Towards cryogenic neutron crystallography on the reduced form of [NiFe]-hydrogenase

Takeshi Hiromoto, Koji Nishikawa, Seiya Inoue, Hiroaki Matsuura, Yu Hirano, Kazuo Kurihara, Katsuhiro Kusaka, Matthew Cuneo, Leighton Coates, Taro Tamada, Yoshiki Higuchi

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Abstract

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.

Original languageEnglish
Pages (from-to)946-953
Number of pages8
JournalActa Crystallographica Section D: Structural Biology
Volume76
DOIs
StatePublished - Oct 1 2020

Funding

Neutron diffraction experiments using iBIX at 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 the SNS at ORNL (IPTS Nos. 19172.1 and 20933.1) was supported by the Scientific User Facilities Division, Office of Basic Energy Sciences, US Department of Energy. X-ray data were collected at BL26B1 of SPring-8 (proposal Nos. 2016B2725, 2017A6744 and 2019A1005) and at NW12A of PF-AR (proposal No. 2019G104). We acknowledge the support of all beamline staff. We also thank K. Hataguchi, K. Matsumoto, Y. Ikeda, Y. Yamada and J. Hiroki for their technical assistance in this study. This study was partly supported by MEXT KAKENHI Grants-in-Aid for Scientific Research on Innovative Areas (Hydrogenomics) 18H05516 (to YH), Scientific Research (A) 19H00984 (to YH), Scientific Research (B) 19H03173 (to TT) and Scientific Research (C) 16K07283 (to TT), Hyogo Science and Technology Association (to TH) and JST CREST grant JPMJCR12M4 (to YH).

Keywords

  • cryo-crystallography
  • hydrogenases
  • neutron diffraction

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