron to the bound CO or O2. More importantly, the reaction rates of the ferrous-oxy intermediate to oxidize L-Trp were increased with the electron-withdrawing ability of the heme-substituents, implying that the higher electron-deficient ferrous-oxy heme is favored for the PaTDO-catalyzed oxygenation. On the basis of these results, we propose that the initial step of the dioxygen activation by PaTDO is a direct electrophilic addition of the heme-bound O2 to the indole ring of L-Trp.
    No preview · Article · May 2015 · Biochemistry
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The direct hydroxylation of alkanes under mild conditions is a key issue in catalytic chemistry that addresses an increasing number of industrial and economic requirements. Cytochrome P450s are monooxygenases that are capable of oxidizing less reactive C–H bonds; however, wild-type P450s are unavailable for many important nonnative substrates such as gaseous alkanes. Here, we report the enhanced hydroxylation activities and crystallographic evidence for the role of decoy molecules in wild-type P450BM3-catalyzed hydroxylation of gaseous ethane and propane by using the next generation of decoy molecule. A cocrystal structure of P450BM3 and a decoy molecule reveals that an N-perfluoroacyl amino acid (decoy molecule) partially occupies the substrate-binding site of P450BM3. This binding of the decoy re-forms the active site pocket to allow the accommodation of small substrates and simultaneously influences the formation of compound I species by expelling water molecules from the active site.
    Full-text · Article · Jan 2015 · ACS Catalysis
  • [Show abstract] [Hide abstract]
    ABSTRACT: Numerous cytochrome P450s are involved in secondary metabolite biosynthesis. The biosynthetic gene cluster for reveromycin A (RM-A), which is a promising lead compound with anti-osteoclastic activity, also includes a P450 gene, revI. To understand the roles of P450revI, we comprehensively characterized the enzyme by genetic, kinetic, and structural studies. The revI gene disruptants (ΔrevI) resulted in accumulation of reveromycin T (RM-T), and revI gene complementation restored RM-A production, indicating that the physiological substrate of P450revI is RM-T. Indeed, the purified P450revI catalyzed the C18-hydroxylation of RM-T more efficiently than the other RM derivatives tested. Moreover, the 1.4-Å resolution co-crystal structure of P450revI with RM-T revealed that the substrate binds the enzyme with a folded compact conformation for C18-hydroxylation. To address the structure-enzyme activity relationship, site-directed mutagenesis was performed in P450revI. Arg190Ala and Arg81Ala mutations, which abolished salt bridge formation with C1 and C24 carboxyl groups of RM-T, respectively, resulted in significant loss of enzyme activity. The interaction between Arg190 and the C1 carboxyl group of RM-T elucidated why P450revI was unable to catalyze both RM-T 1-methyl ester and RM-T 1-ethyl ester. Moreover, the accumulation of RM-T in ΔrevI mutants enabled us to characterize its biological activity. Our results show that RM-T had stronger anticancer activity and isoleucyl-tRNA synthetase inhibition than RM-A. However, RM-T showed much less anti-osteoclastic activity than RM-A, indicating that hemisuccinate moiety is important for the activity. Structure-based P450revI engineering for novel hydroxylation and subsequent hemisuccinylation will help facilitate the development of RM-derivatives with anti-osteoclast activity.
    No preview · Article · Sep 2014 · Journal of Biological Chemistry
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Iron is an essential element for almost all organisms, since iron serves as a catalytic center for redox reactions in many enzymes. Bacterial pathogens need to acquire iron from tissues of host to survive. Heme transport by ATP-binding cassette (ABC) transporter plays a key role in pathological processes. In gram-negative bacteria, the heme or heme protein binds to specific outer membrane receptors on the bacterial surface. The heme is then transported into the cell via ABC transporters. Here, we present the crystal structure of the heme transporter complex BhuUV-T from Burkholderia cenocepacia at 3.5 Å resolution in nucleotide-free state. The permeation pathway created by transmembrane helices of two BhuU subunit exhibits an inward-facing conformation. Comparison with the outward-facing conformation previously reported for the heme transporter HmuUV from Yersinia pestis and homologous vitamin B12 transporter BtuCD-F from E. coli indicates the structural mechanism involving the translational shift of nucleotide binding subunit and repositioning of the helices of permease subunits for substrate translocation. Structure of interface between BhuUV and periplasmic heme-binding protein BhuT suggests that the acidic residues of BhuU at the periplasmic interface may have an important role in releasing the heme from BhuT. We also determined the BhuT in apo and two types of holo form, providing the structural basis for transient and ambiguous heme recognition.
