? This research project involves the study of the membrane-bound respiratory protein succinate:ubiquinone oxidoreductase (Complex II) and its bacterial homologs. The mechanism of electron transfer from the covalently bound flavin cofactor, through the series of iron-sulfur clusters and to a protein bound quinone and heme are a focus of the studies. Mutations in the quinone and heme-binding domains of Complex II have been shown to be linked to premature aging in eukaryotes and the formation of highly vascularized tumors such as paragangliomas and pheochromocytomas in humans. The proposed studies will take advantage of the recently described x-ray crystal structure for Complex II from Escherichia coli which is an excellent model system for the mammalian enzyme. Site-specific amino acid substitutions will be constructed that mimic the similarly conserved residues in the eukaryotic enzymes. Biochemical and biophysical analysis of wild type and mutant enzymes will be done to dissect the effects on electron transfer and catalytic activity. Alteration in activity and the ability to form reactive oxygen species will be correlated with the phenotype found in the eukaryotic and mammalian mutations. The particular focus of these studies will be the quinone and heme-binding domains of Complex II. Studies are designed to investigate if the b heme cofactor is directly involved in the electron transport pathway or if it serves as an electron sink which may only function in reverse electron transfer such as could occur during periods of hypoxia or ischemia. Studies will also be done on the amino acid residues which may stabilize the semiquinone species generated during the catalytic cycle of Complex II. How the iron-sulfur clusters of the enzyme control the electron transfer rate and if this gates electron entry to/from the quinone will also be investigated. During these investigations additional structures of wild-type and mutants of Complex II will be developed to aid in structural analysis and to assist in describing the biochemical defects of Complex II that lead to tumor formation. ? ?
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