Charge recombination between the oxidized high-potential c-type cytochromes and Q−A in reaction centers from Rhodopseudomonas viridis

Isolated reaction centers from Rhodopseudomonas viridis contain two high-potential c-type cytochromes, cytochrome c-559 and cytochrome c-556, and two low potential c-type cytochromes, cytochrome c-552 and cytochrome c-554. At moderate redox potentials, the two low-potential cytochromes are oxidized and are not identifiably involved in light-induced turnover. Following a flash, in the absence of functional secondary acceptor quinone, QB, the charge separation states, C+H2CH1Q−A and C+H2C+H1Q−A are rapidly created, where CH1 is cytochrome c-559 and CH2 is cytochrome c-556. Decay of these two states occurs via slow intramolecular charge recombinations. The rates of recombination for the two states were distinguishable and were pH and temperature dependent. At pH 9 and 296 K, kappH1 = 8.5 ± 0.5 s−1 for the charge recombination of C+H2C+H1Q−A and kappH2 = 0.95 ± 0.05 s−1 for the charge recombination of C+H2CH1Q−A. It was suggested that the mechanism for decay of both cytochrome c+Q−A states is via repopulation of the P+Q−A state, where P is the primary donor, followed by rapid (ms) recombination of this state, with a rate kP. Recovery of C+H2CH1Q−A also requires thermal equilibrium between the two hemes: C+H2CH1 a3 CH2C+H1. In addition to these indirect paths, there is a direct route of recovery for each of the states, C+H2CH1Q−A and C+H2C+H1Q−A. The direct pathways are evident at low temperature when the recovery processes become almost temperature independent, below about 220 K. In 60% ethylene glycol, the limiting rates are: k0H1 = 1.85 s− for C+H2C+H1Q−A, and k0H2 = 0.15 s−1 for C+H2CH1Q−A. That these rates differ by only a factor of 10 is remarkable in view of the 30 A (center-to-center) separation between the two high-potential hemes and the involvement of intermediate states is briefly considered. From the model, the calculated rates, at any temperature, are given by: kappH1 = (kP + Kck0H1)(1 + Kc) for C+H2C+H1Q−A recombination, and kappH2 = [kP + Kc(k0H1 + Kek0H2)][1 + Kc(1 + Ke)] for C+H2CH1Q−A recombination, where Kc is the equilibrium constant for the positive hole to lie on either P or CH1, and Ke is the equilibrium constant for electron transfer between the two high-potential cytochromes, CH1 and CH2. Equilibrium redox titrations distinguished the two high-potential cytochromes c and revealed their distinct pH and temperature dependences. Kc was taken from the difference in the measured equilibrium midpoint potentials (ΔEm) of the C+H1/CH1 and P+/P couples, and Ke was taken from ΔEm for C+H2/CH2 and C+H1/CH1. The rate of P+Q−A decay, kP, was taken from measurements when all c-type cytochromes were chemically oxidized, and this was used to approximate the rate when the high-potential cytochromes were reduced. All three variable parameters were measured over a range of pH and temperature, and the calculated rates were compared to experimental rates determined under these conditions. The agreement between calculated and measured values was good and provides strong support for the proposed mechanism. The adequacy of equilibrium Em values and the measured value of kP is discussed in terms of possible electrostatic interactions between charged redox centers. It is concluded that such interactions are of minor importance for all relevant parameters in isolated reaction centers.