Yu L, McCurley J P, Yu C A
Biochim Biophys Acta. 1987 Aug 12;893(1):75-82. doi: 10.1016/0005-2728(87)90150-2.
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.
在纯的、具有重组活性的琥珀酸脱氢酶中检测到了抗霉素不敏感的琥珀酸 - 细胞色素c还原酶活性。在存在100微摩尔细胞色素c的情况下，该酶催化电子从琥珀酸转移到细胞色素c，速率为每分钟每毫克蛋白质氧化0.7微摩尔琥珀酸。这种活性约为该制剂中重组活性（琥珀酸脱氢酶与辅酶泛醌结合蛋白（QPs）重组形成琥珀酸 - 泛醌还原酶的能力）或琥珀酸 - 吩嗪硫酸甲酯活性的2%，与在亚线粒体颗粒或分离的琥珀酸 - 细胞色素c还原酶中检测到的抗霉素不敏感的琥珀酸 - 细胞色素c还原酶活性不同。前者对细胞色素c的米氏常数过高无法测量。后者的米氏常数约为4.4微摩尔，与分离的琥珀酸 - 细胞色素c还原酶中抗霉素敏感的琥珀酸 - 细胞色素c活性的米氏常数相似，这表明琥珀酸 - 细胞色素c还原酶的抗霉素不敏感的琥珀酸 - 细胞色素c活性可能是由于抗霉素的不完全抑制所致。与琥珀酸脱氢酶的重组活性一样，琥珀酸脱氢酶的抗霉素不敏感的琥珀酸 - 细胞色素c活性对氧气敏感；在0℃、蛋白质浓度为23毫克/毫升时，半衰期约为20分钟。在存在QPs的情况下，琥珀酸脱氢酶的抗霉素不敏感的琥珀酸 - 细胞色素c活性消失，同时出现对噻吩甲酰三氟丙酮敏感的琥珀酸 - 泛醌还原酶活性。这表明当琥珀酸脱氢酶从膜或QPs上脱离时，琥珀酸脱氢酶的抗霉素不敏感的琥珀酸 - 细胞色素c还原酶活性就会出现。具有重组活性的琥珀酸脱氢酶使用琥珀酰化细胞色素c作为电子受体氧化琥珀酸，这表明可能涉及一种低电位中间体（自由基）。通过自旋捕获技术检测到一个未知自由基，证实了这一推测。当将自旋捕获剂α - 苯基 - N - 叔丁基硝酮（PBN）添加到含有具有重组活性的琥珀酸脱氢酶的琥珀酸氧化系统中时，会生成PBN自旋加合物。尽管这种PBN自旋加合物与黄嘌呤氧化酶产生的相同，表明可能涉及过羟基自由基，但这种抗霉素不敏感的琥珀酸 - 细胞色素c还原酶活性对超氧化物歧化酶和氧气不敏感，这对所观察到的自由基的性质提出了疑问。
