Interaction of integral and peripheral membrane proteins: affinity labeling of yeast cytochrome oxidase by modified yeast cytochrome c.
To identify possible substrate-binding subunit(s) of yeast cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1-9-3-1), the purified enzyme was reacted with yeast iso-1-cytochrome c whose single free sulfhydryl group at position 107 had been activated with 5,5'-dithiobis(2-nitrobenzoate). The resulting cytochrome c derivative appeared to function as an "affinity-label" of cytochrome oxidase, since it rapidly inactivated the enzyme. Inactivation was competitively prevented by underivatized cytochrome c. When the "affinity-labeled" oxidase was analyzed by two-dimensional polyacrylamide electrophoresis in dodecyl sulfate (separation in the second dimension being carried out in the presence of excess sulfhydryl compound), it was found that the derivatized cytochrome c had specifically formed a mixed disulfide with the mitochondrially made subunit III (apparent molecular weight 24,000) of the oxidase. Similar results were obtained when underivatized iso-I-cytochrome c was crosslinked to the oxidase by oxidative disulfide bridge formation in the presence of ortho-phenanthroline and Cu++. These data indicate that the hydrophobic mitochondrially made subunit III of yeast cytochrome c oxidase is in close proximity to the cytochrome c binding site on the enzyme. Since cytochrome c and the mitochondrially made cytochrome oxidase subunit III are typical peripheral and integral membrane proteins, respectively, the present study suggests a useful approach for analyzing specific interactions between these different classes of membrane proteins.
D000345:Affinity Labels; D001665:Binding Sites; D002384:Catalysis; D003574:Cytochrome c Group / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000096:biosynthesis / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D013329:Structure-Activity Relationship
D000345:Affinity Labels; D003574:Cytochrome c Group; D008565:Membrane Proteins; D003576:Electron Transport Complex IV