FLAVOPROTEINS IN FATTY ACID METABOLISM

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The long term goal of this research is to gain a deeper understanding of the structure, catalytic mechanism, and metabolic control of flavoproteins involved in fatty acid oxidation. Three proteins form the main focus of this proposal: the medium chain acyl-CoA dehydrogenase and its physiological electron acceptor, electron transferring flavoprotein (ETF) from pig kidney mitochondria, together with the peroxisomal acyl-CoA oxidase from yeast. The chain length discrimination shown by the medium chain dehydrogenase will be compared to that of the mitochondrial short chain enzyme to see whether specificity is dictated by similar features. CoA affinity media, in which the coenzyme is attached to the gel hydrocarbon spacers of various lengths, will be evaluated in the purification of short, medium, and long chain enzymes. The inhibition of the acyl-CoA dehydrogenases by trans-3-enoyl-CoA derivatives will be studied because these thioesters are intermediates in the beta-oxidation of unsaturated fatty acids. The reactivity of the reduced medium chain dehydrogenase and acyl-CoA oxidase toward molecular oxygen will be compared by rapid reaction techniques in the presence or absence of various CoA derivatives to assess the influence of complexation on the rate of reoxidation. Similarly, the role of enoyl-CoA product in facilitating reduction of ETF by the medium chain dehydrogenase will be investigated using redox-inactive thioether analogs. Several approaches will be used to locate the FAD binding site in the heterodimeric electron carrier, ETF. Attempts will be made to identify physiological modulators of ETF activity. Complexes between the dehydrogenase and ETF will be studied by static titrations using flavin analogs as spectrophotometric probes. FAD analogs will also be used to maintain the participants in defined redox states. 2- and 3- alkynoyl-CoA derivatives will be used to label active sites peptides in the oxidized acyl-CoA dehydrogenases and acyl-CoA oxidase. The target residue(s) of these modifications will be identified, and the amino acid sequences of these peptides examined for possible homologies. The stable covalent flavin adduct formed upon inactivation of the reduced medium chain acyl-CoA dehydrogenase with 2-octynoyl-CoA will be characterized.