| Summary: | The Cu$\rm\sb{A}$ center, located in subunit II of cytochrome c oxidase, is the primary site for electron entry from cytochrome c. The recent crystal structure confirms this site to be a binuclear copper center with a dithiolate, dihistidyl coordination. A methionine and the carbonyl oxygen of Glu$\sp{198}$ are also ligands. To further characterize the structure and function of the Cu$\sb{A}$ center, site directed mutagenesis has been implemented in subunit II of S. cerevisiae. Substitutions of direct ligands to the Cu$\rm\sb{A}$ site result in loss of cellular respiration. Cytochrome a from cytochrome c oxidase is not detected in the visible absorption spectrum. Furthermore, subunit II does not accumulate to wild-type levels as observed by immunodetection. These results suggest that the enzyme is not assembling, most likely due to the disruption of copper binding, and that subunit II is being degraded. In contrast, substitutions to Glu$\sp{198},$ whose carbonyl oxygen is a ligand to one of the coppers, merely results in cells with reduced respiratory function. In an attempt to perturb the Cu$\rm\sb{A}$ site without disrupting protein stability, nearby amino acids were substituted. Asp$\sp{158}$ is known to hydrogen bond to the Cu$\rm\sb{A}$ ligand His$\sp{161}$, and Ser$\sp{197}$ is adjacent to the Cu$\rm\sb{A}$ ligand Cys$\sp{196}$. Substitutions at these positions result in reduced cellular respiration and may perturb the Cu$\rm\sb{A}$ site. Substitutions to conserved residues Gly$\sp{194}$ and Gly$\sp{201}$ result in a loss of cellular respiration and the underaccumulation of subunit II, while substitutions at other conserved positions have no observable effect on respiration. These results are discussed in relation to the homologous quinol oxidases and nitrous oxide reductases. To begin to characterize residues which may be involved in binding cytochrome c, substitutions were made to various conserved carboxylates which had been implicated in binding cytochrome c. The recent crystal structure demonstrates that Glu$\sp{198}$ and Asp$\sp{158}$ are not available for direct interaction with cytochrome c. However, the crystal structure does point to other conserved carboxylates. Asp$\sp{112}$ may be at the periphery of a cytochrome c binding site since replacement of this residue by Arg results in only a small decrease in cellular respiration.
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