Abstract

FeFe hydrogenases are the most efficient H2-producing enzymes. However, inactivation by O2 remains an obstacle that prevents them being used in many biotechnological devices. Here, we combine electrochemistry, site-directed mutagenesis, molecular dynamics and quantum chemical calculations to uncover the molecular mechanism of O2 diffusion within the enzyme and its reactions at the active site. We propose that the partial reversibility of the reaction with O2 results from the four-electron reduction of O2 to water. The third electron/proton transfer step is the bottleneck for water production, competing with formation of a highly reactive OH radical and hydroxylated cysteine. The rapid delivery of electrons and protons to the active site is therefore crucial to prevent the accumulation of these aggressive species during prolonged O2 exposure. These findings should provide important clues for the design of hydrogenase mutants with increased resistance to oxidative damage.

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Acknowledgements

The French teams were supported by CNRS, INSA, CEA, Agence Nationale de la Recherche (ANR-12-BS08-0014, ANR-14-CE05-0010) and the A*MIDEX project (n° ANR-11- IDEX-0001-02) funded by the «Investissements d’Avenir» French Government program, managed by the French National Research Agency (ANR). The authors thank R. van Lis for constructing the V296F and F290W mutants. D.D.S. acknowledges support from EPSRC grant no. EP/J016764/1 and an Ikerbasque Research Fellowship. A.K. was supported by EPSRC grant no. EP/J015571/1. R.B.B. was supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health. J.B. thanks the Royal Society for a University Research Fellowship. This work was carried out on the HECToR and Archer computing facilities (Edinburgh), access to which was granted through the Materials Chemistry Consortium (EPSRC grants nos EP/F067496 and EP/L000202). The authors acknowledge the use of the UCL Legion High Performance Computing Facility (Legion@UCL) and associated support services in the completion of this work as well as the computational resources of the NIH HPC Biowulf cluster (http://hpc.nih.gov). D.D.S. acknowledges PRACE for awarding access to the FERMI resource based in Italy at CINECA. D.D.S. thanks A. Szabo and E. Rosta for discussions.

Author information

Author notes

    • Adam Kubas
    • , Christophe Orain
    •  & David De Sancho

    These authors contributed equally to this work

Affiliations

  1. Department of Physics and Astronomy, University College London, Gower Street, London WC1E 6BT, UK

    • Adam Kubas
    •  & Jochen Blumberger
  2. Institute of Physical Chemistry, Polish Academy of Science, ul. Kasprzaka 44/52, 01-224 Warsaw, Poland

    • Adam Kubas
  3. Aix Marseille Univ, CNRS, Laboratoire de Bioénergétique et Ingénierie des Protéines, Institut de Microbiologie de la Méditerranée, Marseille, France

    • Christophe Orain
    • , Matteo Sensi
    • , Carole Baffert
    • , Vincent Fourmond
    •  & Christophe Léger
  4. Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK

    • David De Sancho
  5. CIC nanoGUNE, Tolosa Hiribidea 76, 20018 Donostia-San Sebastián, Spain

    • David De Sancho
  6. IKERBASQUE; Basque Foundation for Science, María Díaz de Haro 3, 48013 Bilbao, Spain

    • David De Sancho
  7. Institut de Biologie et de Technologies de Saclay IBITECS, SB2SM, F-91191 Gif sur Yvette, France

    • Laure Saujet
    •  & Hervé Bottin
  8. Institut de Biologie Intégrative de la Cellule I2BC, UMR 9198, CEA, CNRS, Université Paris Sud, F-91191 Gif sur Yvette, France

    • Laure Saujet
    •  & Hervé Bottin
  9. Université de Toulouse, INSA, UPS, INP, LISBP, INRA:UMR792, CNRS:UMR 5504, 135 avenue de Rangueil, Toulouse 31077 Cedex 04, France

    • Charles Gauquelin
    • , Isabelle Meynial-Salles
    •  & Philippe Soucaille
  10. Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, USA

    • Robert B. Best

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Contributions

A.K., C.O. and D.D.S. contributed equally to this work. All authors discussed the results and commented on the manuscript. A.K., D.D.S., R.B.B. and J.B. performed the calculations and analysed the data. C.O., M.S., C.B., V.F. and C.L. performed the electrochemical measurements and analysed the data. L.S., C.G., I.M.-S., P.S. and H.B. prepared the enzyme samples. A.K., D.D.S., R.B.B., C.B., V.F., J.B. and C.L. co-wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Jochen Blumberger or Christophe Léger.

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DOI

https://doi.org/10.1038/nchem.2592

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