SYMMETRY-RELATED MUTANTS IN THE QUINONE BINDING-SITES OF THE BACTERIAL REACTION-CENTER - THE EFFECTS OF CHANGES IN CHARGE-DISTRIBUTION

To probe the structural elements that contribute to the functional asy mmetries of the two ubiquinone(10) binding pockets in the reaction cen ter of Rhodobacter capsulatus, we targeted the L212Glu-L213Asp (near Q (B)) and the M246Ala-M247Ala (near Q(A)) pairs of symmetry-related res idues for site-specific mutagenesis. We have constructed site-specific mutants that eliminate the sequence differences at these positions (L 212Glu-L213Asp-->Ala-Ala or M246Ala-M247Ala-->Glu-Asp), and have rever sed that asymmetry by constructing a quadruple-mutant strain, 'RQ' 212 Glu-L213Asp-M246Ala-M247Ala-->Ala-Ala-Glu-Asp). The mutations were des igned to change the charge distribution in the quinone-binding region of the reaction center; none of the strains is capable of photosynthet ic growth. In photocompetent phenotypic revertants of the RQ strain, s econd-site mutations which affect Q(B) function are coupled to mutatio ns in the Q(A) site which restore an Ala or substitute a Tyr at the M2 47 site; one strain carries an additional Met-->Leu substitution at M2 60 near Q(A). All of the RQ revertants retain the engineered M246Ala-- >Glu mutation in the Q(A) site as well as the L212Ala-L213Ala mutation s in the Q(B) site. Kinetic characterization of the RQ revertants will give us an idea of what structural and functional elements are import ant for restoring efficiency to electron and proton transfer pathways in the RQ RC, which is far from native. To date, these preliminary res ults underscore the importance of an asymmetric distribution of polar amino acids in the quinone binding pockets and its influence on the fu nctional properties of the reaction center.