(13)C NMR Characterization of an Exchange Reaction between CO and CO(2) Catalyzed by Carbon Monoxide Dehydrogenase

[Image: see text] Carbon monoxide dehydrogenase (CODH) catalyzes the reversible oxidation of CO to CO(2) at a nickel−iron−sulfur cluster (the C-cluster). CO oxidation follows a ping-pong mechanism involving two-electron reduction of the C-cluster followed by electron transfer through an internal electron transfer chain to external electron acceptors. We describe (13)C NMR studies demonstrating a CODH-catalyzed steady-state exchange reaction between CO and CO(2) in the absence of external electron acceptors. This reaction is characterized by a CODH-dependent broadening of the (13)CO NMR resonance; however, the chemical shift of the (13)CO resonance is unchanged, indicating that the broadening is in the slow exchange limit of the NMR experiment. The (13)CO line broadening occurs with a rate constant (1080 s(−1) at 20 °C) that is approximately equal to that of CO oxidation. It is concluded that the observed exchange reaction is between (13)CO and CODH-bound (13)CO(2) because (13)CO line broadening is pH-independent (unlike steady-state CO oxidation), because it requires a functional C-cluster (but not a functional B-cluster) and because the (13)CO(2) line width does not broaden. Furthermore, a steady-state isotopic exchange reaction between (12)CO and (13)CO(2) in solution was shown to occur at the same rate as that of CO(2) reduction, which is approximately 750-fold slower than the rate of (13)CO exchange broadening. The interaction between CODH and the inhibitor cyanide (CN(−)) was also probed by (13)C NMR. A functional C-cluster is not required for (13)CN(−) broadening (unlike for (13)CO), and its exchange rate constant is 30-fold faster than that for (13)CO. The combined results indicate that the (13)CO exchange includes migration of CO to the C-cluster, and CO oxidation to CO(2), but not release of CO(2) or protons into the solvent. They also provide strong evidence of a CO(2) binding site and of an internal proton transfer network in CODH. (13)CN(−) exchange appears to monitor only movement of CN(−) between solution and its binding to and release from CODH.