The C-cluster of carbon monoxide dehydrogenase (CODH) catalyzes the reversible oxidation of CO to form CO2. This study reports electron nuclear double resonance (ENDOR) spectroscopy of the one-electron reduced (C-red1), the CN--inhibited, and the CO (or dithionite)-reduced (C-red2) forms of the C-cluster from Clostridium thermoaceticum CODH (CODHCt). The observed hyperfine interactions of H-1,H-2,N-14, C-13, and Fe-57 support and extend the current Ni-X-[Fe4S4] C-cluster model in which a [Fe4S4] center is linked to a Ni ion through a unique iron, FCII. The unpaired electron spin apparently is localized on the [Fe4S4] component of the cluster, and thus the hyperfine interactions observed by ENDOR most probably reflect species associated with that component. A solvent-exchangeable proton with a maximum hyperfine coupling of A(H-1) = 16 MHz is detected in the C-red1 form, but not in the CN--inhibited or C-red2 forms. The exchangeable proton is assigned to a probable solvent-derived (HxO, x = 1, 2) ligand to FCII of the C-red1 [Fe4S4](1+) center and is predicted to be a substrate in CO/CO2 catalysis. For both C-red1 and C-red2, We find ENDOR features in the region expected for a nitrogen-donor ligand which likely arise from a histidine ligand to the [Fe4S4] center. Fe-57 ENDOR detects at least two classes of Fe in C-red1 that most likely arise from the (Fe2.5+)(2) mixed-valence pair. Their large maximum couplings of A(Fe-57) > 40 MHz support the unusual nature of the cluster; these do not change dramatically between the C-red1 and C-red2 forms of the enzyme. C-red2 formed by reduction with (CO)-C-13 reveals no new C-13 features, strongly suggesting that neither CO nor its oxidized products are bound to the [Fe4S4] center in C-red2 Taken together, these ENDOR assignments suggest that in the C-red1 state, the unique Fe ion of the CODH C-cluster has an available coordination site that stably binds HxO or CN- and that reduction of the C-cluster results in rearrangement at that site, causing loss of the bound aqueous ligand.