A multinuclear ENDOR study of the C-cluster in CO dehydrogenase from Clostridium thermoaceticum: Evidence for H(x)O 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 (C(red1)), the CN^--inhibited, and the CO (or dithionite)-reduced(C(red2)) forms of the C-cluster from Clostridium thermoaceticum CODH (CODH(Ct)). The observed hyperfine interactions of (1.2)H, 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 C(red1) form, but not in the CN^--inhibited or C(red2) forms. The exchangeable proton is assigned to a probable solvent-derived (H(x)O, 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. ⁵⁷Fe ENDOR detects at least two classes of Fe in C(red1) that most likely arise from the (Ee(2.5+))₂ mixed- valence pair. Their large maximum couplings of A(⁵⁷Fe) > 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 ¹³CO reveals no new ¹³C 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 H(x)O or CN^- and that reduction of the C-cluster results in rearrangement at that site, causing loss of the bound aqueous ligand.