Genetic evidence for coenzyme Q requirement in plasma membrane electron transport

Plasma membranes isolated from wild-type Saccharomyces cerevisiae crude mem brane fractions catalyzed NADH oxidation using a variety of electron accept ers, such as ferricyanide, cytochrome c, and ascorbate free radical. Plasma membranes from the deletion mutant strain coq3 Delta, defective in coenzym e Q (ubiquinone) biosynthesis, were completely devoid of coenzyme Q(6) and contained greatly diminished levels of NADH-ascorbate free radical reductas e activity (about 10% of wild-type yeasts). In contrast, the lack of coenzy me Q6 in these membranes resulted in only a partial inhibition of either th e ferricyanide or cytochrome-e reductase. Coenzyme Q dependence of ferricya nide and cytochrome-e reductases was based mainly on superoxide generation by one-electron reduction of quinones to semiquinones. Ascorbate free radic al reductase was unique because it was highly dependent on coenzyme Q and d id not involve superoxide since it was not affected by superoxide dismutase (SOD). Both coenzyme Q6 and NADH-ascorbate free radical reductase were res cued in plasma membranes derived from a strain obtained by transformation o f the coq3 Delta strain with a single-copy plasmid bearing the wild type CO Q3 gene and in plasma membranes isolated form the coq3 Delta, strain grown in the presence of coenzyme Q6 The enzyme activity was inhibited by the qui none antagonists chloroquine and dicumarol, and after membrane solubilizati on with the nondenaturing detergent Zwittergent 3-14. The various inhibitor s used did not affect residual ascorbate free radical reductase of the coq3 Delta strain. Ascorbate free radical reductase was not altered significant ly in mutants atp2 Delta and cor1 Delta which are also respiration-deficien t but not defective in ubiquinone biosynthesis, demonstrating that the lack of ascorbate free radical reductase in coq3 Delta mutants is related solel y to the inability to synthesize ubiquinone and not to the respiratory-defe ctive phenotype. For the first time, our results provide genetic evidence f or the participation of ubiquinone in NADH-ascorbate free radical reductase , as a source of electrons for transmembrane ascorbate stabilization.