title: Mutations in the environment of the primary quinone facilitate proton delivery to the secondary quinone in bacterial photosynthetic reaction centers.
abstract: 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 RQ, two acidic residues near QB, L212Glu and L213Asp, have been mutated to Ala; conversely, in the QA pocket, the symmetry-related residues M246Ala and M247Ala have been mutated to Glu and Asp. We have selected photocompetent phenotypic revertants (designated RQrev3 and RQrev4) that carry compensatory mutations in both the QA and QB pockets. Near QA, 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 substitutions remain in the QB site of both revertants but are accompanied by an additional electrostatic-type mutation. To probe the respective influences of the mutations occurring near the QA and QB sites on electron and proton transfer, we have constructed two additional types of strains. First, "half" revertants were constructed that couple the QB site of the revertants with a wild-type QA site. Second, the QA sites of the two revertants were linked with the L212Glu-L213Asp --> Ala-Ala mutations of the QB site. We have studied the electron and proton-transfer kinetics on the first and second flashes in reaction centers from these strains by flash-induced absorption spectroscopy. Our data demonstrate that substantial improvements of the proton-transfer capabilities occur in the strains carrying the M246Ala --> Glu + M247Ala --> Tyr mutations near QA. Interestingly, this is not observed when only the M246Ala --> Glu mutation is present in the QA pocket. We suggest that the M247Ala --> Tyr mutation in the QA pocket, or possibly the coupled M246Ala --> Glu + M247Ala --> Tyr mutations, accelerates the uptake and delivery of protons to the QB anions. The M247Tyr substitution may enable additional pathways for proton transfer that are located near QA.
authors.affiliation: Centre de Génétique Moléculaire, Centre National de la Recherche Scientifique, Gif/Yvette, France.
authors.forename: M
authors.initials: M
authors.lastname: Valerio-Lepiniec
authors.forename: J
authors.initials: J
authors.lastname: Miksovska
authors.forename: M
authors.initials: M
authors.lastname: Schiffer
authors.forename: D K
authors.initials: DK
authors.lastname: Hanson
authors.forename: P
authors.initials: P
authors.lastname: Sebban
mesh_terms: D000409:Alanine / Q000235:genetics; D016227:Benzoquinones / Q000737:chemistry*; D003574:Cytochrome c Group / Q000737:chemistry / Q000378:metabolism; D004579:Electron Transport / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010641:Phenotype; D010782:Photolysis; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011522:Protons; D016354:Rhodobacter capsulatus; D014443:Tyrosine / Q000235:genetics
chemical_list: D016227:Benzoquinones; D003574:Cytochrome c Group; D045322:Photosynthetic Reaction Center Complex Proteins; D011522:Protons; D018698:Glutamic Acid; C004532:quinone; D014443:Tyrosine; D000409:Alanine