Abstract
To explore the nature of proposed ligands to the CuA center in cytochrome c oxidase, site-directed mutagenesis has been initiated in subunit II of the enzyme. Mutations were introduced into the mitochondrial gene from the yeast Saccharomyces cerevisiae by high velocity microprojectile bombardment. A variety of single amino acid substitutions at each of the proposed cysteine and histidine ligands (His-161, Cys-196, Cys-200, and His-204 in the bovine numbering scheme), as well as at the conserved Met-207, all result in yeast which fails to grow on ethanol/glycerol medium. Similarly, all possible paired exchange Cys,His and Cys,Met mutants show the same phenotype. Furthermore, protein stability is severely reduced as evidenced by both the absence of an absorbance maximum at 600 nm in the spectra of mutant cells and the underaccumulation of subunit II, as observed by immunolabeling of mitochondrial extracts. In the same area of the protein, a variety of amino acid substitutions at one of the carboxylates previously implicated in binding cytochrome c, Glu-198, allow (reduced) growth on ethanol/glycerol medium, with normal intracellular levels of protein. These results suggest that a precise folding environment of the CuA site within subunit II is essential for assembly or stable accumulation of cytochrome c oxidase in yeast.
Highlights
Cytochrome c oxidase accepts electrons from cytochrome c to reduce dioxygen to water in the final step of cellular respiration, according to: Coupled to this electron transfer, the enzyme pumps protons against an electrochemical gradient, and the energy stored in this gradient is subsequently utilized for the production of ATP [1, 2]
Two metal centers serve in electron transfer from cytochrome c to the oxygen binding site
Glu198, which lies in the primary sequence of subunit II between the two targeted cysteines and has been implicated in the binding of cytochrome c, was substituted with single amino acid mutations, as indicated in Table I and Fig. 2
Summary
Site-directed mutagenesis of subunit II in the eukaryotic enzyme has been carried out in the yeast Saccharomyces cerevisiae to test directly the involvement of the four most likely ligands to CuA: His-161, Cys-196, Cys-200, and His-204, as well as the potential ligand Met-207. Single substitutions have been introduced at each site and include mutations intended either to retain or remove the ability to coordinate copper. Double mutations have been prepared which exchange proposed ligand functional groups (e.g. a mutation of Cys to His at one site combined with a mutation of His to Cys at another). To complement studies of the closely spaced Cys residues and to test the role of this region in the binding of the physiological redox partner cytochrome c, the centrally located Glu-198 has been replaced by semiconservative, as well as more dramatic substitutions
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