Destruction and reconstitution of the activity of hydrogenase from Chromatium vinosum

Abstract

The specific activity and the EPR spectra of purified hydrogenase from Chromatium vinosum vary from preparation to preparation. It has been found that both properties are dependent on the redox state of specific redox groups, probably thiols, in the enzyme. In defect enzyme molecules the proposed thiol groups are oxidized to an -S-S- bridge. As a result, the activity of the enzyme in the assay with viologens is minimal and nickel and the iron-sulphur cluster in the oxidized enzyme are present as two non-interacting S = 1 2 systems, Ni(III) and a [3Fe-4S] 1+ cluster. In intact enzyme the -S-S- bridge does not exist. Such enzyme molecules are 15- (H 2-production reaction) or 65- (H 2-uptake reaction) times as active as defect molecules. Intact oxidized enzyme can accomodate a spin-coupled Ni(III)/[4Fe-4S] 3+ pair. It can be converted to defect molecules by partial reduction with phenazine methosulphate plus ascorbate in air at 40–50°C. The reverse transition is induced by anaerobic incubation with a disulphide-reducing agent at 50°C.