An antimycin-insensitive succinate-cytochrome c reductase activity in pure reconstitutively active succinate dehydrogenase.
Antimycin-insensitive succinate-cytochrome c reductase activity has been detected in pure, reconstitutively active succinate dehydrogenase. The enzyme catalyzes electron transfer from succinate to cytochrome c at a rate of 0.7 mumole succinate oxidized per min per mg protein, in the presence of 100 microM cytochrome c. This activity, which is about 2% of that of reconstitutive (the ability of succinate dehydrogenase to reconstitute with coenzyme ubiquinone-binding proteins (QPs) to form succinate-ubiquinone reductase) or succinate-phenazine methosulfate activity in the preparation, differs from antimycin-insensitive succinate-cytochrome c reductase activity detected in submitochondrial particles or isolated succinate-cytochrome c reductase. The Km for cytochrome c for the former is too high to be measured. The Km for the latter is about 4.4 microM, similar to that of antimycin-sensitive succinate-cytochrome c activity in isolated succinate-cytochrome c reductase, suggesting that antimycin-insensitive succinate-cytochrome c activity of succinate-cytochrome c reductase probably results from incomplete inhibition by antimycin. Like reconstitutive activity of succinate dehydrogenase, the antimycin-insensitive succinate-cytochrome c activity of succinate dehydrogenase is sensitive to oxygen; the half-life is about 20 min at 0 degrees C at a protein concentration of 23 mg/ml. In the presence of QPs, the antimycin-insensitive succinate-cytochrome c activity of succinate dehydrogenase disappears and at the same time a thenoyltrifluoroacetone-sensitive succinate-ubiquinone reductase activity appears. This suggests that antimycin-insensitive succinate-cytochrome c reductase activity of succinate dehydrogenase appears when succinate dehydrogenase is detached from the membrane or from QPs. Reconstitutively active succinate dehydrogenase oxidizes succinate using succinylated cytochrome c as electron acceptor, suggesting that a low potential intermediate (radical) may be involved. This suggestion is confirmed by the detection of an unknown radical by spin trapping techniques. When a spin trap, alpha-phenyl-N-tert-butylnitrone (PBN), is added to a succinate oxidizing system containing reconstitutively active succinate dehydrogenase, a PBN spin adduct is generated. Although this PBN spin adduct is identical to that generated by xanthine oxidase, indicating that a perhydroxy radical might be involved, the insensitivity of this antimycin-insensitive succinate-cytochrome c reductase activity to superoxide dismutase and oxygen questions the nature of this observed radical.
D000968:Antimycin A / Q000031:analogs & derivatives* / Q000494:pharmacology; D003579:Cytochrome Reductases / Q000037:antagonists & inhibitors*; D003574:Cytochrome c Group / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D005609:Free Radicals; D009245:NADH Dehydrogenase / Q000037:antagonists & inhibitors*; D010084:Oxidation-Reduction; D013379:Substrate Specificity; D013385:Succinate Dehydrogenase / Q000378:metabolism*; D014451:Ubiquinone / Q000378:metabolism
D003574:Cytochrome c Group; D005609:Free Radicals; C032456:antimycin; D014451:Ubiquinone; D000968:Antimycin A; D013385:Succinate Dehydrogenase; D003579:Cytochrome Reductases; D009245:NADH Dehydrogenase