Characterization of a semi-stable, charge-separated state in reaction centers from Rhodobacter sphaeroides

In reaction centers from Rhodobacter sphaeroides, subjected to continuous illumination in the presence of an inhibitor of the Q(A) to Q(B) electron transfer, the oxidation of P870 consisted of several kinetic phases with a fast initial reaction followed by very slow accumulation of P870(+) with a halftime of several minutes. When the light was turned off, a phase of fast charge recombination was followed by an equally slow reduction of P870+. In reaction centers depleted of Q(B), where forward electron transfer from Q(A) is also prevented, the slow reactions were also observed but with different kinetic properties. The kinetic traces of accumulation and decay of P870(+) could be fitted to a simple three-state model where the initial, fast charge separation is followed by a slow reversible conversion to a long-lived, charge-stabilized state. Spectroscopic examination of the charge-separated, semi-stable state, using optical absorbance and EPR spectroscopy, suggests that the unpaired electron on the acceptor side is located in an environment significantly different from normal. The activation parameters and enthalpy and entropy changes, determined from the temperature dependence of the slow conversion reaction, suggest that this might be coupled to changes in the protein structure of the reaction centers, supporting the spectroscopic results. One model that is consistent with the present observations is that reaction centers, after the primary charge separation, undergo a slow, light-induced change in conformation affecting the acceptor side.