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J. Biol. Chem., Vol. 280, Issue 29, 27458-27465, July 22, 2005

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Uncovering the Molecular Mode of Action of the Antimalarial Drug Atovaquone Using a Bacterial System^*

Michael W. Mather, Elisabeth Darrouzet¶, Maria Valkova-Valchanova||, Jason W. Cooley, Michael T. McIntosh**, Fevzi Daldal, and Akhil B. Vaidya

From the Center for Molecular Parasitology, Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19129 and the Department of Biology, Plant Science Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104

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.

Received for publication, March 1, 2005 , and in revised form, May 19, 2005.

^* This work was supported by National Institutes of Health Grants AI28398 (to A. B. V.) and GM38237 (to F. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Present address: Service de Biochimie Post-génomique et Toxicologie Nucléaire, DIEP, DSV, CEA VALRHO, 30207 Bagnols sur Cèze, France.

^|| Present address: Medarex, Inc., Bloomsbury, NJ 08804.

^** Present address: Dept. Internal Medicine, Yale University, School of Epidemiology and Public Health, 60 College St., P. O. Box 208034, New Haven, CT 06520-8034.

To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Drexel University College of Medicine, 2900 Queen Lane, Philadelphia, PA 19129. Tel.: 215-991-8557; E-mail: av27{at}drexel.edu.

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