Abstract
Atovaquone is a substituted 2-hydroxynaphthoquinone that is used therapeutically to treat Plasmodium falciparum malaria, Pneumocystis carinii pneumonia, and Toxoplasma gondii toxoplasmosis. It is thought to act on these organisms by inhibiting the cytochrome bc1 complex. We have examined the interaction of atovaquone with the bc1 complex isolated from Saccharomyces cerevisiae, a surrogate, nonpathogenic fungus. Atovaquone inhibits the bc1 complex competitively with apparent Ki = 9 nm, raises the midpoint potential of the Rieske iron-sulfur protein from 285 to 385 mV, and shifts the g values in the EPR spectrum of the Rieske center. These results indicate that atovaquone binds to the ubiquinol oxidation pocket of the bc1 complex, where it interacts with the Rieske iron-sulfur protein. A computed energy-minimized structure for atovaquone liganded to the yeast bc1 complex suggests that a phenylalanine at position 275 of cytochrome b in the bovine bc1 complex, as opposed to leucine at the equivalent position in the yeast enzyme, is responsible for the decreased sensitivity of the bovine bc1 complex (Ki = 80 nm) to atovaquone. When a L275F mutation was introduced into the yeast cytochrome b, the sensitivity of the yeast enzyme to atovaquone decreased (Ki = 100 nm) with no loss in activity, confirming that the L275F exchange contributes to the differential sensitivity of these two species to atovaquone. These results provide the first molecular description of how atovaquone binds to the bc1 complex and explain the differential inhibition of the fungal versus mammalian enzymes.
Highlights
Atovaquone is a substituted 2-hydroxynaphthoquinone that is used therapeutically to treat Plasmodium falciparum malaria, Pneumocystis carinii pneumonia, and Toxoplasma gondii toxoplasmosis
Atovaquone inhibits the bc1 complex competitively with apparent Ki ؍9 nM, raises the midpoint potential of the Rieske iron-sulfur protein from 285 to 385 mV, and shifts the g values in the EPR spectrum of the Rieske center. These results indicate that atovaquone binds to the ubiquinol oxidation pocket of the bc1 complex, where it interacts with the Rieske iron-sulfur protein
When a L275F mutation was introduced into the yeast cytochrome b, the sensitivity of the yeast enzyme to atovaquone decreased (Ki ؍100 nM) with no loss in activity, confirming that the L275F exchange contributes to the differential sensitivity of these two species to atovaquone
Summary
Materials—Yeast extract and peptone were from Difco. Nitrogen base without amino acids but with ammonium sulfate was from U.S Biological. DEAE-Biogel A was obtained from Bio-Rad. Diisopropylfluorophosphate, decyl ubiquinone, and dithionite were purchased from Sigma. Purification of Cytochrome bc Complexes—Wild type yeast and the L275F mutant were grown in yeast extract/peptone/dextrose medium and harvested by centrifugation. The L275F mutation was introduced into the mitochondrial cytochrome b gene by biolistic transformation as described elsewhere [15]. Cytochrome bc complexes were isolated from yeast and bovine heart mitochondria as described previously [17, 18]. The cytochrome bc complex was diluted to 2.5 nM in the assay buffer, and the reaction was started by adding 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinol, an analogue of ubiquinol. To determine activities in the presence of various concentrations of atovaquone, inhibitor and substrate were mixed simultaneously into the assay buffer, and the reaction was started by adding enzyme. Assuming rapid equilibrium between enzyme, substrate, and inhibitor, the Ki values for a competitive inhibitor were calculated from the measured IC50 values and the following formula [19]
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