Carbon monoxide dehydrogenase from Rhodospirillum rubrum:: Effect of redox potential on catalysis

The Ni-Fe-S-containing C-cluster of carbon monoxide dehydrogenases is the active site for catalyzing the reversible oxidation of CO to CO2. This cluster can be stabilized in redox states designated C-ox, C-red1, C-int, and C-red2. What had until recently been the best-supported mechanism of catalysis involves a one-electron reductive activation of C-ox to C-red1 and a catalytic cycle in which the C-red1 state binds and oxidizes CO, forming C-red2 and releasing CO2. Recent experiments cast doubt on this mechanism, as they imply that activation requires reducing the C-cluster to a state more reduced than C-red1. In the current study, redox titration and stopped-flow kinetic experiments were performed to assess the previous results and conclusions. Problems in previous methods were identified, and related experiments for which such problems were eliminated or minimized afforded significantly different results. In contrast to the previous study, activation did not correlate with reduction of Fe-S clusters in the enzyme, suggesting that the potential required for activation was milder than that required to reduce these clusters (i.e., E-act(0) > -420 mV vs SHE). Using enzyme preactivated in solutions that were poised at various potentials, lag phases were observed prior to reaching steady-state CO oxidation activities. Fits of the Nernst equation to the corresponding lag-vs-potential plot yielded a midpoint potential of -150 +/- 50 mV. This value probably reflects E-o' for the C-ox/C-red1 couple, and it suggests that C-red1 is indeed active in catalysis.