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 cytochrom e bc(1) complex and, consequently, to the absence of growth on non-fer mentable substrates. We isolated and characterized, from these mutants , fourteen different intragenic pseudo-revertants of various respirato ry sufficient phenotypes. Both first-site and second-site suppressor m utations 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 transmem brane disposition of helices F and G of the current eight-helix cytoch rome 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 o f helices I, II and VII in bacteriorhodopsin, respectively. We observe d from the study of second-site revertants that compensation across th e membrane never occurs. For each revertant, the suppressor mutation a nd the corresponding target mutation are on the same side of the membr ane. This membrane sidedness strengthens the topological constraints i mposed by the Q-cycle, namely the necessity of spatial separation of t wo catalytic reaction sites for ubiquinone.