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

Geier, B.M., Schagger, H., Brandt, U., Colson, A.M., Jagow, G. von.
European journal of biochemistry 1992 v.208 no.2 pp. 375-380
Saccharomyces cerevisiae, mutants, cytochrome b, NADH dehydrogenase, physicochemical properties, enzyme inhibitors, amino acid sequences, mutation, protein structure, catalytic activity
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 Q(0) 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 Q(0) center. This is a new approach to obtain structural information about the interaction of cytochrome b with the iron-sulfur subunit.