Antagonistic Effects of Point Mutations on Charge Recombination and a New View of Primary Charge Separation in Photosynthetic Proteins

Light-induced electron-transfer reactions were investigated in wild-type and three mutant Rhodobacter sphaeroides reaction centers with the secondary electron acceptor (ubiquinone QA) either removed or permanently reduced. Under such conditions, charge separation between the primary electron donor (bacteriochlorophyll dimer, P) and the electron acceptor (bacteriopheophytin, HA) was followed by P+HA– → PHA charge recombination. Two reaction centers were used that had different single amino-acid mutations that brought about either a 3-fold acceleration in charge recombination compared to that in the wild-type protein, or a 3-fold deceleration. In a third mutant in which the two single amino-acid mutations were combined, charge recombination was similar to that in the wild type. In all cases, data from transient absorption measurements were analyzed using similar models. The modeling included the energetic relaxation of the charge-separated states caused by protein dynamics and evidenced the appearance of an intermediate charge-separated state, P+BA–, with BA being the bacteriochlorophyll located between P and HA. In all cases, mixing of the states P+BA– and P+HA– was observed and explained in terms of electron delocalization over BA and HA. This delocalization, together with picosecond protein relaxation, underlies a new view of primary charge separation in photosynthesis.

. Comparison of the 5.5-ps and 160-ps DADS for the AMW RC normalized at ~542 nm.

Comparison of 6-and 7-exponential global fits for the ELL sample
The corresponding 570/590-ps DADS and 4.2/4.5-ns DADS for the ELL RC are of very similar shapes for the 6-and 7-exponential global fits (compare spectra in Figs. S3 and 2C). An extra component of ~75-ps lifetime in the 7-exponential fit caused shortening of the 3.2-ps and 17-ps components (resolved in the 6-exponential fit) to 2.2 and 5.7 ps, respectively (in the 7-exponential fit). Moreover, introduction of the ~75-ps DADS resulted in the shape and lifetime of the 2.2-ps DADS being very similar to those resolved for the three remaining RCs (Fig. 3A). Estimation of the relative contribution of the states P + BAand P + HAwithin the compartment "(P + HA -)1" In the following we describe the way in which we estimated the relative contributions of the states P + BAand P + HAwithin the compartment "(P + HA -)1" in the ELL/AMW RC. Estimations for the remaining RCs were made in an analogous way.
We assumed that the (P + HA -)2 SADS in Fig. S4 represents pure P + HAstate. Consequently, we assumed that the negative band of this SADS at ~540 nm is exclusively due to photobleaching of the HA Qx band whereas the negative band at ~600 nm is exclusively due to photobleaching of the P Qx band. From the relative amplitudes of these bands, ΔAH and ΔAP, using the Lambert-Beer law, we estimated the ratio of differential molar extinction coefficients of these two molecules at the respective wavelengths: ΔεH,540/ΔεP,600 = ΔAH/ΔAP = 1.17. (S1) The method for determining the amplitudes ΔAH and ΔAP shown in Fig. S4 is somewhat arbitrary but we cannot see any clear advantage of different methods to estimate these quantities.
Next we assumed that the "(P + HA -)1" SADS in Fig. S4 represents a mixture of the states P + HAand P + BA -, and that the differential molar extinction coefficient of BA at ~600 nm, ΔεB,600, is the same as that of P at ~600 nm and, for simplicity, assumed to be equal to 1 (arbitrary unit): ΔεB,600 = ΔεP,600 = 1.
We also assumed that the relaxation state of the protein does not affect the values of ΔεH,540 and ΔεP,600, meaning that they are the same for the compartments "(P + HA -)1" and (P + HA -)2.
Under these assumptions we could estimate the relative concentrations cPH and cPB (arbitrary unit) of the two states contributing to the "(P + HA -)1" compartment, P + HAand P + BA -, respectively, from the following set of linear equations obtained from the Lambert-Beer law: -at ~540 nm cPH ΔεH,540 = ΔAH, -at ~600 nm c PH Δε P + c PB (Δε B + Δε P ) = ΔA, where ΔA H and ΔA are amplitudes of the ~540 and ~600-nm bands of the "(P + H A -) 1 " SADS, as shown in Fig. S4.
Finally, we estimated the free energy gap, ΔG1, between the states P + HAand P + BAcontributing to the compartment "(P + HA -)1": where k stands for a Boltzmann constant and T -an absolute temperature (kT ≈ 25 meV).
In the case of WT sample, due to larger amplitude of "(P + HA -)1" SADS at ~540 nm than that of the (P + HA -)2 SADS, the latter one was multiplied by the arbitrarily selected correction factor of 1.3, the minimal value necessary to obtain consistent results. Figure S4. Graphical illustration on the determination of energetic parameters from the negative SADS bands amplitudes associated with the states "(P + HA -)1" and (P + HA -)2 of the ELL/AMW RC (spectra redrawn from Fig. 5D). ΔAHamplitude of the signal at ~540 nm ascribed to photobleaching of the HA Qx band; ΔAPamplitude of the signal at ~600 nm ascribed to photobleaching of the P Qx band. See text for further details.