Time-resolved and Steady-state Spectroscopic Analysis of Membrane-bound Reaction Centers from Rhodobacter Sphaeroides: Comparisons with Detergent-solubilized Complexes

The spectroscopic analysis of the antenna-deficient Rhodobacter sphaeroides strain RCO1 has been extended to an investigation of the kinetics and spectroscopy of primary charge separation. Global analysis of time-resolved difference spectra demonstrated that the rate of charge separation in membrane-bound reaction centers is slightly slower than in detergent-solubilized reaction centers from the same strain. A kinetic analysis of the decay of the primary donor excited state at single wavelengths was carried out using a high repetition rate laser system, with the reaction centers being maintained in the open state using a combination of phenazine methosulfate and horse heart cytochrome c. The kinetics of primary charge separation in both membrane-bound and solubilized reaction centers were found to be non-monoexponential, with two exponential decay components required for a satisfactory description of the decay of the primary donor excited state. The overall rate of charge separation in membrane-bound reaction centers was slowed if the primary acceptor quinone was reduced using sodium ascorbate. This slowing was caused, in part, by an increase in the relative amplitude of the slower of the two exponential components. The acceleration in the rate of charge separation observed on removal of the reaction center from the membrane did not appear to be caused by a significant change in the electrochemical properties of the primary donor. The influence of the environment of the reaction center on primary charge separation is discussed together with the origins of the non-monoexponential decay of the primary donor excited state.