Research Project
3279953
The long term objectives of the proposed research are to: A.) Elucidate mechanisms of electron transfer in dation-reduction reactions catalyzed by flavoproteins; B.) Evaluate the role of flavin in those enzymes where catalysis does not involve a net oxidation-reduction reaction. Studies related to long term objective B will focus on DNA photolyase. This enzyme repairs pyrimidine dimers in UV-damaged DNA and contain chromophores, reduced flavin (FADH-2) and pterin, which act as sensitizers in catalysis. Failure to repair dimers causes mutations, cancer and cell death. We seek to define substrate binding determinants, including contributions due to: 1. the nature of the dimer; 2. interactions with the sugar-phosphate backbone; 3. the nature of bases near the dimer. Information regarding the mechanism of dimer repair will be sought in studies which seek to: Evaluate the catalytic role of the excited singlet state of FADH-2; 2. Determine whether dimer cleavage involves electron transfer and/or formation of covalent flavin-dimer adducts; 3. Determine the catalytic significance of energy transfer from the excited pterin chromophore to FADH-2. Studies related to long-term objective will focus on corynebacterial sarcosine oxidase. This enzyme contains covalent [(8-alpha-N(3)-histidyl)FAD] and noncovalent FAD, 4 different subunits, a single sarcosine site but 2 sites for another substrate, tetrahydrofolate. We seek to investigate the biosynthesis and catalytic significance of the covalent flavin-polypeptide Linkage. The functional significance of the enzyme's complex quaternary structure will be probed studies which seek to identify which subunits are involved in Ligand binding and to determine whether all 4 subunits are essential for activity or partial reactivity. The catalytic mechanism will be investigated in studies which seek to: 1. Elucidate the mechanism of electron transfer from sarcosine to the noncovalent flavin; 2. determine whether a flavin biradical intermediate is formed during interflavin electron transfer and whether he enzyme cycles between fully oxidized and 2-electron reduced forms, as observed with a poor substrate, L-proLine. Achievement of these goals will be facilitated by studies which seek to clone, sequence, and express the genes which code for each of the enzyme's subunits. The proposed research with DNA photolyase and sarcosine oxidase will involve use of modified substrates and flavins, a variety of spectroscopic techniques including time-resolved fluorescence, flash photolysis and stopped flow), action spectra and quantum yield measurements, and recombinant DNA methodology.
