EXPRESSION AND CHARACTERIZATION OF 2 PATHOGENIC MUTATIONS IN HUMAN ELECTRON-TRANSFER FLAVOPROTEIN

Defects in electron transfer flavoprotein (ETF) or its electron accept or, 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 de hydrogenases to the main respiratory chain is impaired, Among these de hydrogenases are the four chain length-specific flavoprotein dehydroge nases of fatty acid beta-oxidation. In this investigation, two mutatio ns in the alpha subunit that have been identified in patients were exp ressed in Escherichia coli, Of the two mutant alleles, alpha T266M and alpha G116R, the former is the most frequent mutation found in patien ts with ETF deficiency, The crystal structure of human ETF shows that alpha G116 lies in a hydrophobic pocket, under a contact residue of th e alpha/beta subunit interface, and that the hydroxyl hydrogen of alph a T266 is hydrogen bonded to N(5) of the FAD; the amide backbone hydro gen of alpha T266 is hydrogen-bonded to C(4)-O of the flavin prostheti c 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 alp ha G116R ETF required coexpression of the chaperonins, GroEL and GroES , alpha G116R ETF folds into a conformation different from the wild ty pe, and is catalytically inactive in crude extracts, It is unstable an d 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, i ts flavin environment is altered as indicated by absorption and circul ar 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 semiqui none of wild type ETF. The mutation has relatively little effect on th e reductive half-reaction of ETF catalyzed by sarcosine and medium cha in acyl CoA dehydrogenases which reduce the flavin to the semiquinone, However, k(cat)/K-m of ETF-QO in a coupled acyl-CoA:ubiquinone reduct ase assay with oxidized alpha T266M ETF as substrate is reduced 33-fol d; this decrease is due in largest part to a decrease in the rate of d isproportionation of the alpha T266M ETF semiquinone catalyzed by ETF- QO.