Specific mutagenesis of the Rieske iron-sulfur protein in Rhodobacter sphaeroides shows that both the thermodynamic gradient and the pK of the oxidized form determine the rate of quinol oxidation by the bc(1) complex

In the Rieske iron-sulfur protein (ISP) of the ubiquinol:cytochrome alpha o xidoreductase (bc(1) complex) of Rhoctobacter sphaeroides, residue Tyr 156 is located close to the iron-sulfur cluster. Previous studies of the equiva lent residue in both Saccharomyces cerevisiae [Denke, E., Merbitz-Zahradnik , T., Hatzfeld, O. M., Snyder, C. H., Link, T. A., and Trumpower, B. L. (19 98) J. Biol. Chem. 273, 9085-9093] and Paracoccus denitrificans [Schroter, T., Hatzfeld, O. M., Gemeinhardt, S., Korn, M., Friedrich, T., Ludwig, B., and Link, T. A. (1998) Eur. J. Biochem. 255, 100-106] have indicated that m utations at this site can lead to modifications in the redox potential of t he ISP. To study the effect of similar modifications on the thermodynamic b ehavior and kinetics of partial reactions of the bc(1) complex upon flash a ctivation, we have constructed four mutant strains of Rb. sphaeroides where Tyr 156 was mutated to His, Leu, Phe, or Trp. The bc(1) complex was assemb led and able to support photosynthetic growth in all mutants. Three substit utions (Leu, Phe, Trp) led to alteration of the midpoint potential (E-m) of the ISP and a slowing in rate of quinol oxidation, suggesting that electro n transfer from quinol to the oxidized ISP controls the overall rate and th at this step includes the high activation barrier. The Trp mutation led to an increase of similar to 1 pH unit in the pK value of the oxidized ISP. Th e pH dependence of the rate of quinol oxidation in this mutant was also shi fted up by similar to 1 pH unit, showing the importance of the protonation state of the ISP for quinol oxidation. This provides support for a model in which the dissociated form of the oxidized ISP is required for formation o f the enzyme-substrate complex [Ugulava, N., and Crofts, A. R. (1998) FEES Lett. 440, 409-413].