Mutations in the environment of the primary quinone facilitate proton delivery to the secondary quinone in bacterial photosynthetic reaction centers

In Rhodobacter capsulatus, we constructed a quadruple mutant that reversed a structural asymmetry that contributes to the functional asymmetry of the two quinone sites. In the photosynthetically incompetent quadruple mutant R Q, two acidic residues near Q(B), L212Glu and L213Asp, have been mutated to Ala; conversely, in the Q(A) pocket, the symmetry-related residues M246Ala and M247Ala have been mutated to Glu and Asp. We have selected photocompet ent phenotypic revertants (designated RQrev3 and RQrev4) that carry compens atory mutations in both the Q(A) and Q(B) pockets. Near Q(A), the M246Ala - -> Glu mutation remains in both revertants, but M247Asp is replaced by Tyr in RQrev3 and by Ala in RQrev4. The engineered L212Ala and L213Ala substitu tions remain in the Q(B) site of both revertants but are accompanied by an additional electrostatic-type mutation. To probe the respective influences of the mutations occurring near the Q(A) and Q(B) sites on electron and pro ton transfer, we have constructed two additional types of strains. First, " half" revertants were constructed that couple the Q(B) site of the revertan ts with a wild-type Q(A) site. Second, the Q(A) sites of the two revertants were linked with the L212Glu-L213Asp --> Ala-Ala mutations of the Q(B) sit e. We have studied the electron and proton-transfer kinetics on the first a nd second flashes in reaction centers from these strains by flash-induced a bsorption spectroscopy. Our data demonstrate that substantial improvements of the proton-transfer capabilities occur in the strains carrying the M246A la --> Glu + M247Ala --> Tyr mutations near Q(A). interestingly, this is no t observed when only the M246Ala --> Glu mutation is present in the Q(A) po cket. We suggest that the M247Ala --> Tyr mutation in the Q(A) pocket, or p ossibly the coupled M246Ala --> Glu + M247Ala --> Tyr mutations, accelerate s the uptake and delivery of protons to the Q(B) anions. The M247Tyr substi tution may enable additional pathways for proton transfer that are located near Q(A).