This proposal is to extend the structural studies of the acyl-CoA dehydrogenases and their physiological electron transfer partner, electron transfer flavoprotein (ETF). Acyl-CoA dehydrogenases are a family of enzymes that are involved in the first oxidative step in the mitochondrial catabolism of fatty acids and in the catabolism of some amino acids. ETF funnels the reducing equivalents to the main mitochondrial respiratory chain via ETF-ubiquinone oxidoreductase (ETF-QO), a membrane-bound, iron-sulfur flavoprotein; it is also proposed to determine the structure of ETF-QO. Fatty acid oxidation is the principal energy-yielding process in the liver, kidney, and skeletal muscle. The rate of this process can be altered by diet, physiological status, and disease, exemplified by starvation, pregnancy, and diabetes. The critical roles played in metabolism by acyl-CoA dehydrogenases, ETF, and ETF-QO, is illustrated by the severity of human diseases resulting from inherited deficiencies of each of these enzymes. Crystal structures have been determined for short- and medium-chain acyl-CoA dehydrogenases and their mutants and isovaleryl- and glutaryl-CoA dehydrogenase (GCD). It is proposed: to continue the studies of mutants of MCAD and its complexes with substrate analog/inhibitors; to determine the structure of human long chain acyl-CoA dehydrogenase in order to confirm the catalytic base and determine the structural basis for long-chain specificity; and to complete the refinement of the structure of GCD in order to study the mechanism of the decarboxylation reaction. GCD catalyzes both the dehydrogenation and decarboxylation reactions. Crystal structures of human ETF and a bacterial ETF have been determined. It is proposed to extend these studies to mutant ETFs to study the interactions with their electron transfer partners, the dehydrogenases and ETF-QO. Pig ETF-QO has been crystallized and its structure determination is in progress, using X-ray diffraction methods. The high resolution structure, together with those of ETF and the dehydrogenases, will enable us to study the molecular basis of electron transfer pathway between these flavoproteins.
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