Identification of essential amino acids within the proposed CuA binding site in subunit II of Cytochrome c oxidase.
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.
Speno
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Department of Chemistry, University of Massachusetts, Amherst 01003-4510, USA.
Taheri
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MR
Sieburth
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Martin
C T
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D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015139:Blotting, Southern; D017124:Conserved Sequence; D003300:Copper / Q000737:chemistry*; D003433:Crosses, Genetic; D004252:DNA Mutational Analysis; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000235:genetics; D005057:Eukaryotic Cells; D008667:Metalloproteins / Q000737:chemistry* / Q000235:genetics; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010101:Oxygen Consumption; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013053:Spectrophotometry
D008667:Metalloproteins; D011994:Recombinant Proteins; D003300:Copper; D003576:Electron Transport Complex IV