INHIBITION BY IODOACETAMIDE AND ACETYLENE OF THE H-D-EXCHANGE REACTION CATALYZED BY THIOCAPSA-ROSEOPERSICINA HYDROGENASE

The kinetics of H-D isotope exchange catalyzed by the thermostable hyd rogenase from Thiocapsa roseopersicina have been studied by analysis o f the exchange between D-2 and H2O. The pH dependence of the exchange reaction was examined between pH 2.5 and pH 11. Over the whole pH rang e, HD was produced at a higher initial velocity than H-2, with a marke d optimum at pH 5.5; a second peak in the pH profile was observed at a round pH 8.5. The rapid formation of H-2 with respect to HD in the D-2 /H2O system is consistent with a heterolytic cleavage of D-2 into D+ a nd an enzyme hydride that can both exchange with the solvent. The H-D- exchange activity was lower in the H-2/D2O system than in the D-2/H2O system. The other reactions catalyzed by the hydrogenase, H-2 oxidatio n and H-2 evolution, are pH dependent; the optimal pH were 9.5 for H-2 uptake and 4.0 for H-2 production. Treatment of the active form of hy drogenase by iodoacetamide led to a slow and irreversible inhibition o f the H-D exchange. When iodo[1-C-14]acetamide was incubated with hydr ogenase, the radioactive labeling of the large subunit was higher for the enzyme activated under H-2 than for the inactive oxidized form. Cy steine residues were identified as the alkylated derivative by amino a cid analysis. Acetylene, which inhibits H-D exchange and abolishes the Ni-C EPR signal, protected the enzyme from irreversible inhibition by iodoacetamide. These data indicate that iodoacetamide can reach the a ctive site of the H-2-activated hydrogenase from T. roseopersicina. Th is was not found to be the case with the seleno hydrogenase from Desul fovibrio baculatus (now Desulfomicrobium baculatus). Cysteine modifica tion by iodoacetamide upon activation of the enzyme concomitant with l oss of H-D exchange indicates that reductive activation makes at least one Cys residue of the active site available for alkylation.