Cytochrome b Mutation Y268S Conferring Atovaquone Resistance Phenotype in Malaria Parasite Results in Reduced Parasite bc1 Catalytic Turnover and Protein Expression

Abstract Atovaquone is an anti-malarial drug used in combination with proguanil (e.g. MalaroneTM) for the curative and prophylactic treatment of malaria. Atovaquone, a 2-hydroxynaphthoquinone, is a competitive inhibitor of the quinol oxidation (Qo) site of the mitochondrial cytochrome bc1 complex. Inhibition of this enzyme results in the collapse of the mitochondrial membrane potential, disruption of pyrimidine biosynthesis, and subsequent parasite death. Resistance to atovaquone in the field is associated with point mutations in the Qo pocket of cytochrome b, most notably near the conserved Pro260-Glu261-Trp262-Tyr263 (PEWY) region in the ef loop). The effect of this mutation has been extensively studied in model organisms but hitherto not in the parasite itself. Here, we have performed a molecular and biochemical characterization of an atovaquone-resistant field isolate, TM902CB. Molecular analysis of this strain reveals the presence of the Y268S mutation in cytochrome b. The Y268S mutation is shown to confer a 270-fold shift of the inhibitory constant (Ki) for atovaquone with a concomitant reduction in the Vmax of the bc1 complex of ∼40% and a 3-fold increase in the observed Km for decylubiquinol. Western blotting analyses reveal a reduced iron-sulfur protein content in Y268S bc1 suggestive of a weakened interaction between this subunit and cytochrome b. Gene expression analysis of the TM902CB strain reveals higher levels of expression, compared with the 3D7 (atovaquone-sensitive) control strain in bc1 and cytochrome c oxidase genes. It is hypothesized that the observed differential expression of these and other key genes offsets the fitness cost resulting from reduced bc1 activity.