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

Authors


  • Note. The novel nucleotide sequence data published here has been deposited with the EMBL sequence data banks and is available under accession number X-05550.

Correspondence to J. A. Berden, E. C. Slater Institute for Biochemical Research, University of Amsterdam, Plantage Muidergracht 12, NL-1018-TV Amsterdam, The Netherlands
Fax:+31 20 5255124.

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.

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