The Ni-Fe-S C-cluster of carbon monoxide dehydrogenases (CODH), which catalyzes the reversible oxidation of CO to CO2, can be stabilized in four redox states: Cox, Cred1, Cint, and Cred2. The best-supported mechanism of catalysis involves a one-electron reductive activation of Cox to Cred1 and a catalytic cycle in which Cred1 binds and oxidizes CO, forming Cred2 and releasing CO2. Recently reported experiments appear to have disqualified this mechanism, as activation was concluded to require reduction to a C-cluster state more reduced than Cred1. The results presented in this dissertation suggest that the activation potential was milder than that required to reduce these clusters. The results support a mechanism in which Cred1 is the form of the cluster that reacts with CO. The structure of the active-site C-cluster in CO dehydrogenase from Carboxydothermus hydrogenoformans (CODHCh) includes a ??2-sulfide ion bridged to the Ni and unique Fe, while the same cluster in enzymes from Rhodospirillum rubrum (CODHRr) and Moorella thermoacetica (CODHMt) lack this ion. This difference was investigated by exploring effects of sulfide on activity and spectral properties. Sulfide partially inhibited CO oxidation activities of CODHRr and CODHMt. Adding sulfide to CODHMt in the Cred1 state caused the gav = 1.82 Electron Paramagnetic Resonance spectroscopy (EPR) signal to decline and new features to appear. Sulfide did not affect the gav = 1.86 signal from the Cred2 state. A model was developed in which sulfide binds reversibly to Cred1, inhibiting catalysis. The results also suggest that the substrate hydroxyl group bridges the Ni and unique Fe. A cysteine residue recently found to form a persulfide bond with the C-cluster was characterized. The Ser mutant of the persulfide-forming Cys316 was inactive and displayed no C-cluster EPR signals. Electronic absorption and metal analysis suggest that the C-cluster is absent in this mutant. The persulfide bond appears to be essential for either the assembly or stability of the C-cluster, and/or for eliciting the redox chemistry of the C-cluster required for catalytic activity.

The Ni-Fe-S C-cluster of carbon monoxide dehydrogenases (CODH), which catalyzes the reversible oxidation of CO to CO2, can be stabilized in four redox states: Cox, Cred1, Cint, and Cred2. The best-supported mechanism of catalysis involves a one-electron reductive activation of Cox to Cred1 and a catalytic cycle in which Cred1 binds and oxidizes CO, forming Cred2 and releasing CO2. Recently reported experiments appear to have disqualified this mechanism, as activation was concluded to require reduction to a C-cluster state more reduced than Cred1. The results presented in this dissertation suggest that the activation potential was milder than that required to reduce these clusters. The results support a mechanism in which Cred1 is the form of the cluster that reacts with CO. The structure of the active-site C-cluster in CO dehydrogenase from Carboxydothermus hydrogenoformans (CODHCh) includes a ??2-sulfide ion bridged to the Ni and unique Fe, while the same cluster in enzymes from Rhodospirillum rubrum (CODHRr) and Moorella thermoacetica (CODHMt) lack this ion. This difference was investigated by exploring effects of sulfide on activity and spectral properties. Sulfide partially inhibited CO oxidation activities of CODHRr and CODHMt. Adding sulfide to CODHMt in the Cred1 state caused the gav = 1.82 Electron Paramagnetic Resonance spectroscopy (EPR) signal to decline and new features to appear. Sulfide did not affect the gav = 1.86 signal from the Cred2 state. A model was developed in which sulfide binds reversibly to Cred1, inhibiting catalysis. The results also suggest that the substrate hydroxyl group bridges the Ni and unique Fe. A cysteine residue recently found to form a persulfide bond with the C-cluster was characterized. The Ser mutant of the persulfide-forming Cys316 was inactive and displayed no C-cluster EPR signals. Electronic absorption and metal analysis suggest that the C-cluster is absent in this mutant. The persulfide bond appears to be essential for either the assembly or stability of the C-cluster, and/or for eliciting the redox chemistry of the C-cluster required for catalytic activity.