Arrangement of subunit IV in beef heart cytochrome c oxidase probed by chemical labeling and protease digestion experiments

Abstract

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.

Radioiodinated, photoactivatable phosphatidylcholine and phosphatidylserine: transfer properties and differential photoreactive interaction with human erythrocyte membrane proteins

“…Therefore, it would seem that the inhibition of labeling obtained with DTT might be a result of its partitioning within the bilayer since DTT is freely soluble in a variety of organic solvents whereas glutathione is not. These observations are further supported by the results of Malatesta et al (1983), who have shown that similarly labeled phosphatidylcholine selectively labels cytochrome c oxidase in its hydrophobic membrane-spanning stretch.…”

The Transmembrane Helices of Beef Heart Cytochrome Oxidase

“…Finally, there is the problem of what the length of a transmembrane helix actually is. We find that 18or 19-residue segments are more useful in locating the probable transmembrane stretches in a membrane protein than longer segments if hydrophobicity profiles are used because charged residues may be present particularly in the lipid head region (3,6,7,15). The transmembrane helices observed in the crystal structure of the photosyn-thetic reaction center of Rhodopseudomonas viridis range in length from 24-30 residues and, therefore, can not be predicted in their entirety from the hydrophobicity profile of a 19-residue moving window using the hydropathy scale of Kyte and Doolittle (5,19) or Table I (3).…”

“…Hydrophobicity sums were obtained for a moving window of 19-residue segments using an adapted computer program from Kyte and Doolittle (5). The transmembrane helices predicted using this scale correspond to the lipid-buried segments of the two transmembrane helices in subunit II and the single transmembrane helix in subunit IV of beef heart COX located by the arylazidophospholipid labeling experiments of Buse et al (6,7).…”

Orientation of the cytoplasmically made subunits of beef heart cytochrome c oxidase determined by protease digestion and antibody binding experiments

“…We have previously used trypsin cleavage, as well as chemical labeling with 3Hand uC-labeled arylazidophospholipids, [3H]-(V-(4-azido-2-nitrophenyl)-2-aminoethanesulfonate and [35S]diazobenzenesulfonate, to examine the topology of C(v in beef heart cytochrome c oxidase (Malatesta et al, 1983). We concluded that this polypeptide was oriented with its N-terminus on the M side and its C-terminus on the C side of the inner membrane, but with the C-terminus mostly buried by protein-protein interactions.…”