研究者業績

Koji Nishikawa

  (西川 幸志)

Profile Information

Affiliation
University of Hyogo
Degree
Ph. D.(Mar, 2010, University of Hyogo)

J-GLOBAL ID
201901015325678030
researchmap Member ID
B000370021

Papers

 26
  • Takeshi Hiromoto, Koji Nishikawa, Seiya Inoue, Hideaki Ogata, Yuta Hori, Katsuhiro Kusaka, Yu Hirano, Kazuo Kurihara, Yasuteru Shigeta, Taro Tamada, Yoshiki Higuchi
    Chemical Science, 14(35) 9306-9315, 2023  Peer-reviewed
    We report the first neutron structure of [NiFe]-hydrogenase in its oxidized state. This study leads to new insights into the oxidized active site and visualization of the protons characteristic of the oxidized enzyme.
  • Takahiro Imanishi, Koji Nishikawa, Midori Taketa, Katsuhiro Higuchi, Hulin Tai, Shun Hirota, Hironobu Hojo, Toru Kawakami, Kiriko Hataguchi, Kayoko Matsumoto, Hideaki Ogata, Yoshiki Higuchi
    Acta crystallographica. Section F, Structural biology communications, 78(Pt 2) 66-74, Feb 1, 2022  Peer-reviewed
    Hydrogenases catalyze the reversible oxidation of H2. Carbon monoxide (CO) is known to be a competitive inhibitor of O2-sensitive [NiFe]-hydrogenases. Although the activities of some O2-tolerant [NiFe]-hydrogenases are unaffected by CO, the partially O2-tolerant [NiFe]-hydrogenase from Citrobacter sp. S-77 (S77-HYB) is inhibited by CO. In this work, the CO-bound state of S77-HYB was characterized by activity assays, spectroscopic techniques and X-ray crystallography. Electron paramagnetic resonance spectroscopy showed a diamagnetic Ni2+ state, and Fourier-transform infrared spectroscopy revealed the stretching vibration of the exogenous CO ligand. The crystal structure determined at 1.77 Å resolution revealed that CO binds weakly to the nickel ion in the Ni-Fe active site of S77-HYB. These results suggest a positive correlation between O2 and CO tolerance in [NiFe]-hydrogenases.
  • Takeshi Hiromoto, Koji Nishikawa, Taro Tamada, Yoshiki Higuchi
    TOPICS IN CATALYSIS, 64(9-12) 622-630, Aug, 2021  Peer-reviewed
    X-ray crystallography is the most powerful tool for obtaining structural information about protein molecules, affording accurate and precise positions for all of the atoms in the protein except for hydrogen. However, hydrogen species play crucial roles in the physiological functions of enzymes, including molecular recognition through hydrogen bonding and catalytic reactions involving proton transfer. Neutron crystallography enables direct identification of the positions of hydrogen species. [NiFe]-hydrogenase from Desulfovibrio vulgaris Miyazaki F is an enzyme that catalyzes the reversible oxidation of molecular hydrogen. It contains a bimetallic Ni-Fe active site for the catalytic reaction and three Fe-S clusters for electron transfer. Previous X-ray structure analyses of the enzyme under various oxidation conditions have revealed that the active site changes its coordination structure depending on the redox state. In the inactive air-oxidized form, an oxygen species was identified between the Ni and Fe atoms, whereas in the active H-2-reduced form, subatomic-resolution X-ray structure analysis and single-crystal EPR analyses indicated a hydride ligand between the two metal atoms. However, the assignment of the hydride moiety by X-ray crystallography remains controversial, and the proton transfer pathways in the molecule are still ambiguous. To allow neutron diffraction experiments, large crystals of [NiFe]-hydrogenase were prepared by the vapor diffusion method with the macroseeding technique according to the two-dimensional phase diagram (protein concentration vs. precipitant concentration). Neutron diffraction data were collected at approximately 2.0 angstrom resolution at cryogenic temperature using a gas-stream cooling system to trap short-lived intermediates in the catalytic reaction.
  • Takeshi Hiromoto, Koji Nishikawa, Seiya Inoue, Hiroaki Matsuura, Yu Hirano, Kazuo Kurihara, Katsuhiro Kusaka, Matthew Cuneo, Leighton Coates, Taro Tamada, Yoshiki Higuchi
    Acta crystallographica. Section D, Structural biology, 76(Pt 10) 946-953, Oct 1, 2020  Peer-reviewed
    A membrane-bound hydrogenase from Desulfovibrio vulgaris Miyazaki F is a metalloenzyme that contains a binuclear Ni-Fe complex in its active site and mainly catalyzes the oxidation of molecular hydrogen to generate a proton gradient in the bacterium. The active-site Ni-Fe complex of the aerobically purified enzyme shows its inactive oxidized form, which can be reactivated through reduction by hydrogen. Here, in order to understand how the oxidized form is reactivated by hydrogen and further to directly evaluate the bridging of a hydride ligand in the reduced form of the Ni-Fe complex, a neutron structure determination was undertaken on single crystals grown in a hydrogen atmosphere. Cryogenic crystallography is being introduced into the neutron diffraction research field as it enables the trapping of short-lived intermediates and the collection of diffraction data to higher resolution. To optimize the cooling of large crystals under anaerobic conditions, the effects on crystal quality were evaluated by X-rays using two typical methods, the use of a cold nitrogen-gas stream and plunge-cooling into liquid nitrogen, and the former was found to be more effective in cooling the crystals uniformly than the latter. Neutron diffraction data for the reactivated enzyme were collected at the Japan Photon Accelerator Research Complex under cryogenic conditions, where the crystal diffracted to a resolution of 2.0 Å. A neutron diffraction experiment on the reduced form was carried out at Oak Ridge National Laboratory under cryogenic conditions and showed diffraction peaks to a resolution of 2.4 Å.
  • Koji Nishikawa, Hideaki Ogata, Yoshiki Higuchi
    CHEMISTRY LETTERS, 49(2) 164-173, Feb, 2020  Peer-reviewed
    Hydrogenases control the proton concentration in cells, which is an essential function for hydrogen metabolism in several microorganisms. Some [NiFe]-hydrogenases are catalytically active under air and are thus of great interest for developing bio-inspired synthetic models and new devices for clean energy conversion. Here, we provide an overview of the structural basis of the reaction mechanism of [NiFe]-hydrogenases, and the recent development of a new assay method which may uncover hidden properties of hydrogenases.

Misc.

 4

Books and Other Publications

 2

Presentations

 17

Research Projects

 1