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Mechanism of hydrogen activation by [NiFe] hydrogenases

Nature Chemical Biology volume 12, pages 46–50 (2016)Cite this article

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Abstract

The active site of [NiFe] hydrogenases contains a strictly conserved arginine that suspends a guanidine nitrogen atom <4.5 Å above the nickel and iron atoms. The guanidine headgroup interacts with the side chains of two conserved aspartic acid residues to complete an outer-shell canopy that has thus far proved intractable to investigation by site-directed mutagenesis. Using hydrogenase-1 from Escherichia coli, the strictly conserved residues R509 and D574 have been replaced by lysine (R509K) and asparagine (D574N) and the highly conserved D118 has been replaced by alanine (D118A) or asparagine (D118N/D574N). Each enzyme variant is stable, and their [(RS)2Niμ(SR)2Fe(CO)(CN)2] inner coordination shells are virtually unchanged. The R509K variant had >100-fold lower activity than native enzyme. Conversely, the variants D574N, D118A and D118N/D574N, in which the position of the guanidine headgroup is retained, showed 83%, 26% and 20% activity, respectively. The special kinetic requirement for R509 implicates the suspended guanidine group as the general base in H2 activation by [NiFe] hydrogenases.

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Figure 1: A stereo image of the active site of [NiFe] hydrogenases as exemplified by E. coli Hyd-1 (PDB 5A4M, oxygenation sites omitted).
Figure 2: Comparison of activities for Hyd-1 and variants investigated in this research.
Figure 3: Protein film electrochemistry of native and variant Hyd-1 enzymes described in this paper.
Figure 4: The crystal structures of the active sites of as-isolated and chemically oxidized enzymes described in this paper.
Figure 5: Proposal for the H2 activation step in [NiFe] hydrogenases using suspended arginine as a general base, as in a frustrated Lewis pair mechanism.

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Acknowledgements

This work was supported by the UK Biological and Biotechnology Sciences Research Council, grants BB/I022309-1 and BB/L009722/1 to F.A.A. and BB/L008521/1 to F.S. A studentship for E.J.B. was supported by grants from Global Innovation Initiative and the UK Engineering and Physical Sciences Research Council. F.A.A. is a Royal Society Wolfson Research Merit Award holder. We thank the Diamond Light Source for beam time proposal mx9306 and the staff of beamlines i02, i04 and i04-1 for their assistance during data collection.

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Authors and Affiliations

  1. Department of Chemistry, University of Oxford, Oxford, UK

    Rhiannon M Evans, Emily J Brooke, Sara A M Wehlin, Elena Nomerotskaia & Fraser A Armstrong

  2. Division of Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee, UK

    Frank Sargent

  3. Research Complex at Harwell, Rutherford Appleton Laboratory, Harwell Oxford, Didcot, UK

    Stephen B Carr & Simon E V Phillips

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Contributions

F.A.A., F.S. and R.M.E. proposed the study. R.M.E., E.J.B. and S.A.M.W. carried out all molecular biology, kinetic and electrochemical characterizations. R.M.E., E.N., E.J.B., S.A.M.W. and S.B.C. produced and purified enzymes. S.B.C. carried out all X-ray data collection, and S.B.C. and S.E.V.P. were responsible for structural determinations. F.A.A., R.M.E., S.E.V.P. and S.B.C. wrote the manuscript with input from other authors.

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Correspondence to Stephen B Carr, Simon E V Phillips or Fraser A Armstrong.

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Supplementary Results, Supplementary Tables 1–4 and Supplementary Figures 1–6. (PDF 2117 kb)

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Evans, R., Brooke, E., Wehlin, S. et al. Mechanism of hydrogen activation by [NiFe] hydrogenases. Nat Chem Biol 12, 46–50 (2016). https://doi.org/10.1038/nchembio.1976

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