ESR-detectable nickel and iron-sulphur centres in relation to the reversible activation of Desulfovibrio gigas hydrogenase

Abstract

The electron spin resonance (ESR) spectra of hydrogenase from Desulfovibrio gigas were observed during the activation of the enzyme in the oxidized, ‘unready’, state by hydrogen. Signals from nickel(III) (Ni-A), and the [3Fe- xS] cluster were reduced within less than 5 min, and a broad ESR signal appeared at the same time. On the basis of simultaneous changes in optical absorption spectrum, it is proposed that the broad ESR signal represents one or possibly both [4Fe-4S] clusters in the reduced state. The increase of enzyme activity was much slower (at 20°C), and was accompanied by the appearance of another type of nickel signal (Ni-C), and a further small decrease and the Ni-C signal became more intense. On further reoxidation by the dye dichlorophenolindophenol at pH above 7.0 the enzyme was converted to the ‘ready’ state, which could now be reactivated much more rapidly by strong reductants. The proportion of the ready state correlated with a third type of nickel signal, Ni-B. The unready enzyme could also be slowly activated by milder reducing conditions which reduced Ni-A and the [3Fe- xS] cluster but did not induce significant amounts of the Ni-C and [4Fe-4S] 1+ signals. The optical absorption changes indicate that the Ni-A is not coupled to an iron-sulphur cluster. It is proposed that the activation of the enzyme involves reduction of the nickel and possibly iron-sulphur centres, followed by a conformational change which alters the coordination state of nickel, and that the unready state contains Ni(III) in the inactive conformation, the ready state Ni(III) in the active conformation, and the active state Ni(I).