Potentiometric titrations of carbon monoxide dehydrogenase and properties of the Ni-labile and nonlabile forms of the acetyl-CoA synthase active sit

Carbon monoxide dehydrogenase (CODH) from Clostridium thermoaceticum is a bifunctional metalloenzyme, catalyzing the synthesis of acetyl-coenzyme A and reversible oxidation of CO to CO$sb2$ at two novel Ni-Fe-S active sites (the A and C-clusters, respectively). CODH also contains an $rmlbrack Fesb4Ssb4rbracksp{2+/1+}$ cube, known as the B-cluster. Midpoint potentials of these clusters were obtained by titrating CODH under equilibrium conditions using various partial pressures of CO in Ar and CO$sb2$ atmospheres, and simulating EPR signal intensities as a function of potential. Simulations assuming n (#e$sp-$ involved in reduction) values larger than expected for the individual reactions generally fit better than those which assumed the expected n, indicating positive redox cooperativity. The presence of CO$sb2$ raised the potentials of the A, B and C clusters, and appeared to increase the strength of CO binding to the reduced A-cluster. CO$sb2$ appears to stabilize an intermediate EPR-silent state of the C-cluster, and alter the saturation/relaxation properties of the reduced B-cluster. CO$sb2$ did not inhibit the removal of the labile Ni, but it did inhibit the CO/acetyl-coenzyme A exchange activity. These effects of CO$sb2$ indicate a significant CO$sb2$-dependent conformational change affecting the properties of all three clusters in both subunits. Since the enzyme operates in a CO$sb2$ environment in vivo, the CO$sb2$-induced conformation may be mechanistically significant.;Populations of the $alpha$ subunit contain two major forms of A-clusters, a catalytically active form called Ni-Labile and an inactive form called Nonlabile. The Ni component of the Ni-labile form could be reduced either by CO and a catalytic amount of native enzyme or by electrochemically-reduced triquat in the presence of CO. CO-binding raised $rm Esp{0prime}sb{Ni2+/1+},$ rendering CO and triquat effective reductants. Dithionite did not reduce the Ni-labile form, though its addition to CO/CODH-reduced Ni-labile clusters caused an intracluster electron transfer from the Ni$sp{1+}$ to the $rmlbrack Fesb4Ssb4rbracksp{2+}$ cluster. Dithionite reduced the $rmlbrack Fesb4Ssb4rbracksp{2+}$ component of the Nonlabile form as well as the cluster of the Ni-labile form once Ni was removed. Ni may not be bridged to the cube in the Nonlabile form. The $rmlbrack Fesb4Ssb4rbracksp{2+}$ may transfer electrons to-and-from the redox-active D site during reductive activation, and when bridged to the Ni of the A-cluster, it may modulate the redox and CO-binding properties of the Ni.