Arrangement of subunit IV in beef heart cytochrome c oxidase probed by chemical labeling and protease digestion experiments.
The arrangement of subunit IV in beef heart cytochrome c oxidase has been explored by chemical labeling and protease digestion studies. This subunit has been purified from four samples of cytochrome c oxidase that had been reacted with N-(4-azido-2-nitrophenyl)-2-aminoethyl[35S]-sulfonate (NAP-taurine), diazobenzene[35S]sulfonate, 1-myristoyl-2-[12-[(4-azido-2-nitrophenyl)amino]lauroyl]-sn-glycero-3- [14C]phosphocholine (I), and 1-palmitoyl-2-(2-azido-4-nitrobenzoyl)-sn-glycero-3-[3H]phosphocholine (II), respectively. The labeled polypeptide was then fragmented by cyanogen bromide, at arginyl side chains with trypsin (after maleylation), and the distribution of the labeling within the sequence was analyzed. The N-terminal part of subunit IV (residues 1-71) was shown to be heavily labeled by water-soluble, lipid-insoluble reagents but not by the phospholipid derivatives. These latter reagents labeled only in the region of residues 62-122, containing the long hydrophobic and putative membrane-spanning stretch. Trypsin cleavage of native cytochrome c oxidase complex at pH 8.2 was shown to clip the first seven amino acids from subunit IV. This cleavage was found to occur in submitochondrial particles but not in mitochondria or mitoplasts. These results are interpreted to show that subunit IV is oriented with its N terminus on the matrix side of the mitochondrial inner membrane and spans the membrane with the extended sequence of hydrophobic lipid residues 79-98 buried in the bilayer.
D000818:Animals; D001120:Arginine; D002417:Cattle; D003488:Cyanogen Bromide; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000302:isolation & purification / Q000378:metabolism*; D008239:Lysine; D046911:Macromolecular Substances; D008929:Mitochondria, Heart / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D013367:Submitochondrial Particles / Q000201:enzymology; D014357:Trypsin
D046911:Macromolecular Substances; D010446:Peptide Fragments; D001120:Arginine; D003576:Electron Transport Complex IV; D014357:Trypsin; D008239:Lysine; D003488:Cyanogen Bromide