ANALYSIS OF REVERTANTS FROM RESPIRATORY DEFICIENT MUTANTS WITHIN THE CENTER-N OF CYTOCHROME-B IN SACCHAROMYCES-CEREVISIAE

Four modified cytochrome b's carrying mononucleotide substitutions aff ecting center N residues were analysed. The mutant carrying a G33D cha nge does not incorporate heme into the apocytochrome b and fails to gr ow on non-fermentable carbon sources. Out of 85 genetically independen t revertants derived from this mutant, 82 were true back-mutants resto ring the wild type sequence (D33G). The remaining three replaced the a spartic acid by an alanine (D33A) indicating that small size residues are best tolerated al this position which is consistent with the perfe ct conservation of the G33 during evolution. This glycine may be of cr ucial importance for helix packing around the hemes. The replacement o f methionine at position 221 by lysine (M221K) produced a non-function al cytochrome b [(1993) J. Biol. Chem. 268, 15626-15632]. Non-native r evertants replacing the lysine 221 by glutamic acid (K221E) or glutami ne (K221Q) expressed a selective resistance to antimycin and antimycin derivatives having a modified dilactone ring moiety. Cytochrome b res idues in 33 and in 221 seemed to contribute to the quinone reduction ( Q(N),) site of the cytochrome be, complex. Possible intramolecular int eractions between the N-terminal region and the loop connecting helice s IV and V of cytochrome b are proposed.