Differential labeling of the subunits of respiratory complex III with [3H]succinic anhydride, [14C]succinic anhydride, and p-diazobenzene-[35S]sulfonate.
Exposure of antimycin-treated Complex III (ubiquinol-cytochrome c reductase) purified from bovine heart mitochondria to [3H]succinic anhydride plus [35S]p-diazobenzenesulfonate (DABS) resulted in somewhat uniform relative labeling of the eight measured subunits of the complex by [3H]succinic anhydride. In contrast, relative labeling by [35S]DABS was similar to [3H]succinic anhydride for the subunits of high molecular mass, i.e., core proteins, cytochromes, and the iron-sulfur protein, but greatly reduced for the polypeptides of molecular mass below 15 kDa. With Complex II depleted in the iron-sulfur protein the relative labeling of core protein I by exposure of the complex to [3H]succinic anhydride was significantly enhanced, whereas labeling of the polypeptides represented by SDS-PAGE bands 7 and 8 was significantly inhibited. Dual labeling of the subunits of Complex III by 14C- and 3H-labeled succinic anhydride before and after dissociation of the complex by sodium dodecyl sulfate, respectively, was measured with the complex in its oxidized, reduced, and antimycin-inhibited states. Subunits observed to be most accessible or reactive to succinic anhydride were core protein II, the iron-sulfur protein, and polypeptides of SDS-PAGE bands 7,8, and 9. Two additional polypeptides of molecular masses 23 and 12kDa, not normally resolved by gel-electrophoresis, were detected. Reduction of the complex resulted in a significant change of 14C/3H labeling ratio of core protein only, whereas treatment of the complex with antimycin resulted in decreases in 14C/3H labeling ratios of core proteins I and II, cytochrome c1, and a polypeptide of molecular mass 13kDa identified as an antimycin-binding protein.
D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D001557:Benzenesulfonates / Q000378:metabolism*; D002250:Carbon Radioisotopes; D002417:Cattle; D003979:Diazonium Compounds / Q000378:metabolism*; D014450:Electron Transport Complex III; D004591:Electrophoresis, Polyacrylamide Gel; D007506:Iron-Sulfur Proteins / Q000378:metabolism; D008929:Mitochondria, Heart / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000378:metabolism*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011808:Quinone Reductases / Q000378:metabolism*; D013386:Succinates / Q000378:metabolism*; D013387:Succinic Anhydrides / Q000378:metabolism*; D013425:Sulfanilic Acids / Q000378:metabolism*; D014316:Tritium
D001557:Benzenesulfonates; D002250:Carbon Radioisotopes; D003979:Diazonium Compounds; D007506:Iron-Sulfur Proteins; D009097:Multienzyme Complexes; C037989:Rieske iron-sulfur protein; D013386:Succinates; D013387:Succinic Anhydrides; D013425:Sulfanilic Acids; D014316:Tritium; D000968:Antimycin A; D011808:Quinone Reductases; D014450:Electron Transport Complex III; C000469:diazobenzenesulfonic acid