EFFECTS OF THIOLS AND MERCURIALS ON THE PERIPLASMIC HYDROGENASE FROM DESULFOVIBRIO-VULGARIS (HILDENBOROUGH)

The H-2-oxidation, H-2-production and H-H-3-exchange activities of the periplasmic hydrogenase from Desulfovibrio vulgaris (Hildenborough) w ere almost completely abolished by Hg(II) and the organic mercurials p -chloromercuribenzoate (pCMB) and p-hydroxymercuriphenylsulphonate. Th e thiol-modifying reagents N-ethylmaleimide, iodoacetate, dithionitrob enzoate and 2-nitro-5-thiocyanobenzoate had no effect on the activitie s. Kinetic and spectroscopic measurements suggest that inactivation by pCMB involves at least two reactions; a rapid reaction that is revers ed by thiols, and a second, slower and irreversible reaction that occu rs at high concentrations of the mercurial. The irreversible reaction was associated with loss of visible absorbance, indicative of a disrup ted iron-sulphur cluster(s). The effects on the H-H-3-exchange activit y indicate that the reversible modification affects the H-2-activating site. Enzyme that had lost activity due to pCMB treatment, or during long-term storage, was reactivated by thiols. This reactivation was fo llowed by a slower irreversible inactivation, as also occurred with na tive enzyme; the inactivation was O2 dependent and it was partly preve nted by catalase, suggesting that H2O2 may be involved.