Acetogenic carbon monoxide dehydrogenases catalyze the reversible oxidation of CO to CO2 and the synthesis of acetyl-coenzyme A, utilizing two novel Ni-Fe-S active sites (the C- and A-clusters, respectively) and an [Fe4S4](2+/1+) cluster (the B-cluster) that serves to transfer electrons. Enzyme samples were titrated under equilibrium conditions using various partial pressures of CO in Ar and CO2 atmospheres. EPR signal intensities from each cluster were analyzed as a function of potential using the Nernst equation. The presence of CO2 raised the reduction potentials of the A-, B- , and C-clusters, and it appeared to increase the strength of CO (substrate for acetyl-CoA synthesis) binding to the reduced A-cluster. Carbon dioxide also appeared to stabilize an intermediate EPR-silent state of the C-cluster and alter the saturation/relaxation properties of the reduced B-cluster. Simulations assuming n values (number of e- involved in reduction) larger than appropriate for the individual reactions generally fit better to the titration data than those which assumed the appropriate n, indicating positive redox cooperativity. Carbon dioxide did not inhibit 1,10- phenanthroline from removing the labile Ni from the A-cluster, but it did inhibit the CO/acetyl-coenzyme A exchange activity, probably by causing CO to bind more tightly to the A-cluster. Taken together, these results indicate a significant CO2-dependent conformational change affecting the properties of all three clusters and both subunits. Since the enzyme operates in vivo in a CO2 environment, the CO2-induced conformation may be mechanistically important.
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