Berry Edward A, Huang Li-Shar, Lee Dong-Woo, Daldal Fevzi, Nagai Kazuo, Minagawa Nobuko
SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, USA.
Biochim Biophys Acta. 2010 Mar;1797(3):360-70. doi: 10.1016/j.bbabio.2009.12.003. Epub 2009 Dec 16.
Ascochlorin is an isoprenoid antibiotic that is produced by the phytopathogenic fungus Ascochyta viciae. Similar to ascofuranone, which specifically inhibits trypanosome alternative oxidase by acting at the ubiquinol binding domain, ascochlorin is also structurally related to ubiquinol. When added to the mitochondrial preparations isolated from rat liver, or the yeast Pichia (Hansenula) anomala, ascochlorin inhibited the electron transport via CoQ in a fashion comparable to antimycin A and stigmatellin, indicating that this antibiotic acted on the cytochrome bc(1) complex. In contrast to ascochlorin, ascofuranone had much less inhibition on the same activities. On the one hand, like the Q(i) site inhibitors antimycin A and funiculosin, ascochlorin induced in H. anomala the expression of nuclear-encoded alternative oxidase gene much more strongly than the Q(o) site inhibitors tested. On the other hand, it suppressed the reduction of cytochrome b and the generation of superoxide anion in the presence of antimycin A(3) in a fashion similar to the Q(o) site inhibitor myxothiazol. These results suggested that ascochlorin might act at both the Q(i) and the Q(o) sites of the fungal cytochrome bc(1) complex. Indeed, the altered electron paramagnetic resonance (EPR) lineshape of the Rieske iron-sulfur protein, and the light-induced, time-resolved cytochrome b and c reduction kinetics of Rhodobacter capsulatus cytochrome bc(1) complex in the presence of ascochlorin demonstrated that this inhibitor can bind to both the Q(o) and Q(i) sites of the bacterial enzyme. Additional experiments using purified bovine cytochrome bc(1) complex showed that ascochlorin inhibits reduction of cytochrome b by ubiquinone through both Q(i) and Q(o) sites. Moreover, crystal structure of chicken cytochrome bc(1) complex treated with excess ascochlorin revealed clear electron densities that could be attributed to ascochlorin bound at both the Q(i) and Q(o) sites. Overall findings clearly show that ascochlorin is an unusual cytochrome bc(1) inhibitor that acts at both of the active sites of this enzyme.
阿斯科氯素是一种由植物致病真菌蚕豆壳二孢产生的类异戊二烯抗生素。与通过作用于泛醇结合结构域特异性抑制锥虫交替氧化酶的阿斯科呋喃酮类似，阿斯科氯素在结构上也与泛醇相关。当添加到从大鼠肝脏或酵母异常毕赤酵母（汉逊酵母）中分离出的线粒体制剂中时，阿斯科氯素以与抗霉素A和柱晶白霉素相当的方式抑制通过辅酶Q的电子传递，表明这种抗生素作用于细胞色素bc(1)复合物。与阿斯科氯素相反，阿斯科呋喃酮对相同活性的抑制作用要小得多。一方面，与Q(i)位点抑制剂抗霉素A和绳菌素一样，阿斯科氯素在异常毕赤酵母中诱导核编码交替氧化酶基因的表达比所测试的Q(o)位点抑制剂强烈得多。另一方面，它在抗霉素A(3)存在下以类似于Q(o)位点抑制剂粘噻唑的方式抑制细胞色素b的还原和超氧阴离子的产生。这些结果表明，阿斯科氯素可能作用于真菌细胞色素bc(1)复合物的Q(i)和Q(o)位点。事实上，在阿斯科氯素存在下，红假单胞菌细胞色素bc(1)复合物的 Rieske 铁硫蛋白的电子顺磁共振（EPR）线形改变，以及光诱导的、时间分辨的细胞色素b和c还原动力学表明，这种抑制剂可以结合到细菌酶的Q(o)和Q(i)位点。使用纯化的牛细胞色素bc(1)复合物的额外实验表明，阿斯科氯素通过Q(i)和Q(o)位点抑制泛醌对细胞色素b的还原。此外，用过量阿斯科氯素处理的鸡细胞色素bc(1)复合物的晶体结构显示出清晰的电子密度，这可归因于结合在Q(i)和Q(o)位点的阿斯科氯素。总体研究结果清楚地表明，阿斯科氯素是一种不寻常的细胞色素bc(1)抑制剂，它作用于该酶的两个活性位点。
