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ABSTRACT Title of Dissertation BENZOYL COA REDUCTASE A BIOLOGICAL BIRCH REDUCTION Steven Thomas Poole Doctor of Philosophy 2003 Dissertation directed by Dr David R Jollie Department of Chemistry Benzoyl CoA reductase isolated from the anaerobic bacterium Thauera aromatica catalyzes the ATP dependent two electron reduction of the aromatic ring of benzoyl CoA A Birch like mechanism which involves two separate one electron and one proton additions to the aromatic ring of benzoyl CoA has been previously proposed for benzoyl CoA reductase The first electron transfer of this reaction which produces a radical anion is thought to be the rate limiting step Other mechanisms such as hydride reduction and catalytic hydrogenation are possible In an effort to determine how the enzyme reduces its substrate several substrate analogues were synthesized and studied using kinetic and or product analysis Of the nitrogen containing heterocyclic analogues only picolinoyl CoA proved to be a substrate for the reductase having a kcat similar to that of benzoylCoA Nicotinoyl CoA did not react with the enzyme and isonicotinoyl CoA was reduced by the electron donor in the absence of the enzyme Mass spectrometric analysis of the products formed by the fluorinated analogues m fluorobenzoylCoA and p fluorobenzoyl CoA indicated that both substrates were defluorinated by benzoyl CoA reductase supporting a Birch like mechanism with the first electron being added to the carbonyl functionality of the thioester Also benzoyl CoA reductase only exhibited a small kinetic isotope effect 1 8 arguing against simultaneous hydrogen and electron transfer and hydride transfer It was also found that under aerobic conditions and without ATP benzoyl CoA reductase could carry out the oxidation of its native reduction product reforming the substrate of the reaction benzoyl CoA Since the reduction capability of benzoyl CoA reductase is quickly and irreversibly inactivated by oxygen it is thought that the enzyme is



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