The long term objectives of the proposed research are: 1) To advance our understanding of the mechanisms for electron transfer in oxidation-reduction reactions catalyzed by flavoenzymes; 2) To evaluate the role of the coenzyme in those flavoproteins where catalysis does not involve a net oxidation-reduction reaction. Studies related to the latter objective will seek to determine the structure of a second chromophore in E. coli DNA photolyase, an enzyme which also contains a blue FAD radical, and to evaluate the role of both chromophores in catalysis. Studies on the mechanism of yeast DNA photolyase will seek to determine the function and the structure of a reduced flavin prosthetic group and of a possible second chromophore. Analogous studies with photolyase from bovine leucocytes and a human promyelocytic cell line will help in defining the diversity in chromophore content and mechansim with enzymes from different sources. Studies with sarcosine oxidase from Corynebacteria will focus on the catalytic functions and properties of its two nonequivalent flavins and will also include experiments to evaluate the possible role of tetrahydrofolate in catalysis. Additional studies will seek to determine whether other flavoproteins from Corynebacteria contain both covalent and noncovalent flavin, similar to sarcosine oxidase. Studies with lactate oxidase will seek to identify an active site base that abstracts the Alpha-proton during lactate oxidation. Evidence regarding the role and origin of a stable flavin radical will be investigated in studies with methanol oxidase which will also seek to characterize the mechanism of methanol oxidation and the nature of a red complex formed with the enzyme and azide. The proposed research will involve kinetic and structural analysis and will include reactions with normal substrates, substrate analogues, inhibitors and modified flavins. The structure of the chromophore(s) in human DNA photolyase could be of therapeutic significance in treating certain forms of xeroderma pigmentosum.
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