Miki T, Miki M, Orii Y
Department of Public Health, Faculty of Medicine, Kyoto University, Japan.
J Biol Chem. 1994 Jan 21;269(3):1827-33.
The cytochrome bc1 complex purified from beef heart mitochondria was incorporated into potassium (K+)-loaded phospholipid vesicles by a cholate dialysis method to study the reverse reaction of electron transfer in the complex. The reduction of cytochrome b in the presence of sodium ascorbate was observed on addition of valinomycin to the K(+)-loaded proteoliposomes in a medium containing no external KCl; it was followed by the gradual oxidation. Nigericin accelerated the reoxidation of reduced cytochrome b, indicating that a K+ diffusion potential (negative inside) induced the reduction of cytochrome b. The extent of the cytochrome b reduction depended on the magnitude of the diffusion potential across the liposomal membranes, and its maximal reduction was attained at more than 210 mV of the diffusion potential. It was cytochrome b562 that was reduced during the establishment of the K+ diffusion potential in the presence of ascorbate, and about 90% of cytochrome b562 was estimated to be reduced. Antimycin A and myxothiazol inhibited the diffusion potential-induced reduction of cytochrome b562, and ubiquinone was proved to be essential for the reversed electron transfer. The K+ diffusion potential also induced the partial reduction of cytochrome b566 when cytochrome b562 had previously been reduced with ascorbate plus tetramethyl-p-phenylenediamine. These results were interpreted well based on the Q cycle scheme which assumed the energy-dependent reduction of ubiquinone at center o. Dicyclohexylcarbodiimide, which did not perturb the ability of proteoliposomes to generate the K+ diffusion potential, inhibited the energy-dependent reduction of cytochrome b562 without a significant loss in the catalytic activity of the complex. The half-inhibition was brought about by 200 mol of dicyclohexylcarbodiimide/mol of cytochrome c1. These results strongly suggest the coupling of a proton flow with the reversed electron transfer in the bc1 complex.
通过胆酸盐透析法将从牛心线粒体中纯化得到的细胞色素bc1复合物整合到负载钾离子（K+）的磷脂囊泡中，以研究该复合物中电子传递的逆向反应。在不含外部氯化钾的培养基中，向负载钾离子的蛋白脂质体中添加缬氨霉素后，观察到在抗坏血酸钠存在下细胞色素b的还原；随后其逐渐氧化。尼日利亚菌素加速了还原型细胞色素b的再氧化，表明钾离子扩散电位（内部为负）诱导了细胞色素b的还原。细胞色素b的还原程度取决于跨脂质体膜的扩散电位大小，在扩散电位超过210 mV时达到最大还原程度。在抗坏血酸存在下建立钾离子扩散电位的过程中被还原的是细胞色素b562，估计约90%的细胞色素b562被还原。抗霉素A和粘噻唑抑制了扩散电位诱导的细胞色素b562的还原，并且已证明泛醌对于逆向电子传递至关重要。当细胞色素b562先前已被抗坏血酸加四甲基对苯二胺还原时，钾离子扩散电位也诱导了细胞色素b566的部分还原。基于假设在中心o处泛醌的能量依赖性还原的Q循环模式，这些结果得到了很好的解释。二环己基碳二亚胺不干扰蛋白脂质体产生钾离子扩散电位的能力，但抑制了细胞色素b562的能量依赖性还原，而该复合物的催化活性没有显著损失。二环己基碳二亚胺的半抑制浓度为每摩尔细胞色素c1 200摩尔。这些结果强烈表明质子流与bc1复合物中的逆向电子传递相偶联。