    Preview · Article · Aug 2014 · Acta Crystallographica Section A: Foundations and Advances
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Iron is one of the essential elements for all living organisms. Pathogenic bacteria acquire heme from the host proteins as an iron source. Gram-negative opportunistic pathogen, Burkholderia cenocepacia have ATP-binding cassette (ABC) transporter BhuUV-T complex to permeate heme through inner membrane. BhuT, periplasmic binding protein (PBP), bind and deliver heme(s) to inner membrane transporter BhuUV complex. BhuUV is 2:2 complex of the transmembrane permease subunit and cytoplasmic ATP-binding subunit which couple ATP hydrolysis to solute translocation. The molecular level mechanism of heme recognition and dissociation by PBP and heme transport by transporter complex are not fully understood. Here we describe the crystal structures of the heme-free and two types of heme-bound state of BhuT. These crystals were obtained in different crystallization conditions. Crystals diffracted to high resolution at SPring-8. BhuT is composed of two globular domains linked by a long a-helix. The transport ligand heme is bound between the two domains. A detailed structural comparison of the conformation of the domain and residues involved in the heme binding will be presented.
    Preview · Article · Aug 2014 · Acta Crystallographica Section A: Foundations and Advances
  • Yoshitsugu Shiro · Takehiko Tosha · Erina Terasaka · Hiroshi Sugimoto

    No preview · Article · Jul 2014 · Biochimica et Biophysica Acta (BBA) - Bioenergetics
  • [Show abstract] [Hide abstract]
    ABSTRACT: Nitric oxide reductase (NOR) catalyzes the generation of nitrous oxide (N2 O) via the reductive coupling of two nitric oxide (NO) molecules at a heme/non-heme Fe center. We report herein on the structures of the reduced and ligand-bound forms of cytochrome c-dependent NOR (cNOR) from Pseudomonas aeruginosa at a resolution of 2.3-2.7 Å, to elucidate structure-function relationships in NOR, and compare them to those of cytochrome c oxidase (CCO) that is evolutionarily related to NOR. Comprehensive crystallographic refinement of the CO-bound form of cNOR suggested that a total of four atoms can be accommodated at the binuclear center. Consistent with this, binding of bulky acetaldoxime (CH3 -CH=N-OH) to the binuclear center of cNOR was confirmed by the structural analysis. Active site reduction and ligand binding in cNOR induced only ~0.5 Å increase in the heme/non-heme Fe distance, but no significant structural change in the protein. The highly localized structural change is consistent with the lack of proton-pumping activity in cNOR, because redox-coupled conformational changes are thought to be crucial for proton pumping in CCO. It also permits the rapid decomposition of cytotoxic NO in denitrification. In addition, the shorter heme/non-heme Fe distance even in the bulky ligand-bound form of cNOR (~4.5 Å) than the heme/Cu distance in CCO (~5 Å) suggests the ability of NOR to maintain two NO molecules within a short distance in the confined space of the active site, thereby facilitating N-N coupling to produce a hyponitrite intermediate for the generation of N2 O. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
    No preview · Article · Jul 2014 · Proteins Structure Function and Bioinformatics
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Nitric oxide reductase (NOR) catalyzes the reduction of nitric oxide to generate nitrous oxide. We recently reported on the crystal structure of a quinol-dependent NOR (qNOR) from Geobacillus stearothermophilus [Y. Matsumoto, T. Tosha, A.V. Pisliakov, T. Hino, H. Sugimoto, S. Nagano, Y. Sugita and Y. Shiro, Nat. Struct. Mol. Biol. 19 (2012) 238–246], and suggested that a water channel from the cytoplasm, which is not observed in cytochrome c-dependent NOR (cNOR), functions as a pathway transferring catalytic protons. Here, we further investigated the functional and structural properties of qNOR, and compared the findings with those for cNOR. The pH optimum for the enzymatic reaction of qNOR was in the alkaline range, whereas Pseudomonas aeruginosa cNOR showed a higher activity at an acidic pH. The considerably slower reduction rate, and a correlation of the pH dependence for enzymatic activity and the reduction rate suggest that the reduction process is the rate-determining step for the NO reduction by qNOR, while the reduction rate for cNOR was very fast and therefore is unlikely to be the rate-determining step. A close examination of the heme/non-heme iron binuclear center by resonance Raman spectroscopy indicated that qNOR has a more polar environment at the binuclear center compared with cNOR. It is plausible that a water channel enhances the accessibility of the active site to solvent water, creating more polar environment in qNOR. This structural feature could control certain properties of the active site, such as redox potential, which could explain the different catalytic properties of the two NORs. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.
