Defects in electron transfer flavoprotein (ETF) or its electron acceptor, electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO), cause the human inherited metabolic disease glutaric acidemia type II, In this disease, electron transfer from nine primary flavoprotein dehydrogenases to the main respiratory chain is impaired, Among these dehydrogenases are the four chain length-specific flavoprotein dehydrogenases of fatty acid beta-oxidation. In this investigation, two mutations in the alpha subunit that have been identified in patients were expressed in Escherichia coli, Of the two mutant alleles, alpha T266M and alpha G116R, the former is the most frequent mutation found in patients with ETF deficiency, The crystal structure of human ETF shows that alpha G116 lies in a hydrophobic pocket, under a contact residue of the alpha/beta subunit interface, and that the hydroxyl hydrogen of alpha T266 is hydrogen bonded to N(5) of the FAD; the amide backbone hydrogen of alpha T266 is hydrogen-bonded to C(4)-O of the flavin prosthetic group (Roberts, D, L,, Frerman, F, E, and Kim, J-J. P, (1996) Proc, Natl, Acad, Sci, U.S.A. 93, 14355-14360). Stable expression of the alpha G116R ETF required coexpression of the chaperonins, GroEL and GroES, alpha G116R ETF folds into a conformation different from the wild type, and is catalytically inactive in crude extracts, It is unstable and could not be extensively purified, The alpha T266M ETF was purified and characterized after stabilization to proteolysis in crude extracts, Although the global structure of this mutant protein is unchanged, its flavin environment is altered as indicated by absorption and circular dichroism spectroscopy and the kinetics of flavin release from the oxidized and reduced protein, The loss of the hydrogen bond at N(5) of the flavin and the altered flavin binding increase the thermodynamic stability of the flavin semiquinone by 10-fold relative to the semiquinone of wild type ETF. The mutation has relatively little effect on the reductive half-reaction of ETF catalyzed by sarcosine and medium chain acyl CoA dehydrogenases which reduce the flavin to the semiquinone, However, k(cat)/K-m of ETF-QO in a coupled acyl-CoA:ubiquinone reductase assay with oxidized alpha T266M ETF as substrate is reduced 33-fold; this decrease is due in largest part to a decrease in the rate of disproportionation of the alpha T266M ETF semiquinone catalyzed by ETF-QO.
