DISTANT ELECTROSTATIC INTERACTIONS MODULATE THE FREE-ENERGY LEVEL OF Q(A)(-) IN THE PHOTOSYNTHETIC REACTION-CENTER

In the reaction centers from the purple photosynthetic bacterium Rhodo bacter capsulatus, we have determined that residue L212Glu, situated n ear the secondary quinone acceptor QB, modulates the free energy level of the reduced primary quinone molecule Q(A)(-) at high pH. Even thou gh the distance between L212Glu and Q(A) is 17 Angstrom, our results i ndicate an apparent interaction energy between them of 30 +/- 18 meV. This interaction was measured by quantitating the stoichiometry of par tial proton uptake upon formation of Q(A)(-) as a function of pH in fo ur mutant strains which lack L212Glu, in comparison with the wild type . These strains are the photosynthetically incompetent site-specific m utants L212Glu --> Gin and L212Glu-L213Asp --> Ala-Ala and the photoco mpetent strains L212Glu --> Ala and L212Ala-L213Ala-M43Asn --> Ala-Ala -Asp. Below pH 7.5, the stoichiometry of proton uptake from all strain s is nearly superimposable with that of the,wild type. However, at var iance 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 th e free energy level of Q(A)(-) at high pH in the L212Glu-modified stra ins is confirmed by the determination of the pH dependence of the rate (k(AP)) of P(+)Q(A)(-) charge recombination in the reaction centers w here the native QA 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, eit her on its own or via interactions with closely associated ionizable g roups, 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 On proteic environment. This long-distance influence of ionization st ates 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 c omplex which spans the distance between them.