Effects of a missense exonic mutation in cytochrome b gene, observed on isolated mitochondrial complex III of Saccharomyces cerevisiae: consequence for the antimycin binding site.

The mitochondrial complex III was isolated from a wild type strain of Saccharomyces cerevisiae PS409 and from two mutants, PS490 and PS493, carrying a missense mutation in the structural gene of cytochrome b (in exons B1 and B4 respectively). These mutants synthesize cytochrome b in variable proportions, but they are unable to grow on a respirable substrate. Strain PS493 does not bind antimycin, whereas strain PS490 contains less cytochromes b and c1 but shows a strong binding to the inhibitor. The complex isolated from the wild type strain or mutant PS493 exhibited a specific cytochrome b and cytochrome c1 heme content of approximately 8 and 4 nmol/mg of protein respectively. This content was about 3 and 2 nmol/mg with PS490, which leads to a molar stoichiometry of 1.3 : 1 for cytochromes b and c1, instead of an 'ideal' ratio of 2 : 1 expected with b-c1 complex, and obtained with the two other strains. This implies that the association (or presence) of b and c1 cytochromes is not pre-requisite for complex III assembly. The wild type complex III isolated from PS409 was found to have a high level of CoQ2H2 activity, using a synthetic coenzyme Q analog as substrate (440 s-1 mol of cytochrome c reduced/mol of cytochrome c1). This activity is fully inhibited by antimycin. The complexes isolated from the two box mutants exhibited no such activity. Analysis of the subunit composition of the three isolated complexes on sodium dodecyl sulfate-gel electrophoresis showed that all the bands belonging to the b-c1 complex were synthesized in both mutants as well as in the wild type strain. Some of them appeared to have slightly diminished, but no specific decrease of a band has been observed in mutant PS493 that does not bind antimycin, with respect to mutant PS490 which binds strongly to the inhibitor. It should be noted that the subunit of about 12-13 kDa, qualified as the antimycin binding protein, is equally present in the three complexes. The results suggest that the loss of antimycin binding in mutant PS493 might be due to conformational perturbations in the modified complex rather than to the disappearance or significant modification of some protein support.

Missense exonic mitochondrial mutation in cytochrome b gene of Saccharomyces cerevisiae, resulting in core protein deficiency in complex III of the respiratory chain.

The level of core protein I and subunit VI of mitochondrial complex III (which are coded by the nuclear genome) was found to be greatly diminished in a yeast strain carrying a mutation (W7) in the mitochondrial gene coding for cytochrome b. This suggests that intricate interactions occur in complex III biogenesis between proteins of cytoplasmic and mitochondrial origin. This mutant was characterized by a low cytochrome b level and a loss of activity in the b-c1 segment of the respiratory chain. It was compared to another mutant showing similar biochemical characteristics, but which had integrated core protein I, as shown by antibody binding experiments. In mutant devoid of core protein I, cytochrome b was found to be reducible by NADH but not by succinate, suggesting two different electron transfer pathways inside comples III from each substrate to cytochrome b heme(s).