Structure of cytochrome c oxidase from baker's yeast - a progress report. Preparation of four subunits for amino acid sequence determination and attempts to localize the cytochrome c binding site.
Cytochrome c oxidase from the inner membrane of yeast mitochondria consists of seven nonidentical protein subunits, three being synthesized on mitochondrial ribosomes (molecular weights I: 43 K, II: 34 K, and III: 24 K) and four being made on cytoplasmic ribosomes (molecular weights IV: 14 K, V: 12 K, VI: 12 K, and VII: 4.5 K). In the present study all four cytoplasmically synthesized subunits of the enzyme were isolated on a large scale using ion exchange chromatography and gel filtration. Their amino acid composition as well as their amino- and carbosy-terminal amino acid residues have been determined. Sequence determinations of subunits IV and VI are already in an advanced state. The sequence of subunit VI is characterized by a large amino-terminal stretch dominated by charged amino acid residues followed by a cluster of hydrophobic amino acids. The binding site of yeast cytochrome oxidase for cytochrome c was studied by chemical crosslinking experiments. The formation of a disulfide bridge between the two proteins was observed by using cytochrome c from yeast modified with 5-thionitrobenzoate at the cysteinyl residue in position 107. Alternatively, a disulfide between yeast cytochrome c and the oxidase could be formed directly by oxidation with copper phenanthroline. Gel electrophoresis of the crosslinked complexes in sodium dodecyl sulfate revealed a new protein band with an apparent molecular weight of 38 K. This new band appears to be derived from cytochrome c and from subunit III of cytochrome oxidase.
D000595:Amino Acid Sequence; D001665:Binding Sites; D003574:Cytochrome c Group; D003576:Electron Transport Complex IV / Q000378:metabolism; D046911:Macromolecular Substances; D008566:Membranes / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D003574:Cytochrome c Group; D046911:Macromolecular Substances; D003576:Electron Transport Complex IV