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Mechanism of O2 diffusion and reduction in FeFe hydrogenases

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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Figure 1: FeFe hydrogenase and its inhibition by O2.
Figure 2: Change in catalytic H2-oxidation current versus time.
Figure 3: Potential dependence of the three rate constants defined in equation (1).
Figure 4: Markov state model for O2 diffusion into Cp hydrogenase.
Figure 5: Graphical representation of the relative free energies of the most important states (1–9) involved in oxygen reduction to water at the active site of the FeFe hydrogenase.
Figure 6: Proposed mechanism for aerobic inhibition of FeFe hydrogenase.

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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.

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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.

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Correspondence to Jochen Blumberger or Christophe Léger.

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Kubas, A., Orain, C., De Sancho, D. et al. Mechanism of O2 diffusion and reduction in FeFe hydrogenases. Nature Chem 9, 88–95 (2017). https://doi.org/10.1038/nchem.2592

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