    Preview · Article · Jul 2014 · Biochimica et Biophysica Acta
  • Source

    Preview · Article · Jul 2014
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A sulfur-modified gold-supported palladium material (SAPd) has been developed bearing palladium nanoparticles on its surface. Herein, we report the for the first time the use of SAPd to affect a Pd-nanoparticle-catalyzed 1,7-Pd migration reaction for the synthesis of benzotriazoles via C-H bond activation. The resulting benzotriazoles were evaluated in terms of their inhibitory activity towards indoleamine 2,3-dioxygenase.
    Full-text · Article · Jun 2014 · ChemInform
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The heme acquisition system A protein secreted by Pseudomonas aeruginosa (HasAp ) can capture several synthetic metal complexes other than heme. The crystal structures of HasAp harboring synthetic metal complexes revealed only small perturbation of the overall HasAp structure. An inhibitory effect upon heme acquisition by HasAp bearing synthetic metal complexes was examined by monitoring the growth of Pseudomonas aeruginosa PAO1. HasAp bound to iron-phthalocyanine inhibits heme acquisition in the presence of heme-bound HasAp as an iron source.
    Full-text · Article · Mar 2014 · Angewandte Chemie International Edition

  • No preview · Article · Mar 2014 · Angewandte Chemie
  • [Show abstract] [Hide abstract]
    ABSTRACT: Cytoglobin (Cgb) was discovered a decade ago and is a fourth member of the group of hexacoordinated globin-folded proteins. Although some crystal structures have been reported and several functions have been proposed for Cgb, its physiological role remains uncertain. In this study, we measured cyanide binding to the ferric state of the wild-type (WT) Cgb, and found that the binding consisted of multiple steps. These results indicated that Cgb may be comprised of several forms, and the presence of monomers, dimers, and tetramers was subsequently confirmed by SDS-PAGE. Remarkably, each species contained two distinguishable forms, and, in the monomer, analyses of alternative cysteine states suggested the presence of an intramolecular disulfide bond (monomer SS form) and a structure with unpaired thiol groups (monomer SH form). These confirmed that forms were separated by gel-exclusion chromatography, and that the cyanide binding of the separated fractions was again measured; they showed different affinities for cyanide, with the monomer fraction showing the highest affinity. In addition, the ferrous state in each fraction showed distinct carbon monoxide (CO)-binding properties, and the affinities for cyanide and CO suggested a linear correlation. Furthermore, we also prepared several variants involving the two cysteine residues. The C38S and C83S variants showed a binding affinity for cyanide similar to the value for the monomer SH form, and hence the fraction with the highest affinity for exogenous ligands was designated as a monomer SS form. We concluded that polymerization could be a mechanism that triggers the exertion of various physiological functions of this protein and that an appropriate disulfide bond between the two cysteine residues was critical for regulating the binding affinity of Cgb, which can act as a ROS scavenger, for exogenous ligands.
    No preview · Article · Mar 2014 · Journal of inorganic biochemistry
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The O2-binding carboxylate-bridged diiron site in DcrH-Hr was engineered in an effort to perform the H2O2-dependent oxidation of external substrates. A His residue was introduced near the diiron site in place of a conserved residue, Ile119. The I119H variant promotes the oxidation of guaiacol and 1,4-cyclohexadiene upon addition of H2O2.
    Full-text · Article · Jan 2014 · Chemical Communications
  • [Show abstract] [Hide abstract]
    ABSTRACT: A nonheme diiron active site in a 13 kDa hemerythrin-like domain of the bacterial chemotaxis protein DcrH-Hr contains an oxo bridge, two bridging carboxylate groups from Glu and Asp residues, and five terminally ligated His residues. We created a unique diiron coordination sphere containing five His and three Glu/Asp residues by replacing an Ile residue with Glu in DcrH-Hr. Direct coordination of the carboxylate group of E119 to Fe2 of the diiron site in the I119E variant was confirmed by X-ray crystallography. The substituted Glu is adjacent to an exogenous ligand-accessible tunnel. UV-vis absorption spectra indicate that the additional coordination of E119 inhibits the binding of the exogenous ligands azide and phenol to the diiron site. The extent of azide binding to the diiron site increases at pH ≤ 6, which is ascribed to protonation of the carboxylate ligand of E119. The diferrous state (deoxy form) of the engineered diiron site with the extra Glu residue is found to react more slowly than wild type with O2 to yield the diferric state (met form). The additional coordination of E119 to the diiron site also slows the rate of reduction from the met form. All these processes were found to be pH-dependent, which can be attributed to protonation state and coordination status of the E119 carboxylate. These results demonstrate that modifications of the endogenous coordination sphere can produce significant changes in the ligand binding and redox properties in a prototypical nonheme diiron-carboxylate protein active site.
