MECHANISM OF CO OXIDATION BY CARBON-MONOXIDE DEHYDROGENASE FROM CLOSTRIDIUM-THERMOACETICUM AND ITS INHIBITION BY ANIONS

Carbon monoxide dehydrogenase (CODH) performs two distinct reactions a t two different metal centers. The synthesis of acetyl-CoA from a meth yl group, CO, and coenzyme A occurs at center A and the oxidation of C O to CO2 occurs at center C. In the work reported here, we have studie d the mechanism of CO oxidation by CODH and its inhibition by thiocyan ate. Our data are consistent with a ping-pong mechanism. A scheme to e xplain the first half-reaction was developed that includes binding of water and CO to the oxidized form of center C, deprotonation of coordi nated water to yield enzyme-bound hydroxyl, nucleophilic attack on coo rdinated CO by OH- to form enzyme-bound carboxyl, and deprotonation an d decarboxylation to form CO2 and the reduced form of center C. In the second half-reaction, the reduced enzyme is reoxidized by an electron acceptor. CO oxidation was pH dependent. The pH dependence of k(cat)/ K-m for CO gave a single pK(a) of 7.7 and a maximum value at 55 degree s C and high pH of 9.1 x 10(6) M(-1) s(-1). The pH dependence of k(cat ) followed a two-phase titration curve with pK(a) values of 7.1 and 9. 5 and maximum value of k(cat) at 55 degrees C and high pH of 3250 s(-1 ) (1310 mu mol of CO oxidized min(-1) mg(-1)). The pH dependencies of k(cat)/K-m and k(cat) are interpreted to reflect the ionization of enz yme-bound water from binary and ternary complexes with center C. React ion with thiocyanate, azide, or cyanate was found to cause a striking shift in the EPR spectrum of center C from g(av) = 1.82 (g = 2.01, 1.8 1, 1.65) to a two-component spectrum with g(av) = 2.15 (g = 2.34, 2.06 7, 2.03) and g(av) = 2.17 (g = 2.34, 2.115, 2.047). Thiocyanate acted as a mixed partial inhibitor with respect to CO. The inhibition consta nts were pH and temperature dependent. The pH dependencies of the inhi bition constants gave pK(a) values of similar to 7.7. Binding of thioc yanate to the oxidized form of center C appears to be favored by a neg ative enthalpy that is offset by a decrease in entropy yielding a slig htly unfavorable free energy of association.