STOICHIOMETRIC REDUCTIVE TITRATIONS OF DESULFOVIBRIO-GIGAS HYDROGENASE

The NiFe hydrogenase from Desulfovibrio gigas contains one [Fe3S4](1+/ 0) cluster, two [Fe4S4](2+/1+) clusters, and one active-site Ni center that can be stabilized in four magnetic states (designated Ni-AB, Ni- SI, Ni-C, and Ni-R). Ni-AB and Ni-SI almost certainly correspond to Ni 3+ and Ni2+ electronic states, respectively, while the electronic desi gnations of Ni-C and Ni-R are uncertain. Ni-C arises from a species co ntaining a photolabile hydrogenic species (H+, H-, or H-2). Stoichiome tric reductive titrations of thionin-oxidized Hase were performed, usi ng the reductant H-2. Titrations were monitored at 310 nm and by EPR. The resulting titration curves were simulated using two models. Each a ssumed that the enzyme contains the redox centers mentioned above, but they differed as to the number of electrons assumed to separate Ni-C from Ni-AB. One model assumed Ni-C was two electrons more reduced than Ni-AB, the other that it was four electrons more reduced. The former model fit the data substantially better than the latter. This restrict s possible Ni-C designations to either a protonated Ni1+ species, a Ni 3+ hydride, a Ni3+(n(2)-H-2) complex, or a Ni2+ species with a ligand radical. An argument is expanded in support of a Ni1+ dithiol designat ion of Ni-C. Partially reduced enzyme also exhibits an unusually compl ex EPR signal (the g = 2.21 signal) that has been proposed to originat e from magnetic coupling between Ni-C and one of the reduced [Fe4S4](1 +) clusters. The analysis presented indicates that the interacting clu ster is the one with the more negative redox potential, designated Fe- 4b. This cluster is almost certainly located nearest to the Ni center. The Ni-C/Ni-R and [Fe-4b](2+/1+) couples are both in odor equilibrium with H-2. A mechanism of catalysis based on the spatial proximity and odor properties of the Ni center and Fe-4b is discussed.