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Relating diffusion along the substrate tunnel and oxygen sensitivity in hydrogenase


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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Figure 1: Structure of D. fructosovorans NiFe hydrogenase depicting the “dry” hydrophobic cavities.
Figure 2: EPR characterization of the NiFe hydrogenase variants.
Figure 3: The inhibition by O2 of D. fructosovorans NiFe hydrogenase selected mutants and the reaction with CO and O2 of the FeFe hydrogenases from C. acetobutylicum and D. desulfuricans.
Figure 4: Kinetic properties of selected NiFe hydrogenase mutants.

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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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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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Liebgott, PP., Leroux, F., Burlat, B. et al. Relating diffusion along the substrate tunnel and oxygen sensitivity in hydrogenase. Nat Chem Biol 6, 63–70 (2010).

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