Benzoyl-CoA Reductase: A Biological Birch Reduction
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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 benzoyl-CoA. 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-fluorobenzoyl-CoA 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 degraded under aerobic conditions. However, these finding suggest that benzoyl-CoA reductase may only be partially degraded by oxygen exposure and that some of its subunits may still retain some of its functionality and structure.