STEADY-STATE KINETICS OF UBIQUINOL CYTOCHROME-C REDUCTASE IN SACCHAROMYCES-CEREVISIAE MITOCHONDRIA - EFFECTS OF FLUIDITY CHANGES OBTAINED BY DIFFERENT GROWTH TEMPERATURES

We have investigated the steady-state kinetics of ubiquinol-cytochrome c reductase using either ubiquinol-1 or -2 as the donor substrate in mitochondria from Saccharomyces cerevisiae either grown at 36°C or brought from 36 to 10°C at the end of the exponential phase ("thermal shock"). The fatty acid composition of the mitochondrial lipids varied under the two conditions, with an increase of the unsaturation degree in the thermally shocked cells. The fluorescence polarization of the membrane probe diphenylhexatriene incorporated into mitochondria was decreased in a wide temperature range in the thermally shocked cells, in accordance with the fatty acid changes. Also the motion and order parameters of two fatty acid spin labels revealed significant changes in the thermally shocked cells, interpreted as an increase of membrane fluidity brought about by the thermal shock. This increase agrees with the finding that the diffusion coefficient of the ubiquinone homolog Q3 in the membrane, investigated by collisional fluorescence quenching of the membrane probe 12-(9-anthroyl) stearic acid, was consistently increased in the mitochondria from the thermally shocked cells. In spite of the physical changes of the membrane and the increase in the diffusion coefficients of the ubiquinone homolog, the steady-state kinetics of ubiquinol cytochrome c reductase showed no change to be interpreted in terms of a diffusion-limited component. An increase of Vmax of the enzyme in the thermally shocked cells was the result of an increased content of the bc1 complex; both the Km for ubiquinols and the turnover number were almost identical under the two growth conditions, so that the kcat/Km ratio, which approaches the second-order rate constant of substrate interaction with the enzyme active site, was unmodified. This is a demonstration that the ubiquinol cytochrome c reductase activity in S. cerevisiae mitochondria is not diffusion-controlled in the interaction of the donor substrate with the enzymatic protein. © 1993 Academic Press, Inc.