Stabilization of reduced primary quinone by proton uptake in reaction centers of Rhodobacter sphaeroides.

Proton binding stoichiometry and kinetics of charge recombination were measured after single flash excitation in reaction centers from the purple photosynthetic bacterium, Rhodobacter sphaeroides strain R-26, where the native ubiquinone in the primary quinone acceptor site QA was 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+/QA-) 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 dependence of the free energy change (stabilization) of the semiquinone was determined by integration of the proton uptake stoichiometry as a function of pH. Ubiquinone had the largest (100 meV at pH 5) and menadione the smallest (49 meV at pH 5) stabilization energy compared to those at very high (> 11) pH. In the case of the anthraquinone-reconstituted reaction center, acceptable agreement was obtained above pH 9 for the stabilization energies derived from energetic parameters of the thermally 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 QB (secondary quinone) pocket (Glu L212) or in the vicinity of the QA binding 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 properties of QA.(ABSTRACT TRUNCATED AT 250 WORDS)