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 protein, CO, and CoA. These reactions occur at metal centers on CODH and involve metal-carbon bond formation and transformation. There are three 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 catalyzes an exchange reaction between CO and acetyl-CoA. Center C is the site of CO oxidation and CO2 reduction. In the work described here, inhibition 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 spectroscopic and steady-state kinetic studies demonstrated that CS2 mimics the binding of CO to the nickel/iron-sulfur cluster at Center A; however, CS2 itself does not undergo oxidation-reduction and does not appear to bind to Center C as does CO. In the isotope exchange reaction between acetyl-CoA and CO, CS2 was found to be a competitive inhibitor with 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 complex has three redox states and that the intermediate state is paramagnetic. A maximum of 0.3-0.4 spins/mol of CODH could be obtained. Fitting this data to the Nernst equation indicated that integral spin intensities could not be obtained because the reduction potentials for the two redox couples were the same (similar to-455 mV). We suggest that similar redox chemistry may limit the spin intensity of the adduct between Center A and CO. Although CS2 did not bind to Center C, it inhibited reactions that occur at Center C. CS2 was a noncompetitive inhibitor us CO2 in CO2 reduction and us CO in CO oxidation.