No abstract

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).

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.

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. Missense mutations in three genetic loci allocated to the exons of the cytochrome b gene, abolish the strong affinity binding of antimycin. 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.

A wild-type strain of Saccharomyces cerevisiae and temperature-dependent revertants of isonuclear box mutants partially or completely devoid of cytochrome h-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 "C, 28 "C and 36 "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"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 "C, 28 "C and 36 "C), were compared with the cytochrome b-565 content.The growth yields showed small variations if compared to the reference strain, when the cytochrome h-565 content was greatly diminished or absent.Thus the ATP production at site I1 is independent of the cytochrome h-565 content. Cytochrome b-565 does not play an essential role in oxidative phosphorylation.Two spectral forms of cytochrome b are present in the mitochondrial inner membrane; several hypotheses have been proposed to interpret results of redox potential, spectrophotometric and kinetic measurements but the question of the role of cytochrome h-565 in oxidative phosphorylation has not been solved.Yeast mitochondrial mutants which are affected in the synthesis of cytochrome b were isolated by Pajot et al. [I]. It was later shown that, in these strains, grown under certain conditions, the amount of cytochrome b-565 was extremely low [2], it seemed therefore possible to use these strains to investigate the Abbreviations and Symbols. Y., molar growth yield in g dry wt/ rnol substrate; EtOH, ethanol; rho", cytoplasmic petite, deleted of all mitochondrial DNA; P/O, mol ATP formed per electron pair transferred to oxygen; respiratory control ratio, ratio of respiratory rate in state 3 (+ADP) to that in state 4 (+ATP); CIPhzC(CN)Z, carbonylcyanide m-chlorophenylhydrazone. particular role of this cytochrome h species in oxidative phosphorylation.The investigations were carried out in vifro and in vivo. Zn vitro, the P/O ratio, respiratory control and respiration of mitochondria were correlated to the cytochrome b-562 and cytochrome b-565 contents. In vivo, the phosphorylation efficiencies of these isonuclear strains (which differ essentially in cytochrome h-565 concentrations) were compared by measuring the growth yields obtained by growing the cells under conditions of limited energy source. MATERIALS AND METHODS Yeast StrainsThe yeast strains used were described in the previous article [ 2 ] ; abbreviated names which refer to the