Identification of ubiquinone-binding proteins in yeast mitochondrial ubiquinol-cytochrome c reductase using an azido-ubiquinone derivative.
An azido-ubiquinone derivative, 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyloctyl)-1,4-benzoquinone, was used to study the ubiquinone-protein interaction and to identify the ubiquinone-binding proteins in yeast mitochondrial ubiquinone-cytochrome c reductase. The phospholipids and Q6 in purified reductase were removed by repeated ammonium sulfate precipitation in the presence of 0.5% sodium cholate. The resulting phospholipid- and ubiquinone-depleted reductase shows no enzymatic activity; activity can be completely restored by the addition of phospholipids and Q6 or Q2. The ubiquinone- and phospholipid-replenished ubiquinonol-cytochrome c reductase is also fully active upon reconstituting with bovine succinate-ubiquinone reductase to form succinate-cytochrome c reductase. When an azido-ubiquinone derivative was added to the ubiquinone and phospholipid-depleted reductase in the dark, followed by the addition of phospholipids, partial reconstitutive activity was restored, while full ubiquinol-cytochrome c reductase activity was observed when Q2H2 was used as substrate in the assay mixture. Apparently, the large amount of Q2H2 present in the assay mixture displaces the azido-ubiquinone in the system. Photolysis of the azido-Q-treated reductase with long-wavelength ultraviolet light abolishes about 70% of both the restored reconstitutive activity and Q2H2-cytochrome c reductase activity. The activity loss is directly proportional to the covalent binding of [3H]azido-ubiquinone to the reductase protein. When the photolyzed, [3H]azido-ubiquinone-treated sample was subjected to SDS-polyacrylamide gel electrophoresis followed by analysis of the distribution of radioactivity among the subunits, the cytochrome b protein and a protein with an apparent molecular weight of 14 000 were heavily labeled. The amount of radioactive labeling in both these proteins was affected by the presence of phospholipids.
D000345:Affinity Labels; D001386:Azides / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D004579:Electron Transport; D014450:Electron Transport Complex III; D008928:Mitochondria / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D010782:Photolysis; D011808:Quinone Reductases / Q000378:metabolism; D013386:Succinates / Q000378:metabolism; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D002352:Carrier Proteins; D009097:Multienzyme Complexes; D010743:Phospholipids; D013386:Succinates; C027208:ubiquinone-binding proteins; D014451:Ubiquinone; C048123:3-azido-2-methyl-5-methoxy-6-(3,7-dimethyloctyl)-1,4-benzoquinone; D011808:Quinone Reductases; D014450:Electron Transport Complex III