These investigations have as the ultimate objective elucidation of the pathway by which certain bacteria grow with CO or CO2 and H2 as the source of carbon and energy. This is a pathway of autotrophic growth which differs from any previously described. It involves reduction of CO2 to methyltetrahydrofolate, then combination of this methyl group with CO and CoASH to form acetyl-CoA which is then used as the source of carbon for anabolic growth. The synthesis of acetyl-CoA from methyltetrahydrofolate, CO and CoASH is the unique portion of this pathway. Homogeneous proteins have been isolated from Clostridium thermoaceticum which catalyze this synthesis. They are CO dehydrogenase methyltransferase, a corrinoid enzyme, ferredoxin, and disulfide reductase. The CO dehydrogenase is the master enzyme of this pathway. It forms the C1 intermediate from CO or CO2 that is the precursor of the carbonyl group of acetyl-CoA, is the acceptor of the methyl from the methyl corrinoid enzyme and through the action of the CO dehydrogenase reductase combines with CoA. It then catalyzes the condensation of these three groups to acetyl-CoA. Our main objective is to elucidate how CO dehydrogenase catalyzes this condensation. We will use electron spin resonance (ESR), electron-nuclear double resonance, Mossbauer, and magnetic circular dichroism techniques to characterize the geometry and redox state of the Ni-Fe-S center of the CO dehydrogenase. We will investigate the mechanism of transfer of the methyl group from the methylated corrinoid protein to CO dehydrogenase with formation of an acetyl intermediate. We hope to detect this methyl-CO dehydrogenase complex using ESR techniques and chemically identify the amino acid sequence at the binding site. Likewise, the sequence near the linkage of CoASH to the enzyme, presumed to be by a disulfide bond, will be investigated. The monomers of the Alpha3Beta3 enzyme will be isolated, their metal content determined and genes for these proteins will be cloned and sequenced. The properties of CO dehydrogenase from certain methanogenic bacteria will be tested for their ability to synthesize acetyl-CoA to determine whether they may use a pathway of acetyl-CoA synthesis similar to that of C. thermoaceticum.
