Proton Transfer and Reactive Oxygen Species in the Cytochrome bc1 Complex

The mitochondrial cytochrome bc₁ complex links electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which ubiquinol is oxidized at center P and ubiquinone is rereduced at center N [1, 2]. E272 of the conserved PEWY loop of most cytochrome b has been suggested as ligand in the enzyme-substrate complex and as proton acceptor in parallel proton-electron transfer towards heme b_L [3]. E272D and E272Q mutations support the importance of the residue for correct ubiquinol oxidation, showing effects such as lowered ubiquinol cytochrome c reductase activity, elevated bypass reactions, and altered K_M for ubiquinol oxidation [4]. However, these effects may also be indirect and the role of E272 as direct ligand of ubiquinol is debated. Furthermore, E272 is not fully conserved across all species. We suggested that in Beta- and Gamma-proteobacteria of which the PEWY glutamate is substituted by valine or leucine, a glutamate equivalent to yeast H253 is conserved, which could take over the proton transfer function. To challenge this hypothesis, single and double substitutions of H253 and E272 have been constructed in Saccharomyces cerevisiae. Eight variants were produced and the detergent-solubilized and purified complexes were characterized. The mutations affect cytochrome c reductase activity and provoke reactive oxygen species production. Combined inhibitor titration, growth analysis of mutants and x-ray structure analysis at 2.7 Å resolution of the E272Q variant co-crystallized with UHDBT in revealed details of the rotational displacement that appears to be important for the catalytic mechanism of quinol oxidation.