Effect of pH on H- 2H exchange, H 2 production and H 2 uptake, catalysed by the membrane-bound hydrogenase of Paracoccus denitrificans

Abstract

(1) The kinetics of isotope exchange catalysed by the membrane-bound hydrogenase of Paracoccus denitrificans have been studied by measuring H 2H, H 2 or 2H 2 produced when the enzyme catalyses the exchange between 2H 2 and H 2O or H 2 and 2H 2O. (2) In the 2H 2-H 2O system the measured rate of H 2 production was always higher than that of H 2H. The H 2 H 2 H ratio remained constant (about 1.70) in the protein concentration range 0.08–1.32 mg. The very rapid formation of H 2 with respect to H 2H is consistent with the hypothesis of a heterolytic cleavage of 2H 2 into a deuteron and an enzyme hydride that can exchange with the solvent. (3) In the H 2- 2H 2O system, the exchange rate was much lower than in the 2H 2-H 2O system, indicating a marked isotopic effect of 2H 2O. (4) The H- 2H exchange activity, determined from the initial velocity of H 2H 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 H 2 production while at pH 8.3–8.5 there is a maximum of H 2 oxidation activity. (5) In ordinary H 2O the K m for hydrogen uptake estimated either from H 2 consumption or from benzyl viologen reduction was 0.06–0.07 μM for both H 2 and 2H 2 indicating a strong affinity of the enzyme for hydrogen at pH 8.3–8.5. Shifting from H 2O to 2H 2O does not affect the K m of the enzyme for H 2 but lowers the V max value about 10-fold. The K m for benzyl viologen and methyl viologen was 0.08 and 2 mM, respectively.