In the reaction centers from the purple photosynthetic bacterium Rhodobacter capsulatus, we have determined that residue L212Glu, situated near the secondary quinone acceptor Q(B), modulates the free energy level of the reduced primary quinone molecule Q(A)- at high pH. Even though the distance between L212Glu and Q(A) is 17 Å, our results indicate an apparent interaction energy between them of 30 ± 18 meV. This interaction was measured by quantitating the stoichiometry of partial proton uptake upon formation of Q(A)- as a function of pH in four mutant strains which lack L212Glu, in comparison with the wild type. These strains are the photosynthetically incompetent site-specific mutants L212Glu → Gln and L212Glu-L213Asp → Ala-Ala and the photocompetent strains L212Glu → Ala and L212Ala-L213Ala-M43Asn → Ala-Ala-Asp. Below pH 7.5, the stoichiometry of proton uptake from all strains is nearly superimposable with that of the wild type. However, at variance with the wild type, reaction centers from all strains that lack L212Glu fail to take up protons above pH 9. The lack of a change in the free energy level of Q(A)- at high pH in the L212Glu-modified strains is confirmed by the determination of the pH dependence of the rate (k(AP)) of P+Q(A)- charge recombination in the reaction centers where the native Q(A) is replaced by quinones having low redox potentials. Contrary to the wild-type reaction centers where k(AP) increases at high pH, almost no pH dependence could be detected in the strains that lack L212Glu. Our data show that the ionization state of L212Glu, either on its own or via interactions with closely associated ionizable groups, is mainly involved in the proton uptake at high pH by reaction centers in the PQ(A)- state. This suggests that the formation of the Q(A)- semiquinone state induces shifts in pK(a)s of residues in the Q(B) proteic environment. This long-distance influence of ionization states is a mechanism which would facilitate electron transfer from Q(A) to Q(B) on the first and second flashes. The functional communication between the two quinone protein pockets may involve the iron- ligand complex which spans the distance between them.
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