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).

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.

Is cytochrome b really the antimycin-binding component of the cytochrome b--c1 complex of yeast mitochondria.

1. The antimycin binding sites were found to be independent of cytochrome(s) b synthesis in pseudo-wild-type revertants of cytochrome b mutants. These revertants, whose primary mutation is located in the introns of the cob-box gene of mitochondrial DNA, have modified contents of cytochromes b-562 and b-565 and fully functional respiratory chain. 2. Missense mutations in three genetic loci allocated to the exons of the cytochrome b gene, abolish the strong affinity binding of antimycin. 3. It is proposed that the antimycin-binding component is not cytochrome b itself, but interacts with it in such a way that an alteration of the cytochrome b structure affects the antimycin-binding site.

Cytochrome b-565 in Saccharomyces cerevisiae: use of mutants in the cob-box region of the mitochondrial DNA to study the functional role of this spectral species of cytochrome b. 1. Measurements of cytochromes b-562 and b-565 and selection of revertants devoid of cytochrome b-565.

In order to study the functional role of the spectral species of cytochrome b-565 observed in mitochondria, genetically manipulated strains of Saccharomyces cerevisiae have been used. Strains have been found which are devoid of cytochrome b-565 under certain conditions and which nevertheless are able to grow on a respirable substrate. Two different methods have been used to determine the cytochrome b-565 content: anaerobic titrations and antimycin-A-induced reduction of cytochrome b-565. Both yield the same results.

Cytochrome b-565 in Saccharomyces cerevisiae: use of mutants in the cob- box region of the mitochondrial DNA to study the functional role of this spectral species of cytochrome b. 2. Relationship between energetic data and cytochrome b-565 content.

A wild-type strain of Saccharomyces cerevisiae and temperature-dependent revertants of isonuclear box mutants partially or completely devoid of cytocheomr b-565, were used to study the role of this cytochrome in oxidative phosphorylation. At the mitochondrial level, the phosphorylating data and the succinate oxidase activity at three different temperatures (16 degrees C, 28 degrees C and 36 degrees C) were measured in these strains showing various cytochrome b-565 contents. It is concluded that this cytochrome b-565 is not in the main pathway of the electron transfer chain. At the optimum growth temperature (28 degrees C), the measurements of the P/O ratio for the wild phenotype strains, with ethanol as substrate, led to the conclusions that two phosphorylation sites of the respiratory chain are functional in these strains. The growth yields for wild phenotype strains and revertants grown in vivo in complex media, with ethanol or galactose as the energy source (at 16 degrees C, 28 degress C and 36 degrees C), were compared with the cytochrome b-565 content. The growth yields showed small variations if compared to the reference strain, when the cytochrome b-565 content was greatly diminished or absent. Thus the ATP production at side II is independent of the cytochrome b-565 content. Cytochrome b-565 does not play an essential role in oxidative phosphorylation.