A region of the C-terminal part of the 11-kDa subunit of ubiquinol-cytochrome-c oxidoreductase of the yeast Saccharomyces cerevisiae contributes to the structure of the Qout reaction domain.
QCR8, the gene encoding the 11-kDa subunit of ubiquinol-cytochrome-c oxidoreductase of the yeast Saccharomyces cerevisiae has been resequenced in the course of a search for mutants disturbed in subunit function. Resequencing shows that the previously published sequence [Maarse A.C. & Grivell L.A. (1987) Eur. J. Biochem 155, 419-425] lacks a C at position 185 of the coding sequence. As a result of this extra nucleotide, the reading frame now contains 285 base pairs and it codes for a protein of 94 amino acids with a calculated molecular mass of 11.0 kDa. Despite the altered C-terminus, similarity to the corresponding beef heart subunit is not significantly altered. One mutant (LTN1), arising from hydroxylamine mutagenesis, has been studied in detail: Assembly of the enzyme appears to be normal, as judged from the levels of the subunits observed in Western blots, while spectral analysis showed that only holo-cytochrome b was lowered to 70% of that of the wildtype. Measurement of the specific activity and calculation of the turnover number of the enzyme showed that these were 45% and 56% of that of the wild type, respectively. Further analysis of the mutant showed that the affinity for the inhibitor myxothiazol was decreased, that the 11-kDa subunit stabilises the enzyme once assembly has occurred, and that the reduction of cytochrome b via the Qout site is impaired. Sequence analysis showed that this mutant carries a deletion of 12 nucleotides at position 206-217 of the coding sequence, resulting in the replacement of residues 69-73 (WWKNG) by a cysteine. These results are discussed in terms of the 11-kDa subunit contributing to the conformation of the Qout binding domain.
E.C. Slater Institute, University of Amsterdam, The Netherlands.
W
W
Hemrika
J A
JA
Berden
L A
LA
Grivell
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D014450:Electron Transport Complex III / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005960:Glucosides; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D010084:Oxidation-Reduction; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013057:Spectrum Analysis; D013379:Substrate Specificity
D005960:Glucosides; D009838:Oligodeoxyribonucleotides; C040358:dodecyl maltoside; D014450:Electron Transport Complex III