CHARACTERIZATION OF A MUTATION THAT ABOLISHES QUINONE REDUCTION BY ELECTRON-TRANSFER FLAVOPROTEIN-UBIQUINONE OXIDOREDUCTASE

Two mutant alleles of the gene encoding electron transfer flavoprotein -ubiquinone oxidoreductase were identified and characterized in fibrob lasts from a patient with glutaric acidemia type II. One of these alle les is a C-T transition in the donor site of an intron that causes ski pping of a 222 bp exon. Included in the missing 74 amino acids is C561 , which is predicted to be one of the four cysteine ligands of the 4Fe 4S cluster. This mutant allele does not encode a stable ETF-QO in huma n fibroblasts but, when expressed in Saccharomyces cerevisiae, the mut ant ETF-QO is relatively stable and properly targeted to and processed by mitochondria. The mutant protein lacks ubiquinone reductase activi ty, but does accept electrons from ETF in the catalyzed disproportiona tion of ETF semiquinone. These data suggest that in the normal protein the flavin center accepts electrons from ETF and that the 4Fe4S clust er reduces ubiquinone. Deleting the 74 amino acids also alters the ass ociation between the protein and membrane suck that the mutant ETF-QO cannot be extracted from the membrane using the same conditions used f or wild type ETF-QO. A site directed mutant that contains only the sin gle amino acid substitution, C561A, exhibits the same catalytic behavi or as the deletion mutant, supporting the hypothesis regarding the spe cific functions of the two redox centers. It is, however, solubilized by the same conditions as wild type ETF-QO.