Water Activity Regulates the QA to QB Electron Transfer in Photosynthetic Reaction Centers from Rhodobacter sphaeroides

We report on the effects of water activity and surrounding viscosity on electron transfer reactions taking place within a membrane protein: the reaction center (RC) from the photosynthetic bacterium Rhodobacter sphaeroides. We measured the kinetics of charge recombination between the primary photoxidized donor (P+) and the reduced quinone acceptors. Water activity (aW) and viscosity (?) have been tuned by changing the concentration of cosolutes (trehalose, sucrose, glucose, and glycerol) and the temperature. The temperature dependence of the rate of charge recombination between the reduced primary quinone, QA -, and P+ was found to be unaffected by the presence of cosolutes. At variance, the kinetics of charge recombination between the reduced secondary quinone (QB -) and P+ was found to be severely influenced by the presence of cosolutes and by the temperature. Results collected over a wide ?-range (2 orders of magnitude) demonstrate that the rate of P+QB - recombination is uncorrelated to the solution viscosity. The kinetics of P+QB - recombination depends on the P+QA -QB T P+QAQB - equilibrium constant. Accordingly, the dependence of the interquinone electron transfer equilibrium constant on T and aW has been explained by assuming that the transfer of one electron from QA - to QB is associated with the release of about three water molecules by the RC. This implies that the interquinone electron transfer involves at least two RC substates differing in the stoichiometry of interacting water molecules. Introduction Proteins are characterized by a complex conformational dynamics. The wide range of internal motions they experience at physiological temperatures originates from rugged energy landscapes, which feature an extremely large number of minima corresponding to different conformational substates, organized in hierarchical tiers.1-3 This ability of the protein to perform structural fluctuations among many different conformational substates appears to be intimately connected to protein function.4,5 The photosynthetic reaction center (RC) from purple bacteria is becoming a paradigmatic system in the study of the relationship between electron transfer processes and protein conformational dynamics. This membrane chromoprotein, following photon absorption by the primary electron donor P (a bacteriochlorophyll dimer), catalyzes a sequential