BINDING OF CARBON-DISULFIDE TO THE SITE OF ACETYL-COA SYNTHESIS BY THE NICKEL-IRON-SULFUR PROTEIN, CARBON-MONOXIDE DEHYDROGENASE, FROM CLOSTRIDIUM-THERMOACETICUM

Carbon monoxide dehydrogenase (CODH) is a key enzyme in the pathway of carbon monoxide and carbon dioxide fixation by anaerobic bacteria. It performs the oxidation of CO to CO2, the reduction of CO2 to CO, and the synthesis of acetyl-CoA from a methylated corrinoid/iron-sulfur pr otein, CO, and CoA. These reactions occur at metal centers on CODH and involve metal-carbon bond formation and transformation. There are thr ee iron-containing centers that play distinct roles in CODH: Centers A , B, and C. Center A is the site of synthesis of acetyl-CoA and cataly zes an exchange reaction between CO and acetyl-CoA. Center C is the si te of CO oxidation and CO2 reduction. In the work described here, inhi bition of CODH by carbon disulfide was studied. CS2 was found to serve as a probe of the interaction of CODH with CO at Center A, EPR spectr oscopic and steady-state kinetic studies demonstrated that CS2 mimics the binding of CO to the nickel/iron-sulfur cluster at Center A; howev er, CS2 itself does not undergo oxidation-reduction and does not appea r to bind to Center C as does CO. In the isotope exchange reaction bet ween acetyl-CoA and CO, CS2 was found to be a competitive inhibitor wi th respect to CO (Ki = 0.47 mM) and a mixed inhibitor with respect to acetyl-CoA (K-i1 = 0.30 and K-i2 = 1.1 mM). The reaction of dithionite -reduced CODH with CS2 resulted in an EPR spectrum with g values of 2. 200, 2.087, and 2.017. This EPR signal from the CS2 adduct with Center A is similar to that assigned to the Ni(I) state of hydrogenases. EPR spectroelectrochemical titrations demonstrated that the CODH-CS2 comp lex has three redox states and that the intermediate state is paramagn etic. A maximum of 0.3-0.4 spins/mol of CODH could be obtained. Fittin g this data to the Nernst equation indicated that integral spin intens ities could not be obtained because the reduction potentials for the t wo redox couples were the same (similar to-455 mV). We suggest that si milar redox chemistry may limit the spin intensity of the adduct betwe en Center A and CO. Although CS2 did not bind to Center C, it inhibite d reactions that occur at Center C. CS2 was a noncompetitive inhibitor us CO2 in CO2 reduction and us CO in CO oxidation.