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 Ni3+ and Ni2+ electronic states, respectively, while the electronic designations of Ni-C and Ni-R are uncertain. Ni-C arises from a species containing a photolabile hydrogenic species (H+, H-, or H-2). Stoichiometric reductive titrations of thionin-oxidized Hase were performed, using the reductant H-2. Titrations were monitored at 310 nm and by EPR. The resulting titration curves were simulated using two models. Each assumed 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 restricts possible Ni-C designations to either a protonated Ni1+ species, a Ni3+ 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 designation of Ni-C. Partially reduced enzyme also exhibits an unusually complex EPR signal (the g = 2.21 signal) that has been proposed to originate from magnetic coupling between Ni-C and one of the reduced [Fe4S4](1+) clusters. The analysis presented indicates that the interacting cluster 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.