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The Challenge of Visualizing the Bridging Hydride at the Active Site and Proton Network of [NiFe]-Hydrogenase by Neutron Crystallography

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Abstract

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 H2-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 Å resolution at cryogenic temperature using a gas-stream cooling system to trap short-lived intermediates in the catalytic reaction.

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Acknowledgements

This study was partly supported by MEXT KAKENHI Grants-in-Aid for Scientific Research on Innovative Areas (Hydrogenomics) 18H05516 (to Y.H.) and for Scientific Research (A) 19H00984 (to Y.H.), (B) 19H03173 (to T.T.,) and (C) 16K07283 (to T.T.); the Hyogo Science and Technology Association (to T.H.); and a JST CREST grant JPMJCR12M4 (to Y.H.). Neutron diffraction experiments using iBIX of J-PARC were performed under user programs (proposal nos. 2014B0312, 2015A0159, 2016A0100, 2017A0036, 2017B0003, and 2018A0042) and the project for the Ibaraki prefectural local government beamline (proposal nos. 2019PX2003 and 2019PX2012). The research at ORNL’s SNS (IPTS nos. 19172.1 and 20933.1) was supported by the Scientific User Facilities Division, Office of Basic Energy Sciences, US Department of Energy. We thank Drs. K. Kusaka, L. Coates, M. Cuneo, Y. Hirano, K. Kurihara, and H. Matsuura for neutron diffraction experiments, and S. Inoue, K. Hataguchi, K. Matsumoto, Y. Ikeda, Y. Yamada, and J. Hiroki for technical assistance in this study.

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Authors and Affiliations

  1. Institute for Quantum Life Science, National Institutes for Quantum and Radiological Science and Technology, 2-4 Shirakata, 319-1106, Tokai, Ibaraki, Japan

    Takeshi Hiromoto & Taro Tamada

  2. Graduate School of Life Science, University of Hyogo, 3-2-1 Koto, 678- 1297, Kamigori, Hyogo, Japan

    Takeshi Hiromoto, Koji Nishikawa & Yoshiki Higuchi

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  1. Takeshi Hiromoto
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  2. Koji Nishikawa
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  4. Yoshiki Higuchi
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Correspondence to Taro Tamada or Yoshiki Higuchi.

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Hiromoto, T., Nishikawa, K., Tamada, T. et al. The Challenge of Visualizing the Bridging Hydride at the Active Site and Proton Network of [NiFe]-Hydrogenase by Neutron Crystallography. Top Catal 64, 622–630 (2021). https://doi.org/10.1007/s11244-021-01417-0

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