Kinetic and spectroscopic studies of the molybdenum-copper CO dehydrogenase from Oligotropha carboxidovorans.

Carbon monoxide dehydrogenase from the aerobic bacterium Oligotropha carboxidovorans catalyzes the oxidation of CO to CO(2), yielding two electrons and two H(+). The steady-state kinetics of the enzyme exhibit a pH optimum of 7.2 with a k(cat) of 93.3 s(-1) and K(m) of 10.7 microM at 25 degrees C. k(red) for the reductive half-reaction agrees well with k(cat) and exhibits a similar pH optimum, indicating that the rate-limiting step of overall turnover is likely in the reductive half-reaction. No dependence on CO concentration was observed in the rapid reaction kinetics, however, suggesting that CO initially binds rapidly to the enzyme, possibly at the Cu(I) of the active site, prior to undergoing oxidation. A Mo(V) species that exhibits strong coupling to the copper of the active center (I = 3/2) has been characterized by EPR. The signal is further split when [(13)C]CO is used to generate it, demonstrating that substrate (or product) is a component of the signal-giving species. Finally, resonance Raman spectra of CODH reveal the presence of FAD, Fe/S clusters, and a [CuSMoO(2)] coordination in the active site, consistent with earlier x-ray absorption and crystallographic results.