Effects of thiols and mercurials on the periplasmic hydrogenase from Desulfovibrio vulgaris (Hildenborough).
The H2-oxidation, H2-production and H-3H-exchange activities of the periplasmic hydrogenase from Desulfovibrio vulgaris (Hildenborough) were almost completely abolished by Hg(II) and the organic mercurials p-chloromercuribenzoate (pCMB) and p-hydroxymercuriphenylsulphonate. The thiol-modifying reagents N-ethylmaleimide, iodoacetate, dithionitrobenzoate and 2-nitro-5-thiocyanobenzoate had no effect on the activities. Kinetic and spectroscopic measurements suggest that inactivation by pCMB involves at least two reactions; a rapid reaction that is reversed by thiols, and a second, slower and irreversible reaction that occurs at high concentrations of the mercurial. The irreversible reaction was associated with loss of visible absorbance, indicative of a disrupted iron sulphur cluster(s). The effects on the H-3H-exchange activity indicate that the reversible modification affects the H2-activating site. Enzyme that had lost activity due to pCMB treatment, or during long-term storage, was reactivated by thiols. This reactivation was followed by a slower irreversible inactivation, as also occurred with native enzyme; the inactivation was O2 dependent and it was partly prevented by catalase, suggesting that H2O2 may be involved.
Fagan
T F
TF
Department of Biochemistry, University College, Dublin, Ireland.
Mayhew
S G
SG
D002729:Chloromercuribenzoates / Q000494:pharmacology*; D003545:Cysteine / Q000494:pharmacology; D016969:Desulfovibrio vulgaris / Q000201:enzymology; D006864:Hydrogenase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008628:Mercury / Q000494:pharmacology*; D010084:Oxidation-Reduction; D010663:Phenylmercury Compounds / Q000494:pharmacology*; D013056:Spectrophotometry, Ultraviolet; D013438:Sulfhydryl Compounds / Q000494:pharmacology*; D020245:p-Chloromercuribenzoic Acid
D002729:Chloromercuribenzoates; D010663:Phenylmercury Compounds; D013438:Sulfhydryl Compounds; C002910:4-hydroxymercuribenzenesulfonate; D020245:p-Chloromercuribenzoic Acid; D006864:Hydrogenase; D008628:Mercury; D003545:Cysteine