A long living charge separated state in photosynthetic Reaction Center in negatively charged proteoliposomes

Reaction centers from the purple bacterium Rhodobacter sphaeroides strain R-26.1 were purified and reconstituted in proteoliposomes formed by the anionic phospholipids phosphatidylglycerol, phosphatidylserine and phosphatidylinositol and by the zwitterionic phospholipid phosphatidylcholine by using the Micelle-to-Vesicle Transition technique coupled with size-exclusion chromatography. The proteoliposomes were prepared either in the dark or under white light illumination. We report the large stabilizing effect induced by anionic phospholipids on the protein charge separated state which results trapped in a long-living (up to tens of minutes) state with a yield up to 80%. The long living charge separated state (LLCS) is fully reversible and is formed in oxygenic conditions regardless the presence of the secondary quinone QB and its lifetime and relative yield increase at low pH. In proteoliposomes formed with QA-depleted RCs the protein results very light-sensitive and the long living charge separated state is not observed. The data collected in negatively charged proteoliposomes are discussed in terms of the electrostatic effect on the primary quinone acceptor and compared with similar long living species reported in literature and obtained in anionic, zwitterionic and non ionic detergents