The structure of the active-site C-cluster in CO dehydrogenase from Carboxydothermus hydrogenoformans includes a mu(2)-sulfide ion bridged to the Ni and unique Fe, whereas the same cluster in enzymes from Rhodospirillum rubrum (CODHRr) and Moorella thermoacetica (CODHMt) lack this ion. This difference was investigated by exploring the effects of sodium sulfide on activity and spectral properties. Sulfide partially inhibited the CO oxidation activity of CODHRr and generated a lag prior to steady-state. CODHMt was inhibited similarly but without a lag. Adding sulfide to CODHMt in the C-red1 state caused the g(av) = 1.82 EPR signal to decline and new features to appear, including one with g = 1.95, 1.85 and (1.70 or 1.62). Removing sulfide caused the g(av) = 1.82 signal to reappear and activity to recover. Sulfide did not affect the g(av) = 1.86 signal from the C-red2 state. A model was developed in which sulfide binds reversibly to C-red1, inhibiting catalysis. Reducing this adduct causes sulfide to dissociate, C-red2 to develop, and activity to recover. Using this model, apparent K-I values are 40 +/- 10 nM for CODHRr and 60 +/- 30 muM for CODHMt. Effects of sulfide are analogous to those of other anions, including the substrate hydroxyl group, suggesting that these ions also bridge the Ni and unique Fe. This proposed arrangement raises the possibility that CO binding labilizes the bridging hydroxyl and increases its nucleophilic tendency toward attacking Ni-bound carbonyl.