De Jong A M, Albracht S P
E. C. Slater Institute, BioCentrum Amsterdam, University of Amsterdam, The Netherlands.
Eur J Biochem. 1994 Jun 15;222(3):975-82. doi: 10.1111/j.1432-1033.1994.tb18948.x.
Until now ubisemiquinones associated with NADH:ubiquinone oxidoreductase (complex I) have been reported to occur in isolated enzyme and in tightly coupled submitochondrial particles. In this report it is shown that ubisemiquinones are always detectable during steady-state electron transfer from NADH to ubiquinone, independent of the type of inner-membrane preparation used. The EPR signal of the rotenone-sensitive ubisemiquinones could be detected not only in coupled MgATP submitochondrial particles, but also in routine preparations of uncoupled submitochondrial particles and in mitochondria. The ubisemiquinone formation in coupled preparations was completely insensitive to uncouplers. The maximal radical concentration during steady-state electron transfer from NADH to quinone was equal to that of iron-sulphur cluster 2. Experiments with antimycin, myxothiazol and 2-thenoyltrifluoroacetone demonstrated that about half of this radical was associated with complex I, giving a ubisemiquinone concentration of about 0.5 mol semiquinone/mol cluster 2. Uncoupled submitochondrial particles, prepared by extensive sonification, never showed radical signals within 100 ms after mixing with NADH. This was due to the reversible inactivation of the enzyme, caused by elevated temperatures during sonification. In preparations with deliberately heat-inactivated complex I, no radical signals were detected within 200 ms after mixing with NADH; at 1 s, however, radical formation was maximal. Yet, depending on the procedure of reactivation of the complex, in preparations previously treated to inactivate them ubisemiquinone concentrations were always less than in untreated particles. When complex I was in the active state the ubisemiquinone signal was maximal within 40 ms. The results described in this report lead to the conclusion that ubisemiquinones form obligatory intermediates in the reaction of NADH dehydrogenase with ubiquinone.
迄今为止，据报道与NADH：泛醌氧化还原酶（复合体I）相关的半泛醌出现在分离出的酶以及紧密偶联的亚线粒体颗粒中。本报告表明，在从NADH到泛醌的稳态电子传递过程中，始终可以检测到半泛醌，这与所使用的内膜制剂类型无关。不仅在偶联了MgATP的亚线粒体颗粒中，而且在未偶联的亚线粒体颗粒和线粒体的常规制剂中，都可以检测到对鱼藤酮敏感的半泛醌的EPR信号。偶联制剂中的半泛醌形成对解偶联剂完全不敏感。从NADH到醌的稳态电子传递过程中的最大自由基浓度与铁硫簇2的浓度相等。用抗霉素、粘噻唑啉和2-噻吩甲酰三氟丙酮进行的实验表明，约一半的这种自由基与复合体I相关，半泛醌浓度约为0.5摩尔半醌/摩尔簇2。通过广泛超声处理制备的未偶联亚线粒体颗粒，在与NADH混合后100毫秒内从未显示出自由基信号。这是由于超声处理过程中温度升高导致酶的可逆失活。在故意热失活复合体I的制剂中，与NADH混合后200毫秒内未检测到自由基信号；然而，在1秒时，自由基形成达到最大值。然而，根据复合体的重新激活程序，在先前经过处理使其失活的制剂中，半泛醌浓度总是低于未处理的颗粒。当复合体I处于活性状态时，半泛醌信号在40毫秒内达到最大值。本报告中描述的结果得出结论，半泛醌在NADH脱氢酶与泛醌的反应中形成必不可少的中间体。
