A Multinuclear ENDOR Study of the C-Cluster in CO Dehydrogenase from Clostridium thermoaceticum: Evidence for HxO and Histidine Coordination to the [Fe4S4] Center

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 (Cred1), the CN--inhibited, and the CO (or dithionite)-reduced (Cred2) forms of the C-cluster from Clostridium thermoaceticum CODH (CODHCt). The observed hyperfine interactions of 1,2H, 14N, 13C, and 57Fe 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(1H) = 16 MHz is detected in the Cred1 form, but not in the CN--inhibited or Cred2 forms. The exchangeable proton is assigned to a probable solvent-derived (HxO, x = 1, 2) ligand to FCII of th...