In hydrogenases and many other redox enzymes, the buried active site is connected to the solvent by a molecular channel whose structure may determine the enzyme's selectivity with respect to substrate and inhibitors. The role of these channels has been addressed using crystallography and molecular dynamics, but kinetic data are scarce. Using protein film voltammetry, we determined and then compared the rates of inhibition by CO and O2 in ten NiFe hydrogenase mutants and two FeFe hydrogenases. We found that the rate of inhibition by CO is a good proxy of the rate of diffusion of O2 toward the active site. Modifying amino acids whose side chains point inside the tunnel can slow this rate by orders of magnitude. We quantitatively define the relations between diffusion, the Michaelis constant for H2 and rates of inhibition, and we demonstrate that certain enzymes are slowly inactivated by O2 because access to the active site is slow.

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This work was funded by the Centre National de la Recherche Scientifique, Commissariat à l'Energie Atomique, Agence Nationale de la Recherche, the University of Provence and the City of Marseilles, and supported by the Pôle de Compétitivité Capénergies. The Groupe de Recherche 2977 (“Bio-hydrogène”) paid the publication fees for this article.

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  1. Centre National de la Recherche Scientifique, Unité Propre de Recherche 9036, Unité de Bioénergétique et Ingénierie des Protéines, Institut Fédératif de Recherche 88, Institut de Microbiologie de la Méditerranée, Marseille, France.

    • Pierre-Pol Liebgott
    • , Fanny Leroux
    • , Bénédicte Burlat
    • , Sébastien Dementin
    • , Carole Baffert
    • , Vincent Fourmond
    • , Pierre Ceccaldi
    • , Bruno Guigliarelli
    • , Patrick Bertrand
    • , Marc Rousset
    •  & Christophe Léger
  2. Aix Marseille Université, Marseille, France.

    • Fanny Leroux
    • , Bénédicte Burlat
    • , Carole Baffert
    • , Pierre Ceccaldi
    • , Bruno Guigliarelli
    •  & Patrick Bertrand
  3. Université de Toulouse; Institut National des Sciences Appliquées, Université Paul Sabatier, Institut National Polytechnique, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés, Toulouse, France.

    • Thomas Lautier
    • , Isabelle Meynial-Salles
    •  & Philippe Soucaille
  4. Institut National de la Recherche Agronomique, Unité Mixte de Recherche 792, Ingénierie des Systèmes Biologiques et des Procédés, Toulouse, France.

    • Philippe Soucaille
  5. Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5504, Toulouse, France.

    • Thomas Lautier
    • , Isabelle Meynial-Salles
    •  & Philippe Soucaille
  6. Laboratoire de Cristallographie et de Cristallogenèse des Protéines, Institut de Biologie Structurale J.P. Ebel, Commissariat à l'Energie Atomique, Centre National de la Recherche Scientifique, Université Joseph Fourier, Grenoble, France.

    • Christine Cavazza
    •  & Juan Carlos Fontecilla-Camps


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P.-P.L. designed mutants of the NiFe enzyme, performed mutagenesis, carried out protein purification, solution assays and electrochemical measurements, and analyzed data, with the support of M.R. and C.L. F.L. performed electrochemical measurements on several forms of the NiFe hydrogenase (WT, L122M V74M, V74M, L122F V74I). B.B. characterized by EPR the NiFe hydrogenase mutants, with the support of B.G. S.D. designed mutants of the NiFe enzyme, performed mutagenesis, carried out protein purification and solution assays, and interpreted studies, with the support of M.R. C.B. performed the electrochemical characterization of the two FeFe hydrogenases and analyzed the data. T.L. purified the FeFe hydrogenase from C. acetobutylicum and assayed its activity, with the support and advice of I.M.-S. and P.S. V.F. contributed to modeling. P.C. characterized the V74W mutant, and analyzed the data. C.C. purified the FeFe hydrogenase from D. desulfuricans, with the support of J.C.F.-C. P.-P.L., S.D., B.B., C.B., M.R., B.G., P.B. and C.L. co-designed research. S.D., P.B. and C.L. conceptualized, analyzed and interpreted all studies and co-wrote the manuscript.

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Correspondence to Christophe Léger.

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