13C NMR characterization of an exchange reaction between CO and CO2 catalyzed by carbon monoxide dehydrogenase

Carbon monoxide dehydrogenase (CODH) catalyzes the reversible oxidation of CO to CO2 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 C-13 NMR studies demonstrating a CODH-catalyzed steady-state exchange reaction between CO and CO2 in the absence of external electron acceptors. This reaction is characterized by a CODH-dependent broadening of the (CO)-C-13 NMR resonance; however, the chemical shift of the (CO)-C-13 resonance is unchanged, indicating that the broadening is in the slow exchange limit of the NMR experiment. The (CO)-C-13 line broadening occurs with a rate constant (1080 s(-1) at 20 degrees C) that is approximately equal to that of CO oxidation. It is concluded that the observed exchange reaction is between (CO)-C-13 and CODH-bound (CO2)-C-13 because (CO)-C-13 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 (CO)-C-13) line width does not broaden. Furthermore, a steady-state isotopic exchange reaction between (CO)-C-12 and (CO2)-C-13) in solution was shown to occur at the same rate as that of CO2 reduction, which is approximately 750-fold slower than the rate of (CO)-C-13 exchange broadening. The interaction between CODH and the inhibitor cyanide (CN-) was also probed by C-13 NMR. A functional C-cluster is not required for (CN-)-C-13 broadening (unlike for (CO)-C-13), and its exchange rate constant is 30-fold faster than that for (CO)-C-13. The combined results indicate that the (CO)-C-13 exchange includes migration of CO to the C-cluster, and CO oxidation to CO2, but not release of CO2 or protons into the solvent. They also provide strong evidence of a CO) binding site and of an internal proton transfer network in CODH. (CN-)-C-13 exchange appears to monitor only movement of CN- between solution and its binding to and release from CODH.