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Topology of the peptides in free and membrane-bound complex III (ubiquinol--cytochrome c reductase) as revealed by lactoperoxidase and p-diazoniumbenzene [35S] sulfonate labelling.

The arrangement of the peptides of free complex III and complex III incorporated into phospholipid vesicles was investigated by labeling with 125I + lactoperoxidase and with p-diazonium-benzene [35S]sulfonate (N = N – PhSO3H). The peptides were separated by sodium dodecylsulfate acrylamide gel electrophoresis and scanned for radioactivity. In free complex III, the most extensive iodination occurred in peptides II and IV, whereas all polypeptides (I – VI), except peptide VII, could be coupled to N = N – PhSO3H. Peptide VII was, however, labeled in the sodium-dodecyl-sulfate-dissociated enzyme. These results show that, with the possible exception of peptide VII, all peptides are at least partially exposed on the surface of the isolated complex. Complex III incorporated into phospholipid vesicles showed a labeling pattern which differed from the free enzyme, both with respect to the total incorporation and to the relative incorporation between the peptides. Total incorporation of N = N – Ph35SO3H and 125I was decreased by 60 – 70% in complex III-vesicles, suggesting that a large number of the binding sites on the free enzyme become buried within the membrane. In addition, peptides I, II, IV and V were relatively less labeled in complex III-vesicles than were peptides III and VI, suggesting a deeper location of the former peptides within the membrane. This is supported by the finding that labeling of deoxycholate-treated complex III-vesicles with N = N – Ph35SO3H results in an incorporation pattern similar to that in free complex III. Binding of N = N – PhSO3H and its effects on enzymatic activity was also studied in free and membrane-bound complex III. Both the binding of N = N – Ph35SO3H and inactivation of duroquinol – cytochrome c reductase were significantly slower in complex III-vesicles. These results indicate that many of the N = N – PhCO3H-binding sites on the free complex are inaccessible when the enzyme is incorporated into a vesicle. This approach was also used to ascertain intactness of the vesicles, and to show that N = N – PhSO3H labeled only those peptides exposed on the outer surface.