BIOCHEMICAL AND MOLECULAR BASIS OF ETF-QO DEFICIENCY

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Glutaric acidemia type II (GA2) is an inherited metabolic disease resulting from deficiency of electron transfer flavoprotein (ETF) or electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO). Because ETF is the electron acceptor for (at least) nine flavoprotein dehydrogenases, deficiency of either of these proteins impairs mitochondrial oxidation pathway. This proposal focuses on ETF-QO, and on its reaction with ETF. Mutations in ETF-QO from GA2 patients with immunologically and enzymatically established ETF-GO deficiency will be identified, using the recently established structure of the ETF-QO gene to amplify individual exons. The interactions between mutant and wild- type ETFs and ETF-QO will be investigated in kinetic experiments. These experiments employ site-directed mutants in two regions that, based on comparisons of the crystal structures of several ETFs (determined in collaboration with Dr. Kim), are predicted to participate in complex formation. The rationale for the site-directed mutations to be studied is also based on previous experiments that implicate specific lysine-containing regions of the beta-ETF subunit in the interaction of ETF with ETF-QO. The three-dimensional structure of ETF-QO will be determined by x-ray crystallography, to better understand the effects of mutations and the functioning of the wild-type protein. Finally, we will establish expression systems to investigate the effects of selected disease- causing mutations on the flavin and iron sulfur redox centers of this integral membrane protein.