DIRECT ELECTROCHEMISTRY OF RHODOCOCCUS-OPACUS HYDROGENASE FOR THE CATALYSIS OF NAD(+) REDUCTION

The catalysis of NAD(+) reduction by Rhodococcus opacus hydrogenase wa s investigated by spectroelectrochemistry in a thin layer cell on a pl atinum electrode. The NADH formation rates were significantly higher t han those obtained with the hydrogenase of Alcaligenes eutrophus which has previously been identified as a good catalyst of this electrochem ical reduction. As a consequence, for the first time to our knowledge, a well-identified current peak which corresponded to NAD(+) reduction was observed on the voltammograms obtained with a platinum cathode. T hanks to the efficiency of this catalysis, it has been possible to imp rove the understanding of the mechanism. A two-step mechanism was assu med, according to the structure of the hydrogenase which is composed o f two dimers with distinct hydrogenase and diaphorase activity. At hig h potentials (above - 0.66 V (SCE)) only the diaphorase dimer was redu ced by direct electron transfer from the electrode, without interventi on of any hydrogen intermediate. For more negative potentials, a reduc ed hydrogen species adsorbed on the electrode surface was involved in the mechanism. In this case, catalysis followed a more classic catalyt ic pathway via the hydrogenase dimer and an intramolecular electron tr ansfer to the diaphorase dimer, which reduced NAD(+).