Analysis of strains of Saccharomyces cerevisiae with amino acid substitutions in the CuA-binding region of subunit II of cytochrome c oxidase

Abstract

Cytochrome c oxidase accepts electrons from cytochrome c and transfers them to oxygen to form water. Electrons enter the complex through the Cu_A site, formed by two copper atoms bound to mitochondrially encoded subunit II. The effect of amino-acid alterations in one of the Cu_A ligands and in an amino acid adjacent to another of the ligands in the yeast enzyme is examined. Substitution of tyrosine for the Cu_A ligand, cysteine 221, completely abolishes enzyme activity. In addition, 19 independent revertants of this mutant yeast strain recover function by restoring the cysteine codon. Replacement of a non-conserved glycine at position 228 by valine, adjacent to the Cu_A-ligand histidine 229, virtually blocks enzyme activity. Activity is restored by inserting alanine or phenylalanine at position 228 or by amino-acid substitutions at nearby codons. Our results demonstrate that the Cu_A ligand appears to be essential for enzyme function while other residues in the copper-binding region are less functionally constrained.