Effect of pH on H-2H exchange,H2 production and H2 uptake,catalysed by the membrane-bound hydrogenase of Paracoccus denitrificans |
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Authors: | Paulette M Vignais Michele-France Henry Yves Berlier Paul Antoine Lespinat |
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Institution: | 1. Biochimie (CNRS/ER 235 - INSERM U.191), Département de Recherche Fondamentale, Centre d''Etudes Nucléaires, 85X, 38041 Grenoble Cedex France;2. Laboratoire commun d''Enzymologie CNRS-CEA, Département de Biologie, Centre d''Etudes Nucléaires de Cadarache, B.P. No. 1, 13115 Saint-Paul-lez-Durance France |
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Abstract: | (1) The kinetics of isotope exchange catalysed by the membrane-bound hydrogenase of Paracoccus denitrificans have been studied by measuring H2H, H2 or 2H2 produced when the enzyme catalyses the exchange between 2H2 and H2O or H2 and 2H2O. (2) In the 2H2-H2O system the measured rate of H2 production was always higher than that of H2H. The ratio remained constant (about 1.70) in the protein concentration range 0.08–1.32 mg. The very rapid formation of H2 with respect to H2H is consistent with the hypothesis of a heterolytic cleavage of 2H2 into a deuteron and an enzyme hydride that can exchange with the solvent. (3) In the H2-2H2O system, the exchange rate was much lower than in the 2H2-H2O system, indicating a marked isotopic effect of 2H2O. (4) The H-2H exchange activity, determined from the initial velocity of H2H formation, is optimal at pH 4.5. A second maximum of activity is observed at pH 8.3. The pH value of 4.5 is also the pH optimum for H2 production while at pH 8.3–8.5 there is a maximum of H2 oxidation activity. (5) In ordinary H2O the Km for hydrogen uptake estimated either from H2 consumption or from benzyl viologen reduction was 0.06–0.07 μM for both H2 and 2H2 indicating a strong affinity of the enzyme for hydrogen at pH 8.3–8.5. Shifting from H2O to 2H2O does not affect the Km of the enzyme for H2 but lowers the Vmax value about 10-fold. The Km for benzyl viologen and methyl viologen was 0.08 and 2 mM, respectively. |
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Keywords: | Hydrogenase Hydrogen exchange pH effect Hydrogen uptake Hydrogen evolution (P denitrificans) Mes Hepes Hipip high-potential iron-sulfur protein |
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