Mechanism of Recombination of the P⁺H_A^– Radical Pair in Mutant Rhodobacter sphaeroides Reaction Centers with Modified Free Energy Gaps Between P⁺B_A^– and P⁺H_A^–

Abstract

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.

Primary Electron Transfer Reactions in Membrane-Bound Open and Closed Reaction Centers from Purple Bacterium Rhodobacter sphaeroides

“…On the other hand, the non-decaying component may have a contribution from non-resolved phases of ≈ 3 and ≈ 12 ns measured for both samples using a slower experimental setup with a much wider temporal window [5,9]. For mbRCs, the amplitudes of both phases were similar to one another (see signals at ≈ 545, ≈ 600, and ≈ 665 nm in Fig.…”

“…5). On the other hand, the relative contributions of fast and slow charge recombination phases depends also on the rate of P + H − A energetic relaxation, the process which was proposed to be responsible for the multiphasic P + H − A → PH A charge recombination [8,9] (Fig. 5).…”

Hopping maps for photosynthetic reaction centers

“…Our map for H L − → B L → P + charge recombination indicates that hopping does not occur (Figure 5), which is hard to understand since it is known that the H L /B L energy gap affects the rate of recombination, as noted above [42]. Hopping is predicted to occur in maps constructed with overall λ > 1 eV, but this is not realistic since reorganization should be the same in forward and reverse directions.…”

“…1 and 2 predict a 300 ps time constant for driving-force-optimized ET, which requires a reorganization energy of 1.1 eV, far greater than for forward ET (≤ 0.2 eV). Several studies suggest that charge recombination does not occur by a simple single-step mechanism [42], and protein conformation could modify the relative energy levels of P, B L and H L [43]. In WT PRCs charge recombination from the P + H L is suggested to at least partially proceed through an activated state resembling P + B L − .…”

“…In WT PRCs charge recombination from the P + H L is suggested to at least partially proceed through an activated state resembling P + B L − . Substituting amino acids near the pigments in the L-branch modulates the relative reduction potentials of H L and B L and thus affects the kinetics of charge recombination [42]. When H L and B L are energetically close (Δ G ° ~ 0), charge recombination is about 10 times more rapid.…”