Point mutation in cytochrome b of yeast ubihydroquinone:cytochrome‐c oxidoreductase causing myxothiazol resistance and facilitated dissociation of the iron‐sulfur subunit

Abstract

Cytochrome-c reductase was isolated from Succhuromyces cerevisiue GM50-3C. A tenth subunit was detected with molecular mass 8.5 kDa on SDSIPAGE.Two yeast mutants selected for resistance to myxothiazol, an inhibitor of the Qo 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 K,,, by fivefold and of the maximal turnover number by fourfold in the N256Y mutant. The K , of the C137R mutant was not affected and the V,,, 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. SDSjPAGE 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 Qo center. This is a new approach to obtain structural information about the interaction of cytochrome b with the iron-sulfur subunit.All mitochondrial ubiquinol : cytochrome-c reductases comprise three proteins carrying prosthetic groups. These are cytochrome b, the iron-sulfur protein and cytochrome el. Depending on the species, h -8 additional subunits are present. In yeast, the primary sequence of nine subunits has been determined by cloning and sequencing the respective genes [I -81. Sequence alignments showed that all subunits of the yeast enzyme possess homologous counterparts in bovine heart, but the latter contains two additional polypeptides that have so far not been detected in yeast [9]. The function of the non-redox subunits was analyzed by studying the effect of deletion of the particular gene [6. 8, 101. Most of them seem lo be involved in the assembly of mitochondrial cytochromec reductase [I 11.As has been known for a long time, cytochrome h is the only subunit of mitochondrial origin, but deletion of the gene C.'orrespondence to C. von Jagow. Universitatsklinikum, Institut fur Therapeutische Biochemic, lheodor-Stern-Kai 7 Haus 25B, W-6000 Frankfurt 70, Fcderal Rcpublic of Germany Ahhreviutions. NBH, nonylbenzohydroquinone; Q, ubiquinone; TCso, mcdian inhibitory concentration. also causes defects in thc iron-sulfur protein, and the 14-kDa and 11-kDa subunits [12, 131. A different approach to elucidate the structure and function of cytochro...

Kinetic Properties and Ligand Binding of the Eleven‐subunit Cytochrome‐c Oxidase from Saccharomyces cerevisiae Isolated with a Novel Large‐Scale Purification Method

“…Mitochondria1 membranes were isolated according to Geier et al [21], frozen rapidly in liquid nitrogen and stored at -80°C. The mitochondrial membranes were diluted to approximately 20 mg/ml protein in 1.1 % Triton X-100, 200 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PhMeSO,F), 10 mh4 potassium phosphate, pH 7.2, and centrifuged for l h at 100000 g. Complexes I11 and IV of the mitochondrial respiratory chain were extracted with 2.2% Triton X-100, 600 mM NaC1, 5 mM EDTA, 1 mM PhMeSO,F, 10 mM potassium phosphate, pH 7.2.…”

Domain movement of iron sulfur protein in cytochrome bc1 complex is facilitated by the electron transfer from cytochrome bL to bH

“…These results support the speculation that the driving force for the movement of the reduced head domain of ISP is the molecular event of electron transfer from b L to b H during the catalytic cycle. It should be mentioned that the redox state dependent binding of stigmatellin or MOAS to the cytochrome bc 1 complex has been investigated by several laboratories [23][24][25][26][27]. A relative wide range of binding or dissociation constants has been obtained with different explanations.…”

Mutational Analysis of Cytochrome b at the Ubiquinol Oxidation Site of Yeast Complex III

“…The side chain of Phe 129 is in hydrophobic interaction with the tail of stigmatellin and probably with that of the substrate. This residue is a locus of center P inhibitor resistance both in yeast and bacterial QCR (17)(18)(19). Mutational analysis of the equivalent residue in bacterial QCR (Phe 144 ) indicated that replacement of Phe 144 affects ubiquinol binding and oxidation (9).…”