We report the observation of two conformational states of closed RCs from Rhodobacter sphaeroides characterized by different P(+)H(A)(-) --> PH(A) charge recombination lifetimes, one of which is of subnanosecond value (700 +/- 200 ps). These states are also characterized by different primary charge separation lifetimes. It is proposed that the distinct conformations are related to two protonation states either of reduced secondary electron acceptor, Q(A)(-), or of a titratable amino acid residue localized near Q(A). The reaction centers in the protonated state are characterized by faster charge separation and slower charge recombination when compared to those in the unprotonated state. Both effects are explained in terms of the model assuming modulation of the free energy level of the state P(+)H(A)(-) by the charges on or near Q(A) and decay of the P(+)H(A)(-) state via the thermally activated P(+)B(A)(-) state.

Transient absorption spectroscopy with a time resolution of approximately 1 ns was applied to study the decay of the primary radical pair P+H- in Rhodobacter sphaeroides R-26 reaction centers with blocked electron transfer from H- to QA. The block in the electron transfer was realized in two ways: by either reducing or removing QA. We found very different kinetics of the P+H- decay in these two cases. Convolution of the multiexponential decay with the instrument response function allowed resolution of as many as three kinetic components of <1-, 3-4-, and 9-12-ns lifetimes in chromatophores with QA reduced and in isolated reaction centers both with QA either reduced or removed (with or without o-phenanthroline) but with variable relative amplitudes. Removing QA or adding o-phenanthroline to isolated reaction centers increased the amplitude of the slowest decay phase relative to that of the fastest phase. On the basis of these observations, we propose that reaction centers adopt three conformational states characterized by different decay kinetics of P+H-. These conformational states appear to be controlled by the charges in the vicinity of the QA site as revealed by the effects of QA reduction and o-phenanthroline-mediated protonation of the sites close to QA.

The kinetics of recombination of the P(+)H(A)(-) radical pair were compared in wild-type reaction centers from Rhodobacter sphaeroides and in seven mutants in which the free energy gap, ΔG, between the charge separated states P(+)B(A)(-) and P(+)H(A)(-) was either increased or decreased. Five of the mutant RCs had been described previously, and X-ray crystal structures of two newly constructed complexes were determined by X-ray crystallography. The charge recombination reaction was accelerated in all mutants with a smaller ΔG than in the wild-type, and was slowed in a mutant having a larger ΔG. The free energy difference between the state P(+)H(A)(-) and the PH(A) ground state was unaffected by most of these mutations. These observations were consistent with a model in which the P(+)H(A)(-) → PH(A) charge recombination is thermally activated and occurs via the intermediate state P(+)B(A)(-), with a mean rate related to the size of the ΔG between the states P(+)B(A)(-) and P(+)H(A)(-) and not the ΔG between P(+)H(A)(-) and the ground state. A more detailed analysis of charge recombination in the mutants showed that the kinetics of the reaction were multiexponential, and characterized by ~0.5, ~1-3, and 7-17 ns lifetimes, similar to those measured for wild-type reaction centers. The exact lifetimes and relative amplitudes of the three components were strongly modulated by the mutations. Two models were considered in order to explain the observed multiexponentiality and modulation, involving heterogeneity or relaxation of P(+)H(A)(-) states, with the latter model giving a better fit to the experimental results.

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In contrast with findings on the wild-type Rhodobacter sphaeroides reaction center, biexponential P+HA− → PHA charge recombination is shown to be weakly dependent on temperature between 78 and 298 K in three variants with single amino acids exchanged in the vicinity of primary electron acceptors. These mutated reaction centers have diverse overall kinetics of charge recombination, spanning an average lifetime from ~2 to ~20 ns. Despite these differences a protein relaxation model applied previously to wild-type reaction centers was successfully used to relate the observed kinetics to the temporal evolution of the free energy level of the state P+HA− relative to P+BA−. We conclude that the observed variety in the kinetics of charge recombination, together with their weak temperature dependence, is caused by a combination of factors that are each affected to a different extent by the point mutations in a particular mutant complex. These are as follows: (1) the initial free energy gap between the states P+BA− and P+HA−, (2) the intrinsic rate of P+BA− → PBA charge recombination, and (3) the rate of protein relaxation in response to the appearance of the charge separated states. In the case of a mutant which displays rapid P+HA− recombination (ELL), most of this recombination occurs in an unrelaxed protein in which P+BA− and P+HA− are almost isoenergetic. In contrast, in a mutant in which P+HA− recombination is relatively slow (GML), most of the recombination occurs in a relaxed protein in which P+HA− is much lower in energy than P+HA−. The weak temperature dependence in the ELL reaction center and a YLH mutant was modeled in two ways: (1) by assuming that the initial P+BA− and P+HA− states in an unrelaxed protein are isoenergetic, whereas the final free energy gap between these states following the protein relaxation is large (~250 meV or more), independent of temperature and (2) by assuming that the initial and final free energy gaps between P+BA− and P+HA− are moderate and temperature dependent. In the case of the GML mutant, it was concluded that the free energy gap between P+BA− and P+HA− is large at all times.Electronic supplementary materialThe online version of this article (doi:10.1007/s11120-016-0239-9) contains supplementary material, which is available to authorized users.