title: Interaction and identification of ubiquinone-binding proteins in ubiquinol-cytochrome c reductase by azido-ubiquinone derivatives.
abstract: Various azido-ubiquinone derivatives were synthesized and characterized. 3-Azido-2-methyl-5-methoxy-6-(3,7-dimethyloctyl)-1,4-benzoquinone was found to be suitable for the study of specific interaction between ubiquinone (Q) and protein. It was synthesized with high specific radioactivity and used to identify the Q-binding proteins in purified ubiquinol-cytochrome c reductase. This azido-Q derivative showed partial efficiency in restoring activity to the Q- and phospholipids-depleted ubiquinol-cytochrome c reductase in the absence of light. Azido-Q derivative treated samples, however, became completely inactivated upon photolysis, and the inactivation was not reversed by addition of Q derivatives. The redox state of the azido-Q derivative has little effect on the Q-binding affinity. Two protein subunits with Mr = 37,000 and 17,000 were found to be heavily labeled when depleted ubiquinol-cytochrome c reductase was treated with [3H] azido-Q derivative followed by photolysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amount of radioactive labeling of the Mr = 17,000 protein was proportional to the degree of inactivation and affected by the presence of phospholipids. The radioactive labeling of the Mr = 37,000 protein subunit, however, showed no correlation with degree of inactivation and was not affected by phospholipids. Since the radiolabeling at the Mr = 17,000 protein subunit was affected by phospholipids and correlated with the enzymatic activity, this subunit is probably the Q-binding protein in this enzyme complex (QPc). The inhibition of enzymatic activity by n-heptyl-4-hydroxyquinoline-N-oxide was easily reversed by addition of the azido-Q derivative. The distribution of radioactivity among the subunits of ubiquinol-cytochrome c reductase was not affected by the presence of antimycin A, 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole or n-heptyl-4-hydroxyquinoline-N-oxide, suggesting that the binding site(s) of these inhibitors are not the Q-binding site.
authors.forename: L
authors.initials: L
authors.lastname: Yu
authors.forename: F D
authors.initials: FD
authors.lastname: Yang
authors.forename: C A
authors.initials: CA
authors.lastname: Yu
mesh_terms: D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000138:chemical synthesis / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D014450:Electron Transport Complex III; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D010084:Oxidation-Reduction; D010743:Phospholipids / Q000494:pharmacology; D010782:Photolysis; D011808:Quinone Reductases / Q000378:metabolism*; D013997:Time Factors; D014451:Ubiquinone / Q000031:analogs & derivatives*
chemical_list: D000345:Affinity Labels; D001386:Azides; D002352:Carrier Proteins; D009097:Multienzyme Complexes; D010743:Phospholipids; C027208:ubiquinone-binding proteins; D014451:Ubiquinone; D009247:NADH, NADPH Oxidoreductases; D011808:Quinone Reductases; D014450:Electron Transport Complex III