Reduction of cytochrome b in mitochondria from yeast lacking coenzyme Q

Abstract

Mitochondria isolated from coenzyme Q deficient yeast cells had no detectable NADH:cytochrome c reductase or succinate:cytochrome c reductase but had comparable amounts of cytochromes b and c1 as wild-type mitochondria. Addition of succinate to the mutant mitochondria resulted in a slight reduction of cytochrome b; however, the subsequent addition of antimycin resulted in a biphasic reduction of cytochrome b, leading to reduction of 68% of the total dithionite-reducible cytochrome b. No "red" shift in the absorption maximum was observed, and no cytochrome c1 was reduced. The addition of either myxothiazol or alkylhydroxynaphthoquinone blocked the reduction of cytochrome b observed with succinate and antimycin, suggesting that the reduction of cytochrome b-562 in the mitochondria lacking coenzyme Q may proceed by a pathway involving cytochrome b at center o where these inhibitors block. Cyanide did not prevent the reduction of cytochrome b by succinate and antimycin the the mutant mitochondria. These results suggest that the succinate dehydrogenase complex can transfer electrons directly to cytochrome b in the absence of coenzyme Q in a reaction that is enhanced by antimycin. Reduced dichlorophenolindophenol (DCIP) acted as an effective bypass of the antimycin block in complex III, resulting in oxygen uptake with succinate in antimycin-treated mitochondria. By contrast, reduced DCIP did not restore oxygen uptake in the mutant mitochondria, suggesting that coenzyme Q is necessary for the bypass. The addition of low concentrations of DCIP to both wild-type and mutant mitochondria reduced with succinate in the presence of antimycin resulted in a rapid oxidation of cytochrome b perhaps by the pathway involving center o, which does not require coenzyme Q.

Ubiquinol-Cytochrome c Oxidoreductase

“…Like Ernster et al (43) before us, we found in antimycin-treated, Q-extracted bovine SMP that succinate addition results in a slow reduction of cytochrome b, which could be considered due to the presence in these particles of a residual amount of Q. However, similar results have been reported by Clejan and Beattie (25) with Q-deficient yeast mitochondria and have been found by ourselves with Q-deficient yeast SMP, except that in our experiments the extent of succinate-induced b reduction was less. It is possible that in the absence of Q the low potential b 560 of complex II can slowly reduce the b of complex III.…”

“…Preparation of SMP from Wild-type and Ubiquinone-deficient Yeast (Saccharomyces cerevisiae)-The wild-type yeast strain (D273-10B) and the ubiquinone-deficient strain E3-24 (24) were generous gifts of Dr. Diana Beattie, West Virginia University, Morgantown, WV. Each strain was spread on an agar plate, and the cells were grown from a single colony in a medium containing galactose as carbon source as described in the literature (25). Mitochondria were prepared in a medium containing 0.6 M sorbitol and 10 mM potassium phosphate, pH 7.4, after treatment of the cells with zymolyase according to Daum et al (26).…”

“…6, trace D), which agrees with the finding of Tsai and Palmer (46) that addition of Q 6 to Q-depleted yeast complex III promotes antimycin binding. Because of the presence in Q-extracted SMP of Յ0.025 nmol of Q/mg of protein, additional experiments were done with SMP prepared from mitochondria isolated from a Q-deficient yeast mutant (25,47). Before use, SMP from the wild-type and Q-deficient yeast were also assayed for their Q content, but none was detected in the mutant SMP.…”