The functional role of conserved acidic residues of the Qcr7 protein of the cytochrome bc(1) complex in Saccharomyces cerevisiae

The 14-kDa Qcr7 protein represents one of the 10 subunits that are componen ts of a functional cytochrome bc(1) complex in Sacharomyces cerevisiae. Pre vious studies have shown that the N-terminus of the Qcr7 protein may be inv olved in the assembly of the cytochrome bc(1), complex and its C-terminus b y interacting with cytochrome b and QCR8 proteins. It has also been suggest ed that Qcr7 protein may be involved in proton pumping. The coding sequence for two highly conserved aspartate residues, D46 and D47, in the QCR7 gene was altered by site-directed mutagenesis and the mutated genes expressed i n cells lacking a functional QCR7 gene. Mutants D46E, D46G, D46N, and D47E were comparable to wild type in growth phenotype on nonfermentable carbon s ources. Mutants D47G and D47N were respiratory deficient and analysis of co mplex components by immunoblotting and spectral analysis of cytochrome b su ggests defective assembly. Despite being respiratory competent and having n ormal electron transport rates in broken mitochondria, the mutant D46G had markedly reduced ATP synthesis from electron transport reactions catalyzed by complexes II plus III of the respiratory chain. This suggests that the g eometry of proton uptake by the bc(1), complex is disturbed by the mutation in D46. (C) 2001 Academic Press.