Heterogeneity of kinetics and electron transfer equilibria in the bacteriopheophytin and quinone electron acceptors of reaction centers from Rhodopseudomonas viridis

The decay of the charge separated state, P + Q A Q − B , produced in a flash, was measured in reaction centers from Rhodopseudomoas viridis supplemented with ubiquinone to reconstitute secondary quinone (Q B ) activity. The decay occurs by recombination via the state P + Q − A Q B which has an inherent recombination rate constant ( k QA of about 10 3 s −1 . The observed rate of decay of P + Q A Q − B is therefore controlled by the electron transfer equilibrium ( K 2 ) between P + Q A Q − B and P + Q − A Q B : k QB = k QA [1 + K 2 −1 . The decay of P + Q A Q − B was biphasic in the same manner as previously found for the much faster recombination of P + Q − A in reaction centers lacking functional Q B (Sebban, P. and Wraight, C.A. (1989) Biochim. Biophys. Acta 974, 54–65). The decay kinetics of both processes were well fit by sums of two exponential components with rate constants that differed by a factor of about 5. Comparison of the fast phase of P + Q A Q − B decay ( k fast QB ) with the fast phase of P + Q − A decay ( k fast QA ), or of k slow QB with k slow QA , yielded very similar values for K 2 over a wide range of pH, temperature and salt concentration. Thus, the source of the heterogeneity in the recombination kinetics does not significantly perturb the energetics of the Q A to Q B electron transfer equilibrium. As a function of pH, K 2 decreased from a value of about 10 3 at pH 5 to a minimum of 10 2 at pH 8. It then increased to about 250 at pH 10, before decreasing again at higher pH. This complex behavior was satisfactorily described by the influence of four independent ionizable groups with differential effects on the stability of the P + Q − A Q B and P + Q A Q − B states, i.e., p K QA − ≠ p K QB − . The temperature dependence of K 2 , at pH 8.5, revealed Δ H ° = −0.35 eV and ΔS° = −0.75 meV/deg (- TΔS ° = 0.22 eV at 296 K). This large entropy change is most likely related to proton binding events accompanying the electron transfer. The salt dependence of K 2 was substantial due to opposing effects on the recombination rates: k QB decreased with increasing salt concentration, while k QA increased. This is interpreted as reflecting the influence of surface pH and solvent dielectric on the species I/I − , Q A /Q − A and Q B /Q − B . The relative amplitudes of the fast and slow components of the P + Q A Q − B recombination were also dependent on pH, temperature and salt concentration and followed closely the heterogeneity of the P + Q − A recombination. The source of the components is ascribed to a slow, proton-linked equilibrium between two conformational states of the protein (conformers), C f C s , that differ in their P + Q − A recombination rates ( k fast QA and k slow QA ). The equilibrium between the states is established before the flash and readjusts, after the flash, in a few seconds — a time scale longer than the P + Q A Q − B lifetime (≤1 s). The conformational equilibrium constant, K cs , varied with pH in a complex way that roughly correlated with the pH dependence of K 2 , suggesting that the quinone electron transfer equilibrium and the interconversion of the two conformers (fast and slow) may be sensitive to some of the same protonation events. The temperature dependence of K cs was slight but corresponded to ΔH° = −60 meV and ΔS° = −0.17 meV/deg (− TΔS ° = 50 meV at 296 K). It is suggested that the fast and slow conformers may differ in the energy gap between P + Q − A and P + I − , but differences in the intrinsic decay rate of P + I − may also contribute. If the source of the heterogeneity is energetic, the major contribution to the variation is likely to be the energy level of P + I − . Variability in the kinetic behavior was observed in different reaction center preparations, some exhibiting rates of P + Q − A recombination (both phases) and Cyt c + Q − A decay that are 2−2.5-fold faster, without any corresponding acceleration of the P + Q A Q − B decay rates. The origin of this effect is unknown but it is suggested that it reflects an increase in the free energy level of the P + Q − A state by 20–25 meV, simultaneously decreasing the gap between P + Q − A and P + I − and increasing that between P + Q − A Q B and P + Q A Q − B .