    No preview · Article · Nov 2013 · Inorganic Chemistry
  • [Show abstract] [Hide abstract]
    ABSTRACT: FixL is a heme-based oxygen-sensing histidine kinase that induces expression of nitrogen fixation genes under hypoxic conditions. Oxygen binding to heme iron in the sensor domain of FixL initiates protein conformational changes that are transmitted to the histidine kinase domain, inactivating autophosphorylation activity. Although FixL also can bind other diatomic ligands such as CO, the CO-bound FixL represents incomplete inhibition of kinase activity. Ultraviolet resonance Raman (UVRR) spectra revealed that oxygen binding to the truncated sensor domain of FixL markedly decreased the intensity of the Y8a band arising from Fα-10 Tyr. In contrast, no appreciable change in intensity of the Y8a band occurred after CO-binding, and time-resolved UVRR spectra of the sensor domain of FixL upon O2 dissociation indicated that structural changes near Fα-10 Tyr occurred at ~0.1 µs. These results suggest that oxygen binding to FixL changes protein conformation near the Fα-10 Tyr residue within a microsecond. The conformational changes of FixL upon O2 dissociation and underlying sensing mechanism also are discussed.
    No preview · Article · Aug 2013 · The Journal of Physical Chemistry B
  • [Show abstract] [Hide abstract]
    ABSTRACT: Resonance Raman spectra of ligand-bound complexes including the 4-phenylimidazole complex and of free and L-Trp-bound forms of indoleamine 2, 3-dioxygenase in the ferric state were examined. Effects on the vinyl and propionate substituent groups of the heme were detected in a ligand-dependent fashion. The effects of phenyl group of 4-phenylimidazole on the vinyl and propionate Raman bands were evident when compared with the case of imidazole ligand. Substrate binding to the ferrous protein caused an upshift of the iron–histidine stretching mode by 3 cm−1, indicating an increase in negativity of the imidazole ring, which favors the O–O bond cleavage. The substrate binding event is likely to be communicated from the heme distal side to the iron–histidine bond through heme substituent groups and the hydrogen-bond network which includes water molecules, as identified in an X-ray structure of a 4-phenylimidazole complex. The results provide evidence for fine-tuning of the reactivity of O–O bond cleavage by the oxygenated heme upon binding of L-Trp.
    No preview · Article · Jun 2013 · Chemical Physics
  • Takehiko Tosha · Yoshitsugu Shiro
    [Show abstract] [Hide abstract]
    ABSTRACT: Respiration is an essential biological process to get bioenergy, ATP, for all kingdoms of life. Cytochrome c oxidase (COX) plays central role in aerobic respiration, catalyzing the reduction of O(2) coupled with pumping proton across the biological membrane. Nitric oxide reductase (NOR) involved in anaerobic nitrate respiration is suggested to be evolutionary related to COX and share the same progenitor with COX, on the basis of the amino acid sequence homology. Contrary to COX, NOR catalyzes the reduction of nitric oxide and shows no proton pumping ability. Thus, the respiratory enzyme acquires (or loses) proton pumping ability in addition to the conversion of the catalytic property along with the environmental change on earth. Recently, we solved the structures of two types of NORs, which provides novel insights into the functional conversion of the respiratory enzymes. In this review, we focus on the structural similarities and differences between COXs and NORs and discuss possible mechanism for the functional conversion of these enzymes during molecular evolution. © 2013 IUBMB Life, 2013.
    No preview · Article · Mar 2013 · International Union of Biochemistry and Molecular Biology Life

Publication Stats

5k Citations
917.91 Total Impact Points


  • 2003-2015
    • RIKEN
      • Biometal Science Laboratory
      Вако, Saitama, Japan
    • Brandeis University
      Waltham, Massachusetts, United States
    • University of Tsukuba
      • Institute of Applied Biochemistry
      Tsukuba, Ibaraki, Japan
  • 2014
    • Hokkaido University
      • Faculty of Pharmaceutical Sciences
      Sapporo, Hokkaidō, Japan
  • 2010
    • The University of Tokushima
      • Department of Life Systems
      Tokusima, Tokushima, Japan
  • 2007
    • Nagoya University
      • Research Center for Materials Science
      Nagoya, Aichi, Japan
  • 2004
    • Toyota Physical and Chemical Institute
      Seto, Aichi, Japan
  • 2003-2004
    • Himeji Institute of Technology
      • Faculty of Science
      Himezi, Hyōgo, Japan
  • 2001
    • Keio University
      • School of Medicine
      Edo, Tokyo, Japan
  • 2000
    • University of Illinois, Urbana-Champaign
      • Beckman Institute for Advanced Science and Technology
      Urbana, Illinois, United States
  • 1979-1995
    • Kyoto University
      • • Department of Molecular Engineering
      • • Department of Energy and Hydrocarbon Chemistry
      Kioto, Kyōto, Japan