TEMPERATURE-JUMP AND POTENTIOMETRIC STUDIES ON RECOMBINANT WILD-TYPE AND Y143F AND Y254F MUTANTS OF SACCHAROMYCES-CEREVISIAE FLAVOCYTOCHROME B(2) - ROLE OF THE DRIVING-FORCE IN INTRAMOLECULAR ELECTRON-TRANSFERKINETICS

The kinetics of intramolecular electron transfer between flavin and he me in Saccharomyces cerevisiae flavocytochrome b(2) were investigated by performing potentiometric titrations and temperature-jump experimen ts on the recombinant wild type and Y143F and Y254F mutants, The midpo int potential of heme was determined by monitoring redox titrations sp ectrophotometric ally, and that of semiquinone flavin/reduced flavin ( Fsq/Fred) and oxidized flavin (Fox)/Fsq couples by electron paramagnet ic resonance experiments at room temperature. The effects of pyruvate on the kinetic and thermodynamic parameters were also investigated. At room temperature, pH 7.0 and I = 0.1 M, the redox potential of the Fs q/Fred, Fox/Fsq, and oxidized heme/reduced heme (Hox/Hred) couples wer e -135, -45, and -3 mV, respectively, in the wild-type form. Although neither the mutations nor excess pyruvate did appreciably modify the p otential of the heme or that of the Fsq/Fred couple, they led to varia ble positive shifts in the potential of the Fox/Fsq couple, thus modul ating the driving force chat characterizes the reduction of heme by th e semiquinone in the -42 to +88 mV range. The relaxation rates measure d at 16 degrees C in temperature-jump experiments were independent of the protein concentrations, with absorbance changes corresponding to t he reduction of the heme. Two relaxation processes were clearly resolv ed in wild-type flavocytochrome b(2) (1/tau(1) = 1500 s(-1), 1/tau(2) = 200 +/- 50 s(-1)) and were assigned to the reactions whereby the hem e is reduced by Fred and Fag, respectively. The rate of the latter rea ction was determined in the whole series of proteins. Its variation as a function of the driving force is well described by the expression o btained from electron-transfer theories, which provides evidence that the intramolecular electron transfer is not controlled by the dynamics of the protein.