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Mechanism of hydrogen activation by [NiFe] hydrogenases

Nature Chemical Biology volume 12, pages 4650 (2016) | Download Citation

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 Using hydrogenase-1 from Escherichia coli, the strictly conserved residues R509 and D574 have been replaced by lysine (R509K) and asparagine (D574N) and the highly conserved D118 has been replaced by alanine (D118A) or asparagine (D118N/D574N).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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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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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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The authors declare no competing financial interests.

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

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https://doi.org/10.1038/nchembio.1976

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