Hatefi Y, Yagi T
Biochemistry. 1982 Dec 7;21(25):6614-8. doi: 10.1021/bi00268a045.
It has been shown that in bovine heart submitochondrial particles, antimycin and 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) inhibit the oxidation of NADH, succinate, and reduced ubiquinone incompletely, the uninhibited rate being about 20-40 nmol of substrate oxidized min-1 (mg of protein)-1. By contrast, rotenone, cyanide, BAL (2,3-dimercaptopropanol), and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole [Trumpower, B. L., & Haggerty, J. G. (1980) J. Bioenerg. Biomembr. 12, 151-164] caused essentially complete inhibition when added alone or after maximal inhibition by antimycin or HQNO. Having thus ascertained that the electron leak through the antimycin block appeared to follow the normal path through complex III (ubiquinol: cytochrome c oxidoreductase) and cytochrome oxidase, the reduction of the b cytochromes by substrates and their oxidation through the leak in the antimycin block by molecular oxygen were studied. It was shown that at normal electron flux from NADH and succinate, both cytochromes b562 and b566 were reduced in antimycin-treated submitochondrial particles. Their oxidation after substrate exhaustion was biphasic, however. At 565 minus 575 nm, 56% of the total reduced cytochrome b was oxidized through the leak in the antimycin block at a more rapid rate, while the remaining 44% was oxidized about 10 times slower. When electron flux from substrates to complex III was slowed down by the use of inhibitors or substrates at less than or equal to 0.1 Km concentration, then only reduced b562 accumulated in antimycin-treated particles. The oxidation of b562 after substrate exhaustion or inhibition of substrate oxidation by an appropriate inhibitor occurred at a rate comparable to that of the slower reoxidation phase described above. These results indicated, therefore, that cytochromes b566 and b562 are oxidized through the leak in the antimycin block at two different rates, the reoxidation rate of b566 being about 10 times faster than that of b562. The implications of these findings on the kinetic relationship of these two cytochromes in the respiratory chain have been discussed.
研究表明，在牛心亚线粒体颗粒中，抗霉素和2-庚基-4-羟基喹啉N-氧化物（HQNO）对NADH、琥珀酸和还原型泛醌的氧化抑制不完全，未受抑制的速率约为20 - 40 nmol底物氧化·分钟⁻¹·（毫克蛋白质）⁻¹。相比之下，鱼藤酮、氰化物、二巯丙醇（BAL）和5-正十一烷基-6-羟基-4,7-二氧代苯并噻唑[特朗普沃尔，B. L.，& 哈格蒂，J. G.（1980年）《生物能量学与生物膜杂志》12卷，151 - 164页]单独添加或在抗霉素或HQNO最大抑制后添加时，基本能完全抑制。由此确定通过抗霉素阻断的电子泄漏似乎遵循通过复合物III（泛醇：细胞色素c氧化还原酶）和细胞色素氧化酶的正常路径后，研究了底物对b型细胞色素的还原以及分子氧通过抗霉素阻断中的泄漏对其的氧化。结果表明，在来自NADH和琥珀酸的正常电子流情况下，抗霉素处理的亚线粒体颗粒中的细胞色素b562和b566均被还原。然而，底物耗尽后它们的氧化是双相的。在565减去575纳米处，总共还原的细胞色素b的56%通过抗霉素阻断中的泄漏以更快的速率被氧化，而其余44%的氧化速度约慢10倍。当通过使用抑制剂或浓度小于或等于0.1 Km的底物使从底物到复合物III的电子流减慢时，那么在抗霉素处理的颗粒中仅积累还原型b562。底物耗尽或用适当抑制剂抑制底物氧化后，b562的氧化速率与上述较慢的再氧化阶段相当。因此，这些结果表明细胞色素b566和b562通过抗霉素阻断中的泄漏以两种不同速率被氧化，b566的再氧化速率比b562快约10倍。已讨论了这些发现对呼吸链中这两种细胞色素动力学关系的影响。
