Point mutation in cytochrome b of yeast ubihydroquinone:cytochrome-c oxidoreductase causing myxothiazol resistance and facilitated dissociation of the iron-sulfur subunit.
Cytochrome-c reductase was isolated from Saccharomyces cerevisiae GM50-3C. A tenth subunit was detected with molecular mass 8.5 kDa on SDS/PAGE. Two yeast mutants selected for resistance to myxothiazol, an inhibitor of the Q0 center (Q, ubiquinone) of cytochrome-c reductase, were analysed. The single amino acid substitution in the cytochrome-b subunit, N256Y in the mutant Myx-119 and G137R in the mutant Myx-118, caused a general resistance to all methoxyacrylate inhibitors to about fivefold higher concentrations. The kinetic measurements with the substrate analogue nonylbenzohydroquinone revealed a decrease in the Km by fivefold and of the maximal turnover number by fourfold in the N256Y mutant. The Km of the G137R mutant was not affected and the Vmax was 50% higher. Cytochrome-c reductase was isolated from mutants to allow determination of the Kd values of methoxyacrylate-stilbene and myxothiazol by means of fluorescence-quench and red-shift titration. Changes in the structure of the multisubunit complex due to a single amino acid exchange became obvious during the purification procedure. SDS/PAGE of the purified enzyme revealed that the substitution N256Y in cytochrome b led to a loss of the iron-sulfur protein and the fifth small subunit with no change in the pattern of the remaining eight subunits. The subunit pattern of the G137R mutant was identical to the wild type. This is the first report of a single amino acid exchange in the catalytic subunit of cytochrome b, greatly affecting the iron-sulfur protein, the second important catalytic subunit of the Q0 center. This is a new approach to obtain structural information about the interaction of cytochrome b with the iron-sulfur subunit.
Geier
B M
BM
Universitätsklinikum Frankfurt, Institut für Therapeutische Biochemie, Federal Republic of Germany.
Schägger
H
H
Brandt
U
U
Colson
A M
AM
Von Jagow
G
G
D000595:Amino Acid Sequence; D002384:Catalysis; D003573:Cytochrome b Group / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004352:Drug Resistance, Microbial / Q000235:genetics; D014450:Electron Transport Complex III / Q000737:chemistry / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D007425:Intracellular Membranes / Q000201:enzymology; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D007700:Kinetics; D008689:Methacrylates; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013844:Thiazoles / Q000494:pharmacology
D003573:Cytochrome b Group; D007506:Iron-Sulfur Proteins; D008689:Methacrylates; D010446:Peptide Fragments; D013844:Thiazoles; C030517:myxothiazol; D014450:Electron Transport Complex III