Mather Michael W, Darrouzet Elisabeth, Valkova-Valchanova Maria, Cooley Jason W, McIntosh Michael T, Daldal Fevzi, Vaidya Akhil B
Center for Molecular Parasitology, Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19129, USA.
J Biol Chem. 2005 Jul 22;280(29):27458-65. doi: 10.1074/jbc.M502319200. Epub 2005 May 24.
Atovaquone is an antiparasitic drug that selectively inhibits electron transport through the parasite mitochondrial cytochrome bc1 complex and collapses the mitochondrial membrane potential at concentrations far lower than those at which the mammalian system is affected. Because this molecule represents a new class of antimicrobial agents, we seek a deeper understanding of its mode of action. To that end, we employed site-directed mutagenesis of a bacterial cytochrome b, combined with biophysical and biochemical measurements. A large scale domain movement involving the iron-sulfur protein subunit is required for electron transfer from cytochrome b-bound ubihydroquinone to cytochrome c1 of the cytochrome bc1 complex. Here, we show that atovaquone blocks this domain movement by locking the iron-sulfur subunit in its cytochrome b-binding conformation. Based on our malaria atovaquone resistance data, a series of cytochrome b mutants was produced that were predicted to have either enhanced or reduced sensitivity to atovaquone. Mutations altering the bacterial cytochrome b at its ef loop to more closely resemble Plasmodium cytochrome b increased the sensitivity of the cytochrome bc1 complex to atovaquone. A mutation within the ef loop that is associated with resistant malaria parasites rendered the complex resistant to atovaquone, thereby providing direct proof that the mutation causes atovaquone resistance. This mutation resulted in a 10-fold reduction in the in vitro activity of the cytochrome bc1 complex, suggesting that it may exert a cost on efficiency of the cytochrome bc1 complex.
阿托伐醌是一种抗寄生虫药物，它能选择性抑制寄生虫线粒体细胞色素bc1复合物的电子传递，并在远低于影响哺乳动物系统的浓度下使线粒体膜电位崩溃。由于这种分子代表了一类新型抗菌剂，我们试图更深入地了解其作用方式。为此，我们采用了细菌细胞色素b的定点诱变技术，并结合生物物理和生化测量方法。从细胞色素b结合的泛醇向细胞色素bc1复合物的细胞色素c1进行电子转移，需要涉及铁硫蛋白亚基的大规模结构域移动。在这里，我们表明阿托伐醌通过将铁硫亚基锁定在其与细胞色素b结合的构象中来阻断这种结构域移动。基于我们的疟疾阿托伐醌抗性数据，产生了一系列细胞色素b突变体，预计它们对阿托伐醌的敏感性会增强或降低。将细菌细胞色素b的ef环改变为更类似于疟原虫细胞色素b的突变，增加了细胞色素bc1复合物对阿托伐醌的敏感性。与抗疟原虫相关的ef环内的一个突变使该复合物对阿托伐醌产生抗性，从而直接证明该突变导致阿托伐醌抗性。这种突变导致细胞色素bc1复合物的体外活性降低了10倍，这表明它可能会对细胞色素bc1复合物的效率产生代价。
