ELECTRON-TRANSFER FROM CYTOCHROME-C(2) TO THE PRIMARY DONOR OF RHODOBACTER-SPHAEROIDES REACTION CENTERS - A TEMPERATURE-DEPENDENCE STUDY

Kinetics of flash-induced electron transfer from the soluble cytochrome c2 to the primary donor (P) of the reaction center purified from the purple bacterium Rhodobacter sphaeroides R-26 were investigated by time-resolved absorption spectroscopy. Re-reduction of P+ induced by a laser pulse was measured at 1283 nm both in isolated reaction centers and in reconstituted proteoliposomes reproducing the lipid composition of the native membrane. The effects of temperature (230-300 K) and of the cytochrome c2/reaction center stoichiometry were examined. At room temperature, over a wide range of cytochrome c2 to reaction center molar ratios, the biphasic kinetics of cytochrome c2 oxidation in the microsecond-to-millisecond time scale could be accurately described by a minimum reaction scheme which includes a second-order collisional process (k = 1.4 X 10(9) M-1 s-1 and k = 2.4 X 10(9) M-1 s-1 in isolated and reconstituted reaction centers, respectively) and a first-order intracomplex electron donation (t1/2 = 590 +/- 110 ns in isolated reaction centers; t1/2 = 930 +/- 140 ns in proteoliposomes). At cytochrome C2 to reaction center molar ratios exceeding 5, the monomolecular process almost completely accounts for P+ re-reduction. At lower stoichiometries, the relative contribution of the two parallel reaction pathways is modulated by a single binding equilibrium between cytochrome c2 and reaction centers, yielding a binding constant of 3.5 X 10(5) M-1 in both systems. In the 230-300 K range, the kinetics of the mono- and bimolecular reactions are markedly affected by temperature, following Arrhenius behavior with activation energies of 4.9-5.8 and 6.5 kcal mol-1, respectively. At low temperature, in isolated reaction centers (but not in the reconstituted system), a minor microsecond phase of P+ re-reduction was detected, attributed to formation of the triplet-state P-3. Upon lowering the temperature, the relative contribution of the fast monomolecular oxidation of cytochrome c2 decreased rather abruptly below 260 K and essentially vanished at 230 K. Mechanisms leading to this dramatic impairment are discussed in relation to medium reorganization coupled to electron transfer. A reorganization energy of 25 kJ mol-1 has been estimated for electron donation within the cytochrome c2-reaction center complex.