Molecular basis for atovaquone binding to the cytochrome bc1 complex

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Atovaquone is a therapeutic agent targeting Plasmodium falciparum malaria, Pneumocystis carinii pneumonia, and Toxoplasma gondii toxoplasmosis.
• Its mechanism involves inhibiting the cytochrome bc1 complex in these pathogens.

Purpose of the Study:

• To investigate the molecular interaction between atovaquone and the cytochrome bc1 complex.
• To elucidate the structural basis for differential sensitivity of fungal and mammalian bc1 complexes to atovaquone.

Main Methods:

• Biochemical assays using isolated Saccharomyces cerevisiae bc1 complex.
• Electron Paramagnetic Resonance (EPR) spectroscopy to study the Rieske iron-sulfur protein.
• Computational modeling and site-directed mutagenesis (L275F) in yeast cytochrome b.

Main Results:

• Atovaquone competitively inhibits the yeast bc1 complex (Ki = 9 nm).
• It alters the redox potential and EPR spectrum of the Rieske iron-sulfur protein, indicating binding to the ubiquinol oxidation pocket.
• A phenylalanine at position 275 in bovine cytochrome b, versus leucine in yeast, confers lower sensitivity (Ki = 80 nm).
• Introducing the L275F mutation in yeast cytochrome b decreased sensitivity (Ki = 100 nm).

Conclusions:

• Atovaquone binds to the ubiquinol oxidation pocket of the bc1 complex, interacting with the Rieske iron-sulfur protein.
• The L275F substitution in cytochrome b is a key determinant of differential atovaquone sensitivity between fungal and mammalian bc1 complexes.
• This study provides a molecular understanding of atovaquone's binding and differential inhibition mechanism.