The C-terminal half of the 11-kDa subunit VIII is not necessary for the enzymic activity of yeast ubiquinol:cytochrome-c oxidoreductase.
Inactivation of the gene encoding the 11-kDa subunit VIII of yeast ubiquinol:cytochrome c oxidoreductase leads to an inactive complex, which lacks detectable cytochrome b [Maarse, A. C., De Haan, M., Schoppink, P. J., Berden, J. A. and Grivell, L. A. (1988) Eur. J. Biochem. 172, 179-184] and in which the steady-state levels of the Fe-S protein and the 14-kDa subunit VII are severely reduced. When the 11-kDao mutant is transformed with a gene encoding a protein consisting of the 11-kDa protein minus its last 11 amino acids and fused to a 7-amino-acid sequence encoded by a stop oligonucleotide, the complex is assembled normally. Enzyme activity is similar to that of the wild type, as is also the sensitivity of the complex to antimycin and myxothiazol. Transformation of the mutant with a gene encoding a protein consisting of the 11-kDa protein lacking the last 43 amino acids (i.e. almost half the protein) and fused to the same 7-amino-acid sequence as above, gives partial restoration of the complex. The Fe-S protein and the 14-kDa subunit VII still exhibit low steady-state levels, but cytochrome b is present again, albeit at a strongly reduced level. Electron transport activity is also partially restored and correlates with the level of cytochrome b indicating that the turnover number of the complex is similar to that of wild-type complex III. These findings demonstrate the important role played by the 11-kDa protein in the stabilization of cytochrome b. They also imply that at least the C-terminal half of the 11-kDa protein is not part of an ubiquinol-binding site. Moreover, since the deletion has no effect on the sensitivity of the complex to myxothiazol and antimycin, at least this part of the protein is probably not involved in binding of these inhibitors.
Schoppink
P J
PJ
Laboratory of Biochemistry, University of Amsterdam, The Netherlands.
De Jong
M
M
Berden
J A
JA
Grivell
L A
LA
D000968:Antimycin A / Q000031:analogs & derivatives / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites / Q000187:drug effects; D003001:Cloning, Molecular; D014450:Electron Transport Complex III / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D005800:Genes, Fungal; D008689:Methacrylates; D009154:Mutation; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013844:Thiazoles / Q000494:pharmacology; D014170:Transformation, Genetic
D008689:Methacrylates; D013844:Thiazoles; C032456:antimycin; D000968:Antimycin A; C030517:myxothiazol; D014450:Electron Transport Complex III