title: Subunit 6 regulates half-of-the-sites reactivity of the dimeric cytochrome bc1 complex in Saccharomyces cerevisiae.
abstract: We have characterized the activities of the cytochrome bc1 complex in mitochondrial membranes from a yeast strain in which we deleted the nuclear gene (QCR6, COR3) which codes for the highly acidic subunit 6 of the bc1 complex. The chromosomal copy of QCR6 was replaced with a plasmid derived copy of QCR6, in which the entire coding region of QCR6 was replaced with the yeast LEU2 gene. The resulting deletion strain, MES8, contained no detectable mRNA for QCR6, and the cytochrome bc1 complex purified from the deletion strain lacked subunit 6. The deletion strain respired and grew on nonfermentable carbon sources such as ethanol and glycerol. Ubiquinol-cytochrome c reductase activity of mitochondria from the deletion strain was decreased 50% under conditions where the activity is zero order with respect to cytochrome c, and there was a similar decrease in the first-order rate constant for cytochrome c reduction. The loss of bc1 complex activities, observed at physiological ionic strengths, was reversible. Both the zero order rate and the first-order rate constant for cytochrome c reduction could be recovered to those of the parental strain by measuring these activities in mitochondrial membranes under conditions of low ionic strength. The zero order rate and first-order rate constant for cytochrome c reduction in membranes from the parent, wild-type yeast showed essentially no change coincident with this change in ionic strength. The 50% drop in both turnover number and first-order rate constant of ubiquinol-cytochrome c reductase activity indicates that half of the cytochrome bc1 complexes are inactive in the deletion strain at physiological ionic strengths. Inhibition by myxothiazol of cytochrome c reductase activity of mitochondrial membranes from the deletion strain showed an ionic strength-dependent lag in the titration curve that extended to the point where half of the inhibitor sites are filled. This lag was not observed with membranes from the wild-type, parent strain. This response to the inhibitor is consistent with half of the cytochrome bc1 complexes being inactive in mitochondria from the deletion strain at physiological ionic strength, but with both active and inactive complexes still able to bind inhibitor. The reversible, half-of-the-sites reactivity indicates that the bc1 complex must be dimeric in situ, in agreement with previous findings that the complexes isolated from fungal (Leonard, K., Wingfield, P., Arad, T., and Weiss, H. (1981) J. Mol. Biol. 149, 259-274) and mammalian (Nalecz, M. J., Bolli, R., and Azzi, A. (1985) Arch. Biochem. Biophys. 236, 619-628) mitochondria are structural dimers.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03756.
authors.forename: M E
authors.initials: ME
authors.lastname: Schmitt
authors.forename: B L
authors.initials: BL
authors.lastname: Trumpower
mesh_terms: D002872:Chromosome Deletion; D003574:Cytochrome c Group / Q000378:metabolism; D014450:Electron Transport Complex III / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D046911:Macromolecular Substances; D008961:Models, Structural; D008970:Molecular Weight; D010957:Plasmids; D012331:RNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000254:growth & development; D013367:Submitochondrial Particles / Q000201:enzymology
chemical_list: D003574:Cytochrome c Group; D046911:Macromolecular Substances; D012331:RNA, Fungal; D014450:Electron Transport Complex III