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The crystal structure of an oxygen-tolerant hydrogenase uncovers a novel iron-sulphur centre


Hydrogenases are abundant enzymes that catalyse the reversible interconversion of H2 into protons and electrons at high rates1. Those hydrogenases maintaining their activity in the presence of O2 are considered to be central to H2-based technologies, such as enzymatic fuel cells and for light-driven H2 production2. Despite comprehensive genetic, biochemical, electrochemical and spectroscopic investigations3,4,5,6,7,8, the molecular background allowing a structural interpretation of how the catalytic centre is protected from irreversible inactivation by O2 has remained unclear. Here we present the crystal structure of an O2-tolerant [NiFe]-hydrogenase from the aerobic H2 oxidizer Ralstonia eutropha H16 at 1.5 Å resolution. The heterodimeric enzyme consists of a large subunit harbouring the catalytic centre in the H2-reduced state and a small subunit containing an electron relay consisting of three different iron-sulphur clusters. The cluster proximal to the active site displays an unprecedented [4Fe-3S] structure and is coordinated by six cysteines. According to the current model, this cofactor operates as an electronic switch depending on the nature of the gas molecule approaching the active site. It serves as an electron acceptor in the course of H2 oxidation and as an electron-delivering device upon O2 attack at the active site. This dual function is supported by the capability of the novel iron-sulphur cluster to adopt three redox states at physiological redox potentials7,8,9. The second structural feature is a network of extended water cavities that may act as a channel facilitating the removal of water produced at the [NiFe] active site. These discoveries will have an impact on the design of biological and chemical H2-converting catalysts that are capable of cycling H2 in air.

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Figure 1: Overall structure of the membrane-bound hydrogenase from R. eutropha.
Figure 2: Metal cofactors of the MBH.
Figure 3: Architecture of the proximal [4Fe-3S] cluster.
Figure 4: Proposed water/proton transfer pathway from the MBH active site to the protein surface.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the reported structure have been deposited in the Protein Data Bank with the accession code 3RGW.


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We are grateful to U. Müller, M. Weiss and the scientific staff of the BESSY-MX/Helmholtz Zentrum Berlin für Materialien und Energie at beamlines BL 14.1 and BL 14.2, D. von Stetten and A. Royant of the ID29S-Cryobench (ESRF, Grenoble) and the European Synchrotron Radiation Facility (ESRF, Grenoble) at beamlines ID23-1, ID23-2, ID14-1 and ID 14-4, where the data were collected, for support. This work was supported by the EU/FP7 programme Solar-H2 (to J.F.), the DFG Cluster of Excellence ‘Unifying Concepts in Catalysis’ (to S.F., B.F., O.L.), and the Sfb740 (to C.M.T.S.). P.S. acknowledges K. P. Hofmann and his advanced investigator ERC grant (ERC-2009/249910—TUDOR) for support.

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Authors and Affiliations



J.F. and P.S. are joint first authors. J.F. optimized cell growth conditions as well as the MBH purification procedure. P.S. conducted the crystallization screening; P.S., J.F. and S.F. optimized MBH crystallization conditions. P.S. and S.K. collected the X-ray diffraction data. P.S. performed data processing, solved and refined the MBH structure. B.F., O.L. and P.S. coordinated the project. J.F., P.S., S.F. and O.L. analysed data. J.F., P.S., S.F., B.F., O.L. and C.M.T.S. wrote the manuscript.

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Correspondence to Patrick Scheerer, Oliver Lenz or Christian M. T. Spahn.

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The authors declare no competing financial interests.

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Fritsch, J., Scheerer, P., Frielingsdorf, S. et al. The crystal structure of an oxygen-tolerant hydrogenase uncovers a novel iron-sulphur centre. Nature 479, 249–252 (2011).

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