B-side Electron Transfer in Bacterial Photosynthetic Reaction Centers Revealed by a Few-Cycle Pulse Laser

B-side electron transfer in wide-type reaction centers from Rhodobacter sphaeroides 2.4.1 was investigated by broadband real-time vibrational spectroscopy. HB ̄ was found to be generated in less than 50 fs after the excitation of 6.3 fs laser pulse, and its decay lifetime was determined to be ~280fs and 1.4ps, which is extremely fast compared with the one occurring in A-side. Considering the ultrafast of the generation time of H B BB ̄, BB rather than P was found to be the initial electron donor to the H. Hence, the initial charge-separated state is determined to be B B


Introduction
The symmetric structure of the reaction center (RC) provides two possible electron-transfer pathways.However, the functional asymmetry has been discovered and only the A-branch is active in charge transfer to form P⁺H A ˉ as the initial charge-separated state when the primary electron donor (P) is excited.The function of the B-branch cofactor is not yet clearly understood and only limited research result has been reported [1][2].According to these previous studies, the B-branch function is very complicated and it electron transfer pathway is discovered to be dependent on the excitation wavelength, i.e. dependent on whether P or B (a monomer bacteriochlorophyll) or H (a bacteriopheophytin) is excited or to which excited state (Q y , Q x , or Soret band) they are excited.
In the present study, we utilize the ultrashort laser pulse at visible range to investigate the kinetic of B-side electron transfer and properties of the initially generated charge pair state.B B was found to act as the initial electron donor rather than P. Electron transfer takes place from B B B B to H B in less than 50 fs, and decay components of the charge-separated B B B B ⁺H B ¯ state are determined to be ~280fs and 1.4ps, which is extremely fast compared with the one occurring in A-side.

B 2 Experimental method and material
Both pump and probe pulses (7.2 fs, 525nm to 723nm) were generated from a noncollinear optical parametric amplifier laser system [3].All the experiment was performed at a constant temperature EPJ Web of Conferences (293K).RCs from Rhodobacter sphaeroides 2.4.1 were prepared as described previously [4].The anaerobically grown wide-type (WT) RCs contain spheroidene.RCs were suspended in a buffer solution of 20mM Tris-HCl (PH 8.0) with 0.1% N, N-Dimethyldodecylamine-N-oxide.Fig. 1a shows the two-dimensionally plotted difference absorption spectra.In Fig. 1b, we plot the probe photon energy dependence of time-resolved spectra from 75 to 1775 fs with an integration width of 50 fs.A negative signal at 595 nm is due to the ground-state bleaching of P and/or B. There is a strong photon induced absorption (PIA) bands located around 630 nm.According to previous studies, this PIA signal is attributed to formation of the H B anion [1][2].Then what is the electron donor for this charge separation?As shown in Fig. 1b, H B B B ¯ is formed just after the excitation of fewcycle visible laser (shorter than 50 fs), which is much faster than the one generated under excitation at 390nm with laser pulse duration of hundred femtosecond [1].Thus, the P⁺ seems unlikely to be the initial electron donor, because the formation of P⁺ from P* usually occurs on the 1~2 ps time scale, which is too slow to explain the ultrafast electron transfer to H B .Because we excited the Q B x state of both B and P, considering the proximity of B B B to H B , the B B B B is much more likely to be the initial electron donor.Thus the corresponding charge-separated state initially formed can be assigned to be BB B ⁺H B B ¯, which is similar with the one excited under 390 nm [1][2].Femtosecond decay component of 280fs and picosecond one of 10ps (maximum delay time is 50ps) have been determined for the B B ⁺H B B B ¯ state.The advantage of real-time vibrational spectroscopy is that both the electronic and vibrational dynamics can been observed at the same time under exactly same experimental condition.To gain a 08012-p.2better understanding of the vibronic coupling mechanisms, the vibrational mode frequencies have been realized by the fast Fourier transform (FFT) analysis of ΔA, as shown in Fig. 2. The vibrational mode with frequency of 1239 cm -1 can uniquely refer to the cis nature of the 15,15' carbon-carbon double bond as characteristic for spheroidene in the RC [5], which provides a fingerprint by which the carotenoid molecule can be identified.Therefore, it is reasonable to assign the vibrational mode at 1240 cm -1 observed in the range of 570-590nm in Fig. 2 due to the carotenoid.Thus, the one located at 1530 cm -1 and 1162 cm -1 can be attributed to C=C and C-C stretching in the carotenoid, respectively.However spheroidene seems can not be excited directly, the observed signal may be explained by the energy transfer from B B to carotenoid S B 1 state during ~50fs, which has been proved to be an effective path in purple-bacterial photosynthetic core antenna [6].

Conclusion
Electron at the B-side is found to transfer from B B to H B B B in less than 50 fs after the excitation using femtosecond real-time vibrational spectroscopy.Decay components of the initial charge-separated BB B ⁺H B B ¯ state are determined to be 280fs and 1.4ps, which is extremely fast in contrast to the processes in A-side.Such short formation and decay time of the electron transfer may arise from the requirement of preventing the photodamage in RCs.Since the A-branch cofactor absorbs the infrared light to undergo electron transfer to convert the light to chemical potential energy, meanwhile, the Bbranch will absorb the light at residual range, i.e., the visible and/or UV light, to avoid the photon damage of these light to the A-branch cofactors and guarantee the successful electron transfer.

Fig. 1 .
Fig. 1.(a) Two-dimensional (probe photon energy versus probe delay time) pseudo-color display of the time dependence of the absorbance changes ΔA.(b) The time-resolved difference absorption spectrum probed at delays from 75 to 1775 fs.

Fig. 2 .
Fig. 2. Two-dimensional contour map of FT amplitude spectra of the pump-probe signal.