Atovaquone represents a class of antimicrobial agents with a broad‐spectrum activity against various parasitic infections, including malaria, toxoplasmosis and Pneumocystis pneumonia. In malaria parasites, atovaquone inhibits mitochondrial electron transport at the level of the cytochrome bc1 complex and collapses mitochondrial membrane potential. In addition, this drug is unique in being selectively toxic to parasite mitochondria without affecting the host mitochondrial functions. A better understanding of the structural basis for the selective toxicity of atovaquone could help in designing drugs against infections caused by mitochondria‐containing parasites. To that end, we derived nine independent atovaquone‐resistant malaria parasite lines by suboptimal treatment of mice infected with Plasmodium yoelii; these mutants exhibited resistance to atovaquone‐mediated collapse of mitochondrial membrane potential as well as inhibition of electron transport. The mutants were also resistant to the synergistic effects of atovaquone/ proguanil combination. Sequencing of the mitochondrially encoded cytochrome b gene placed these mutants into four categories, three with single amino acid changes and one with two adjacent amino acid changes. Of the 12 nucleotide changes seen in the nine independently derived mutants 11 replaced A:T basepairs with G:C basepairs, possibly because of reactive oxygen species resulting from atovaquone treatment. Visualization of the resistance‐conferring amino acid positions on the recently solved crystal structure of the vertebrate cytochrome bc1 complex revealed a discrete cavity in which subtle variations in hydrophobicity and volume of the amino acid side‐chains may determine atovaquone‐binding affinity, and thereby selective toxicity. These structural insights may prove useful in designing agents that selectively affect cytochrome bc1 functions in a wide range of eukaryotic pathogens.

In crystals of the key respiratory and photosynthetic electron transfer protein called ubihydroquinone:cytochrome (cyt) c oxidoreductase or cyt bc 1, the extrinsic [2Fe2S] cluster domain of its Fe-S subunit assumes several conformations, suggesting that it may move during catalysis. Herein, using Rhodobacter capsulatus mutants that have modifications in the hinge region of this subunit, we were able to reveal this motion kinetically. Thus, the bc1 complex (and possibly the homologous b6f complex in chloroplasts) employs the [2Fe2S] cluster domain as a device to shuttle electrons from ubihydroquinone to cyt c1 (or cyt f ). We demonstrate that this domain movement is essential for cyt bc 1 function, because a mutant enzyme with a nonmoving Fe-S subunit has no catalytic activity, and one with a slower movement has lower activity. This motion is apparently designed with a natural frequency slow enough to assure productive Qo site charge separation but fast enough not to be rate limiting. These findings add the unprecedented function of intracomplex electron shuttling to large-scale domain motions in proteins and may well provide a target for cyt bc 1 antibiotics.Rhodobacter capsulatus ͉ photosynthetic and respiratory electron transfer ͉ mitochondrial complex III ͉ protein domain motion ͉ Rieske Fe-S subunit W hen different crystal structures reveal dramatically different protein conformations, large amplitude domain movements are often inferred. However, in only a few cases such as myosin (1), flagellar motor (2), and ATP synthase (3, 4) have such movements been visualized. The cytochrome (cyt) bc 1 (or its cyt b 6 f counterpart in chloroplasts) is a key component of respiratory and photosynthetic electron transfer chains (5, 6). Recent crystal structures of the mitochondrial cyt bc 1 have revealed that the extrinsic [2Fe2S] cluster domain of the Fe-S subunit occupies various locations within this enzyme complex (7-10). It has been observed in either a position proximal to the ubihydroquinone (QH 2 ) oxidation catalytic site (Q o position) from which it takes electrons or a position close to cyt c 1 subunit (c 1 position) to which it donates electrons (Fig. 1). Because of the large distances observed between the electron-donating and electron-accepting cofactors of the cyt bc 1 in the different structures, no one of these locations can support sufficiently rapid electron tunneling (11) to meet the observed turnover rates (12, 13) and the specific substrate-product interactions (14) that occur at the QH 2 oxidation site. Thus, an unprecedented intracomplex electron shuttle motion to transfer electrons during catalysis has been suggested (8). However, neither the presumably essential movement nor the electron transfer associated with it has been visualized before this work.In light-activated energy transduction systems, such as the one provided by the photosynthetic bacterium R. capsulatus, a short flash of light (Ͻ10-s duration) can activate the photochemical reaction center, thereby inducing oxidation of two equival...

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