CO CO2 POTENTIOMETRIC TITRATIONS OF CARBON-MONOXIDE DEHYDROGENASE FROM CLOSTRIDIUM-THERMOACETICUM AND THE EFFECT OF CO2/

Acetogenic carbon monoxide dehydrogenases catalyze the reversible oxid ation of CO to CO2 and the synthesis of acetyl-coenzyme A, utilizing t wo novel Ni-Fe-S active sites (the C- and A-clusters, respectively) an d an [Fe4S4](2+/1+) cluster (the B-cluster) that serves to transfer el ectrons. Enzyme samples were titrated under equilibrium conditions usi ng various partial pressures of CO in Ar and CO2 atmospheres. EPR sign al intensities from each cluster were analyzed as a function of potent ial using the Nernst equation. The presence of CO2 raised the reductio n potentials of the A-, B-, and C-clusters, and it appeared to increas e the strength of CO (substrate for acetyl-CoA synthesis) binding to t he reduced A-cluster. Carbon dioxide also appeared to stabilize an int ermediate EPR-silent state of the C-cluster and alter the saturation/r elaxation 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 dat a than those which assumed the appropriate n, indicating positive redo x cooperativity. Carbon dioxide did not inhibit 1,10-phenanthroline fr om removing the labile Ni from the A-cluster, but it did inhibit the C O/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 proper ties of all three clusters and both subunits. Since the enzyme operate s in vivo in a CO2 environment, the CO2-induced conformation may be me chanistically important.