Raman and infrared spectroscopy of cyanide-inhibited CO dehydrogenase/acetyl-CoA synthase from Clostridium thermoaceticum: Evidence for bimetallic enzymatic CO oxidation

Clostridium thermoaceticum and other autotrophic anaerobic bacteria contain a bifunctional enzyme, carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS), that catalyzes two reactions of CO at two separate Ni-FeS clusters. Oxidation of CO to CO2 is catalyzed by Cluster C, while incorporation of CO into acetyl-CoA occurs at Cluster A. In this study, resonance Raman [RR] and infrared [IR] spectroscopy are applied to the adduct of Cluster C with cyanide, a selective inhibitor of CO oxidation. The RR spectra reveal that CN- binds simultaneously to Fe and Ni, because bands whose C-13 and N-15 shifts identify them as cyanide-metal stretching and bending modes are sensitive to incorporation of both Fe-54 and Ni-64 into the enzyme. The IR spectrum reveals a low frequency, 2037 cm(-1), for the C-N stretch, indicative of Fe-II binding via the C end. Vibrational modeling of the frequencies and isotope shifts indicates a bent Fe-CN-Ni bridging geometry, with a similar to 140 degrees C-N-Ni angle. This geometry of the inhibitory adduct suggests that CO oxidation involves a bimetallic mechanism. It is proposed that pre-organization of the metal ions by the enzyme promotes CO oxidation by Ni-II-OH- attack on Fe-II-CO, followed by Ni-FeS reduction as CO2 is released. This chemistry is analogous to the metal-catalyzed water-gas shift reaction.