Symmetry-related mutants in the quinone binding sites of the bacterial reaction center – the effects of changes in charge distribution

Abstract

To probe the structural elements that contribute to the functional asymmetries of the two ubiquinone₁₀ binding pockets in the reaction center of Rhodobacter capsulatus, we targeted the L212Glu–L213Asp (near Q_B) and the M246Ala-M247Ala (near Q_A) pairs of symmetry-related residues for site-specific mutagenesis. We have constructed site-specific mutants that eliminate the sequence differences at these positions (L212Glu–L213Asp→Ala-Ala or M246Ala–M247Ala→Glu-Asp), and have reversed that asymmetry by constructing a quadruple-mutant strain, ‘RQ’ (L212Glu–L213Asp-M246Ala–M247Ala→Ala-Ala-Gl u-Asp). The mutations were designed to change the charge distribution in the quinone-binding region of the reaction center; none of the strains is capable of photosynthetic growth. In photocompetent phenotypic revertants of the RQ strain, second-site mutations which affect Q_B function are coupled to mutations in the Q_A site which restore an Ala or substitute a Tyr at the M247 site; one strain carries an additional Met→Leu substitution at M260 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 mutations in the Q_B site. Kinetic characterization of the RQ revertants will give us an idea of what structural and functional elements are important for restoring efficiency to electron and proton transfer pathways in the RQ RC, which is far from native. To date, these preliminary results underscore the importance of an asymmetric distribution of polar amino acids in the quinone binding pockets and its influence on the functional properties of the reaction center.