Investigation of the structural interactions between the myxothiazol binding and the ubiquinol oxidation sites in the bc1 complex of S. cerevisiae.

In order to characterize the structural and functional alterations in the catalytic properties of the bcl complex that are associated with specific amino acid replacements in center P cytochrome b deficient mutants, we investigated the enzymatic and myxothiazol inhibition properties of two yeast mutants (1,2), some of their revertants (3,4,5) and those of the parental strain. In the framework of the hypothesis that the mechanism involved in the quinol cytochrome c reductase complex is of the ping pong type [6], the experimental kmin (Vm/Km) values obtained corresponds to the apparent second-order rate constant characteristic of the arrival and binding of the substrate at its site. The interaction of exogenous ubiquinols with the bcl complex involves both the center N and center P sites; however the reaction of the reduced substrate with this latter site seems to be more rate determining [6]; With most of the mutated strains (G137E + C133S; G137E + I147F; G137E + N256K C133Y + A126T) the catalytic efficiency (kmin) of the bcl complex for ubiquinol is not much altered (tablel). Some of these mutants exhibit a decrease in their turnover number (G137E + C133S; G137E + I147F; G137E + N256K) probably due to a decrease in the rate of an intermolecular electron transfer step at center P which would become the rate determining step (unpublished results). Surprisingly two mutated strains exhibited a modified kmin altough the Vm value were identical to that of the parental strain (table 1): this suggests that 1the corresponding mutations induce a large change in the interaction between the bcl complex and the quinol molecules and that 2the encounter between the enzyme and the substrate was not the rate limiting step in the overall reaction catalysed by the complex, both in the parental strain and in the two mutants. Only two strains (G137E, C133Y) exhibited a decrease in both the kmin and Vm values, but the magnitude of the &in decrease (table 1) does not seem to suffice to explain the Vm decrease, that could be also due to a decrease in the electron transfer, at either P and/or N center, observed in these mutants (unpublished results). Mutations in cytochrome b which confer resistance to myxothiazol involve two regions of the apoprotein; one of them is located in the termini of helice 3 [7]. The mutants studied here exhibited one or two mutations located in this region. Some of them exhibited myxothiazol resistance (mutants G137E (I50=2ON); G137E+C133S (I50=5.5N); C133Y (I50=2N)) whereas others exhibited a greater sensitivity towards this inhibitor than the parental strain (A126T+Y133S (I50=0.5N)) (table 1). Among the mutantdrevertants studied four exhibited a decrease in the kmin values, indicating a sigmficative change in the affinity of the quinol for its site; the mutations are located in the region between amino-acid 126 and 137; among these, the mutation G137E induces a high level of resistance to Table 1: Catalytic properties of the ubiquinol cytochrome c reductase in mitochondria of various yeast strains. The reductase activity was measured with DBH2 (2,3dimethoxy-5-methyl-6ndecyl-l,4 benzoquinol) as subtrate. I50 represents the concentration of inhibitor which decreases the rate of succinate cytochrome c reductase by 50%. For the wild type strain: Vm=65.5 s-1; Km=15.6 1M; Kmin=40.1@ M-1s-1; I50=N=0.5 nmoles/nmoles cytochrome b.