Mössbauer Characterization of the Iron−Sulfur Clusters in Desulfovibrio vulgaris Hydrogenase

Abstract

The periplasmic hydrogenase of Desulfovibrio vulgaris (Hildenbourough) is an all Fe-containing hydrogenase. It contains two ferredoxin type [4Fe-4S] clusters, termed the F clusters, and a catalytic H cluster. Recent X-ray crystallographic studies on two Fe hydrogenases revealed that the H cluster is composed of two sub-clusters, a [4Fe-4S] cluster ([4Fe-4S]_H) and a binuclear Fe cluster ([2Fe]_H), bridged by a cysteine sulfur. The aerobically purified D. vulgaris hydrogenase is stable in air. It is inactive and requires reductive activation. Upon reduction, the enzyme becomes sensitive to O₂, indicating that the reductive activation process is irreversible. Previous EPR investigations showed that upon reoxidation (under argon) the H cluster exhibits a rhombic EPR signal that is not seen in the as-purified enzyme, suggesting a conformational change in association with the reductive activation. For the purpose of gaining more information on the electronic properties of this unique H cluster and to understand further the reductive activation process, variable-temperature and variable-field Mössbauer spectroscopy has been used to characterize the Fe−S clusters in D. vulgaris hydrogenase poised at different redox states generated during a reductive titration, and in the CO-reacted enzyme. The data were successfully decomposed into spectral components corresponding to the F and H clusters, and characteristic parameters describing the electronic and magnetic properties of the F and H clusters were obtained. Consistent with the X-ray crystallographic results, the spectra of the H cluster can be understood as originating from an exchange coupled [4Fe-4S]−[2Fe] system. In particular, detailed analysis of the data reveals that the reductive activation begins with reduction of the [4Fe-4S]_H cluster from the 2+ to the 1+ state, followed by transfer of the reducing equivalent from the [4Fe-4S]_H subcluster to the binuclear [2Fe]_H subcluster. The results also reveal that binding of exogenous CO to the H cluster affects significantly the exchange coupling between the [4Fe-4S]_H and the [2Fe]_H subclusters. Implication of such a CO binding effect is discussed.