Uncovering the molecular mode of action of the antimalarial drug atovaquone using a bacterial system.
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.
Mather
Michael W
MW
Center for Molecular Parasitology, Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19129, USA.
Darrouzet
Elisabeth
E
Valkova-Valchanova
Maria
M
Cooley
Jason W
JW
McIntosh
Michael T
MT
Daldal
Fevzi
F
Vaidya
Akhil B
AB
D000962:Antimalarials / Q000494:pharmacology*; D053626:Atovaquone; D001426:Bacterial Proteins / Q000187:drug effects*; D045303:Cytochromes b / Q000187:drug effects / Q000235:genetics; D045304:Cytochromes c / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance / Q000235:genetics; D004579:Electron Transport / Q000187:drug effects; D014450:Electron Transport Complex III / Q000378:metabolism; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009285:Naphthoquinones / Q000494:pharmacology*; D010084:Oxidation-Reduction
D000962:Antimalarials; D001426:Bacterial Proteins; D009285:Naphthoquinones; D045304:Cytochromes c; D045303:Cytochromes b; D014450:Electron Transport Complex III; D053626:Atovaquone