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

Eur J Biochem. 1993 Aug 1;215(3):601-9. doi: 10.1111/j.1432-1033.1993.tb18071.x.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Binding Sites
  • Blotting, Western
  • Electron Transport Complex III / antagonists & inhibitors
  • Electron Transport Complex III / genetics
  • Electron Transport Complex III / metabolism*
  • Glucosides
  • Mitochondria / enzymology
  • Molecular Sequence Data
  • Mutation
  • Oligodeoxyribonucleotides
  • Oxidation-Reduction
  • Protein Conformation
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae / genetics
  • Sequence Homology, Amino Acid
  • Spectrum Analysis
  • Substrate Specificity

Substances

  • Glucosides
  • Oligodeoxyribonucleotides
  • dodecyl maltoside
  • Electron Transport Complex III

Associated data

  • GENBANK/X05550