SPECTROSCOPY OF PORPHYRIN AND QUINONE COFACTORS

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The objectives of this research proposal are the structural identification of the resting and catalytic-intermediate states of organic redox cofactors in proteins. The major focus will be on the heme and chlorin cofactors of cytochrome d oxidase and cytochrome cd1 nitrite reductase. This project builds upon the successful identification of the novel hydroporphyrin cofactors of these two enzymes as 5,6-dihydroxyprotochlorin and 1,3- porphyrindione-6-acrylate, respectively, as well as the total synthesis and metalation of both cofactors. Assisted with these unique model compounds, the exploration of the accessible reaction intermediates of these enzymes will be facilitated, and the characterization of their oxidation, spin, and coordination states by resonance Raman spectroscopy will be carried out. A significant preliminary finding that will be fully explored is the resonance Raman spectroscopic identification of a highly stable oxoferryl intermediate on the chlorin d of cytochrome d oxidase. Studies will be extended to characterize the formation of other possible chlorin d- associated intermediates in the catalytic cycle, such as oxy, peroxo, or ferryl pi-cation radical species. A site-directed mutant lacking heme b558, one of the three porphyrinic cofactors in the enzyme, will be investigated as a trap for these alternate intermediate states. The second aim of this proposal is to conduct a systematic investigation of the two heme cofactors of the dissimilatory nitrite reductase. Through resonance Raman spectroscopy, the coordination chemistry and ligand-binding properties of heme c and the substrate-binding dione will be elucidated. Of particular interest will be the characterization of reaction intermediates of the dioneheme d1 with NOx-type ligands. Research will also be initiated on the novel quinone cofactors pyrroloquinoline quinone of methanol and glucose dehydrogenases, as well as the amino-acid derived quinones tryptophan tryptophylquinone of methylamine dehydrogenase and trihydroxyphenylalanine quinone of amine oxidase. Preliminary studies have demonstrated that resonance Raman spectroscopy successfully differentiates among all three of these redox cofactors in their native, underivatized states. The common reaction mechanisms of quinone cofactors will be investigated using synthetic models, substrate analogs, and enzyme reconstitution.