GENETIC-ANALYSIS OF THE FOLDED STRUCTURE OF YEAST MITOCHONDRIAL CYTOCHROME-B BY SELECTION OF INTRAGENIC 2ND-SITE REVERTANTS

The mutations C133 --> Y133, L282 --> F282 and G340 --> E340 in yeast mitochondrial cytochrome b each lead to a dysfunction of the cytochrome bc(1) complex and, consequently, to the absence of growth on non-fermentable substrates. We isolated and characterized, from these mutants, fourteen different intragenic pseudo-revertants of various respiratory sufficient phenotypes. Both first-site and second-site suppressor mutations were found. A novel type of suppressor mutation consisted of the three-base-pair deletion of the parental mutated codon (E340(Delta)). The results provide, for the first time, evidence for the transmembrane disposition of helices F and G of the current eight-helix cytochrome b model. These two helices are presumably in contact with helix C in the folded protein. A simple modelisation study suggests that the packing of helices C, F and G in cytochrome b may be similar to that of helices I, II and VII in bacteriorhodopsin, respectively. We observed from the study of second-site revertants that compensation across the membrane never occurs. For each revertant, the suppressor mutation and the corresponding target mutation are on the same side of the membrane. This membrane sidedness strengthens the topological constraints imposed by the Q-cycle, namely the necessity of spatial separation of two catalytic reaction sites for ubiquinone.