The aromatic domain 66(YWYWW)70 of subunit VIII of the yeast ubiquinol-cytochrome c oxidoreductase is important for both assembly and activity of the enzyme

Abstract

The aromatic character of the region 66(YWYWW)70 of the 11-kDa subunit VIII of ubiquinol-cytochrome c oxidoreductase (bc1 complex) of the yeast Saccharomyces cerevisiae has previously been demonstrated to be important for assembly of a functional complex [Hemrika et al. (1994) FEBS Lett. 344, 15-19]. Especially the aromatic nature of residue 66 appeared to be relevant, as the very low level (5%) of bc1 complex in the mutant 66(SASAA)70 was restored to nearly 70% of the wild-type level in a phenotypic revertant with the sequence 66(FASAA)70. In the present study, three other site-directed mutants (66(SAYAA)70, 66(SASAW)70 and 66(SWYWW)70) were constructed and analysed. The data indicate that the presence of one aromatic residue is enough for a substantial level of assembly and that its position modulates the level of both assembly and electron transfer activity. The results also confirm the relevance of this region of subunit VIII for the formation of the Q(out) reaction domain, as reported by Hemrika et al. [(1993) Eur. J. Biochem. 215, 601-609]. It is further shown that the lowered specific activity of the mutant described by these authors is not due to the introduction of a cysteine in the sequence of subunit VIII.