STABILIZATION OF REDUCED PRIMARY QUINONE BY PROTON UPTAKE IN REACTIONCENTERS OF RHODOBACTER-SPHAEROIDES

Proton binding stoichiometry and kinetics of charge recombination were measured after single flash excitation in reaction centers from the p urple photosynthetic bacterium, Rhodobacter sphaeroides strain R-26, w here the native ubiquinone in the primary quinone acceptor site Q(A) w as removed and replaced by (benzo-, naphtho-, and anthra-) quinones of various structures and redox midpoint potentials. The observed proton binding stoichiometry was small (0.2-0.4 H+/Q(A)(-)) and not specific to quinones in the acidic and neutral pH ranges. Above pH 9, however, significant differences were detected; reaction centers reconstituted by menadione failed to take up protons above pH 9.5. The pH dependenc e of the free energy change (stabilization) of the semiquinone was det ermined by integration of the proton uptake stoichiometry as a functio n of pH. Ubiquinone had the largest (100 meV at pH 5) and menadione th e smallest (49 meV at pH 5) stabilization energy compared to those at very high (> 11) pH. In the case of the anthraquinone-reconstituted re action center, acceptable agreement was obtained above pH 9 for the st abilization energies derived from energetic parameters of the thermall y activated electron transfer (back reaction) and from proton binding stoichiometries. The stabilization at high pH could be attributed to a single protonatable amino acid, which might be either in the Q(B) (se condary quinone) pocket (Glu L212) or in the vicinity of the Q(A) bind ing domain (Tyr H40). It was shown that this residue had a negligible energy of interaction with bacteriopheophytin and that its coupling to the semiquinone was sensitive to the structure and physicochemical pr operties of Q(A). The possibilities are discussed in terms of long-ran ge electrostatic interactions and solvent accessibility based on the t hree-dimensional structure of the reaction center.