Muller Florian L, Roberts Arthur G, Bowman Michael K, Kramer David M
Institute of Biological Chemistry, Washington State University, 289 Clark Hall, Pullman, Washington 99164-6340, USA.
Biochemistry. 2003 Jun 3;42(21):6493-9. doi: 10.1021/bi0342160.
Although several X-ray structures have been determined for the mitochondrial cytochrome (cyt) bc(1) complex, none yet shows the position of the substrate, ubiquinol, in the quinol oxidase (Q(o)) site. In this study, the interaction of molecular oxygen with the reactive intermediate Q(o) semiquinone is used to probe the Q(o) site. It has been known for some time that partial turnover of the cyt bc(1) complex in the presence of antimycin A, a Q(i) site inhibitor, results in accumulation of a semiquinone at the Q(o) site, which can reduce O(2) to superoxide (O(2)()(-)). It was more recently shown that myxothiazol, which binds close to the cyt b(L) heme in the proximal Q(o) niche, also induces O(2)()(-) production. In this work, it is shown that, in addition to myxothiazol, a number of other proximal Q(o) inhibitors [including (E)-beta-methoxyacrylate-stilbene, mucidin, and famoxadone] also induce O(2)()(-) production in the isolated yeast cyt bc(1) complex, at approximately 50% of the V(max) observed in the presence of antimycin A. It is proposed that proximal Q(o) site inhibitors induce O(2)()(-) production because they allow formation, but not oxidation, of the semiquinone at the distal niche of the Q(o) site pocket. The apparent K(m) for ubiquinol at the Q(o) site in the presence of proximal Q(o) site inhibitors suggests that the "distal niche" of the Q(o) pocket can act as a fully independent quinol binding and oxidation site. Together with the X-ray structures, these results suggest substrate ubiquinol binds in a fashion similar to that of stigmatellin with H-bonds between H161 of the Rieske iron-sulfur protein and E272 of the cyt b protein. When modeled in this way, mucidin and ubiquinol can bind simultaneously to the Q(o) site with virtually no steric hindrance, whereas progressively bulkier inhibitors exhibit increasing overlap. The fact that partial turnover of the Q(o) site is possible even with bound proximal Q(o) site inhibitors is consistent with the participation of two separate functional Q(o) binding niches, occupied simultaneously or sequentially.
尽管已经确定了线粒体细胞色素（cyt）bc(1)复合物的几种X射线结构，但尚无一种结构显示底物泛醇在喹啉氧化酶（Q(o)）位点的位置。在本研究中，利用分子氧与反应中间体Q(o)半醌的相互作用来探测Q(o)位点。一段时间以来已知，在Q(i)位点抑制剂抗霉素A存在下，cyt bc(1)复合物的部分周转会导致Q(o)位点积累半醌，其可将O(2)还原为超氧化物（O(2)()(-)）。最近表明，在近端Q(o)微环境中靠近细胞色素b(L)血红素结合的粘噻唑也会诱导O(2)()(-)的产生。在这项工作中表明，除了粘噻唑之外，许多其他近端Q(o)抑制剂[包括（E）-β-甲氧基丙烯酸酯-芪、粘菌素和恶唑菌酮]在分离的酵母cyt bc(1)复合物中也会诱导O(2)()(-)的产生，其产量约为抗霉素A存在下观察到的V(max)的50%。有人提出，近端Q(o)位点抑制剂诱导O(2)()(-)产生是因为它们允许在Q(o)位点口袋的远端微环境中形成半醌，但不允许其氧化。在存在近端Q(o)位点抑制剂的情况下，Q(o)位点处泛醇的表观K(m)表明，Q(o)口袋的“远端微环境”可作为一个完全独立的喹啉结合和氧化位点。与X射线结构一起，这些结果表明底物泛醇的结合方式类似于鱼藤酮，在 Rieske 铁硫蛋白的H161和细胞色素b蛋白的E272之间形成氢键。以这种方式建模时，粘菌素和泛醇几乎没有空间位阻地同时结合到Q(o)位点，而体积逐渐增大的抑制剂显示出越来越多的重叠。即使存在结合的近端Q(o)位点抑制剂，Q(o)位点的部分周转也是可能的，这一事实与两个独立的功能性Q(o)结合微环境的参与一致，它们可以同时或依次被占据。
