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Structural basis for a [4Fe-3S] cluster in the oxygen-tolerant membrane-bound [NiFe]-hydrogenase

Nature volume 479, pages 253–256 (2011)Cite this article

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Abstract

Membrane-bound respiratory [NiFe]-hydrogenase (MBH), a H2-uptake enzyme found in the periplasmic space of bacteria, catalyses the oxidation of dihydrogen: H2 → 2H+ + 2e− (ref. 1). In contrast to the well-studied O2-sensitive [NiFe]-hydrogenases (referred to as the standard enzymes), MBH has an O2-tolerant H2 oxidation activity2,3,4; however, the mechanism of O2 tolerance is unclear5. Here we report the crystal structures of Hydrogenovibrio marinus MBH in three different redox conditions at resolutions between 1.18 and 1.32 Å. We find that the proximal iron-sulphur (Fe-S) cluster of MBH has a [4Fe-3S] structure coordinated by six cysteine residues—in contrast to the [4Fe-4S] cubane structure coordinated by four cysteine residues found in the proximal Fe-S cluster of the standard enzymes—and that an amide nitrogen of the polypeptide backbone is deprotonated and additionally coordinates the cluster when chemically oxidized, thus stabilizing the superoxidized state of the cluster. The structure of MBH is very similar to that of the O2-sensitive standard enzymes except for the proximal Fe-S cluster. Our results give a reasonable explanation why the O2 tolerance of MBH is attributable to the unique proximal Fe-S cluster; we propose that the cluster is not only a component of the electron transfer for the catalytic cycle, but that it also donates two electrons and one proton crucial for the appropriate reduction of O2 in preventing the formation of an unready, inactive state of the enzyme.

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Figure 1: Overall structure of MBH heterotetramer.
Figure 2: Structural comparison of the proximal cluster in different redox conditions.
Figure 3: A comparison of charged residues around the proximal Fe-S cluster in MBH and the standard [NiFe]-hydrogenase from D. gigas.
Figure 4: Proposed proton-transfer pathways.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the reported structures have been deposited in the Protein Data Bank with the accession codes 3AYX, 3AYY and 3AYZ.

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Acknowledgements

We thank K. Hagiya for technical assistance at Ibaraki University. The synchrotron radiation experiments were performed at the BL41XU (proposal no. 2010A1223) and BL44XU (proposal no. 2010A/B6520) with the approval of JASRI. The CCD detector MX225-HE (Rayonix) at BL44XU was financially supported by Academia Sinica and National Synchrotron Radiation Research Center (Taiwan). This work was supported by a grant-in-aid for Scientific Research from MEXT (20051022 (Y.S.), 22770111 (Y.S.), 18GS0207 (Y.H.)), grant-in-aid for Scientific Research from JSPS (20580094 (H.N.), 22370061 (Y.H.) and 22657031 (Y.H.)), grant-in-aid for research and education from University of Hyogo (Y.S.), and grant-in-aid for young scientists from Hyogo Science and Technology Association (Y.S.). This work was also partially supported by the GCOE Program (Y.H.), the Japanese Aerospace Exploration Agency Project (Y.H.), and the basic research programs CREST type, ‘Development of the Foundation for Nano-Interface Technology’ from JST, Japan (Y.H.).

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Author notes

  1. Ki-Seok Yoon

    Present address: Present address: Department of Chemistry and Biochemistry, Graduate School of Engineering, Kyushu University, 744 Moto-oka, Nishi-ku, Fukuoka 819-0395, Japan.,

Authors and Affiliations

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

    Yasuhito Shomura & Yoshiki Higuchi

  2. RIKEN SPring-8 Center, 1-1-1 Koto, Sayo-gun, Sayo-cho, Hyogo 679-5148, Japan

    Yasuhito Shomura & Yoshiki Higuchi

  3. Department of Bioresource Science, College of Agriculture, Ibaraki University, 3-21-1 Chu-ou, Ami-machi, Inashiki-gun, Ibaraki 300-0393, Japan

    Ki-Seok Yoon & Hirofumi Nishihara

  4. Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Kawaguchi Center Building, 4-1-8 Honcho, Kawaguchi-shi, Saitama 332-0012, Japan

    Yoshiki Higuchi

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Contributions

K.-S.Y. and H.N. performed bacterial culture and protein purification under management by H.N. Y.S. performed crystallization, X-ray data collection and structure determination. Y.S. and Y.H. prepared the manuscript with contributions from all authors. The overall project management was done by Y.H.

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Correspondence to Yoshiki Higuchi.

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Shomura, Y., Yoon, KS., Nishihara, H. et al. Structural basis for a [4Fe-3S] cluster in the oxygen-tolerant membrane-bound [NiFe]-hydrogenase. Nature 479, 253–256 (2011). https://doi.org/10.1038/nature10504

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