CHARACTERIZATION OF THE METAL CENTERS OF THE NI FE-S COMPONENT OF THECARBON-MONOXIDE DEHYDROGENASE ENZYME COMPLEX FROM METHANOSARCINA-THERMOPHILA/

Methanosarcina thermophila contains a multienzyme complex called the c arbon-monoxide dehydrogenase complex, which has been resolved into a n ickel/iron-sulfur and a corrinoid/iron-sulfur component. This complex plays a central role in acetoclastic methanogenesis. The Ni/Fe-S compo nent catalyzes CO oxidation and has been proposed to be involved in cl eavage of acetyl-CoA into its methyl, carbonyl, and CoA moieties. In t he work reported here, three metal centers in the Ni/Fe-S component we re characterized by electron paramagnetic resonance (EPR) spectroscopy and spectroelectrochemistry and pre-steady state kinetics. Center A c ontains nickel and iron and forms an EPR active adduct with CO, which is called the NiFeC species. The EPR spectrum of the NiFeC species has g values of 2.059, 2.051, and 2.029 and is observable at temperatures as high as 150 K. This signal had previously been observed only in th e carbon-monoxide dehydrogenase complex of M. thermophila and the acet yl-CoA synthase from acetate-producing bacteria. Incubation of the CO- reduced Ni/Fe-S component with acetyl-CoA resulted in an increase in i ntensity of the NiFeC signal, which supports a role for the component in the cleavage of acetyl-CoA. Generation of the NiFeC EPR signal occu rs with a rate constant of 0.4 s-1, a result that demonstrates the kin etic competence of this species in the acetyl-CoA cleavage reaction bu t rules it out as the site of oxidation of CO to CO2. Center B is like ly to be a [4Fe-4S]2+1/1+ center with g values of 2.04, 1.93, and 1.89 (g(av) = 1.95) and a standard reduction potential (E'0) of -444 mV. A t potentials less than -500 mV, another EPR signal develops that appea rs to originate from another state of Center B. Center C is a fast rel axing center with g values of 2.02, 1.88, and 1.71 (g(av) = 1.87) and an E'0, of -154 mV.