ANALYSIS OF OXIDATIVE TITRATIONS OF DESULFOVIBRIO-GIGAS HYDROGENASE -IMPLICATIONS FOR THE CATALYTIC MECHANISM

The oxidative titrations of hydrogenase (Hase) from Desulfovibrio giga s [Barondeau, D. P., Roberts, L. M., & Lindahl, P. A. (1994) J. Am. Ch em. Soc. 116, 3442] were simulated using model descriptions of the red ox reactions in the enzyme. The data fit best to a model that assumed Hase contains one [Fe3S4](1+/0) cluster, two [Fe4S4](2+/1+) clusters, and a Ni center stable in four redox states (Ni-B, Ni-SI, Ni-C, and Ni -R), each separated by one electron. A model in which Ni-SI, Ni-C, and Ni-R correspond to Nickel(2+) dithiolate, nickel(1+) dithiol, and nic kel(2+) dithiol hydride, respectively, is compatible with all establis hed relevant properties of the Ni center. This model and the concept o f redox microstates were employed to define electronic states of the e nzyme and to reformulate the catalytic mechanism initially proposed by Cammack et al. [Cammack, R., Patil, D. S., Hatchikian, E. C., & Ferna ndez, V. M. (1987) Biochim. Biophys. Acta 912, 98] into three intercon nected catalytic cycles. These cycles differ in the average oxidation level of the Fe4S4 clusters. The cycle with the most reduced clusters appears to operate reversibly (catalyzing both H-2 oxidation and H+ re duction), while those with more oxidized clusters function only to oxi dize H-2. The difference in reversibility is explained by assuming tha t Ni-R prefers to reduce an [Fe4S4](2+) cluster instead of H+ and that H+ is reduced only when that Fe4S4 cluster is in its reduced state.