Site-directed mutations in the mitochondrially encoded subunits I and III of yeast cytochrome oxidase

Since yeast is amenable to mitochondrial transformation, designed mutations can be introduced in the mitochondrially encoded subunits of the respirato ry complexes. In the present work, six mutations have been introduced by th e biolistic method into yeast (Saccharomyces cerevisiae) cytochrome oxidase subunits I and III. The effects of these mutations on respiratory growth c ompetence, cytochrome oxidase activity and optical properties were then cha racterized. Firstly, the conserved glutamate Glu-243 in the D-channel of su bunit I was replaced by an asparagine or an aspartate residue. The effects of the mutations showed that Glu-243, which is essential for proton movemen t in bacterial oxidases, is also required for the activity of the eukaryoti c enzyme. Secondly, four mutations associated with human disease were intro duced in yeast, allowing detailed analysis of their deleterious effects on cytochrome oxidase function: Met-273-->Thr, Ile-280-->Thr and Gly-317-->Ser , affecting residues located in or near the K-channel in subunit I, and a s hort in-frame deletion comprising residues Phe-102 to Phe-106 in subunit II I (Delta F102-F106). The subunit III mutation was highly deleterious and ab olished enzyme assembly. The change GIy-317 -->Ser had no effect on respira tory function. However, mutations Met-273-->Thr and IIe-280-->Thr were mild ly deleterious, decreased cytochrome oxidase activity and slightly perturbe d the properties of the binuclear centre.