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 affecting center N residues were analysed. The mutant carrying a G33D change does not incorporate heme into the apocytochrome b and fails to grow on non-fermentable carbon sources. Out of 85 genetically independent revertants derived from this mutant, 82 were true back-mutants restoring the wild type sequence (D33G). The remaining three replaced the aspartic acid by an alanine (D33A) indicating that small size residues are best tolerated at this position which is consistent with the perfect conservation of the G33 during evolution. This glycine may be of crucial importance for helix packing around the hemes. The replacement of methionine at position 221 by lysine (M221K) produced a non-functional cytochrome b [(1993) J. Biol. Chem. 268, 15626-15632]. Non-native revertants replacing the lysine 221 by glutamic acid (K221E) or glutamine (K221Q) expressed a selective resistance to antimycin and antimycin derivatives having a modified dilactone ring moiety. Cytochrome b residues in 33 and in 221 seemed to contribute to the quinone reduction (QN) site of the cytochrome bc1 complex. Possible intramolecular interactions between the N-terminal region and the loop connecting helices IV and V of cytochrome b are proposed.
Coppée
J Y
JY
Laboratoire de Génétique Microbienne, Université Catholique de Louvain, Louvain-La-Neuve, Belgium.
Tokutake
N
N
Marc
D
D
di Rago
J P
JP
Miyoshi
H
H
Colson
A M
AM
D000968:Antimycin A / Q000031:analogs & derivatives / Q000494:pharmacology; D001483:Base Sequence; D002874:Chromosome Mapping; D003062:Codon; D003573:Cytochrome b Group / Q000737:chemistry / Q000235:genetics*; D004271:DNA, Fungal / Q000737:chemistry; D004272:DNA, Mitochondrial / Q000737:chemistry; D004352:Drug Resistance, Microbial; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D009154:Mutation; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017422:Sequence Analysis, DNA
D003062:Codon; D003573:Cytochrome b Group; D004271:DNA, Fungal; D004272:DNA, Mitochondrial; D009097:Multienzyme Complexes; C032456:antimycin; D000968:Antimycin A; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases