Cytochrome c oxidase from bakers' yeast. Photolabeling of subunits exposed to the lipid bilayer.
Yeast mitochondria and purified yeast cytochrome c oxidase incorporated into micelles of the nonionic detergent Tween 80 were equilibrated with the hydrophobic aryl azides 5-[125I]iodonaphthyl-1-azide or S-(4-azido-2-nitrophenyl)-[35S]thiophenol. The azides were then converted to highly reactive nitrenes by flash photolysis or by illumination for 2 min and the derivatized cytochrome c oxidase subunits were identified by gel electrophoresis and radioactivity measurements. 5-[125I]Iodonaphthyl-1-azide labeled mainly the three mitochondrially made Subunits I to III and the cytoplasmically made Subunit VII. Subunits IV to VI or cytochrome c bound to the purified enzyme were labeled 9- to 90-fold less. Essentially the same result was obtained with S-(4-azido-2-nitrophenyl)-[35S]thiophenol except that Subunit V was labeled as well. In contrast, all seven subunits as well as cytochrome c were heavily labeled when the enzyme was dissociated with dodecyl sulfate prior to photolabeling with either of the two probes. These data indicate that all subunits of yeast cytochrome c oxidase except Subunits IV and VI are at least partly embedded in the lipid bilayer of the mitochondrial inner membrane.
D000345:Affinity Labels; D001386:Azides; D003576:Electron Transport Complex IV / Q000378:metabolism*; D007425:Intracellular Membranes / Q000201:enzymology*; D007700:Kinetics; D008823:Micelles; D008928:Mitochondria / Q000201:enzymology; D009281:Naphthalenes; D010782:Photolysis; D011136:Polysorbates; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D000345:Affinity Labels; D001386:Azides; D008823:Micelles; D009281:Naphthalenes; D011136:Polysorbates; D003576:Electron Transport Complex IV