Identification of the first steps in charge separation in bacterial photosynthetic reaction centers of Rhodobacter sphaeroides by ultrafast mid-infrared spectroscopy: electron transfer and protein dynamics

Abstract

Time-resolved visible pump/mid-infrared (mid-IR) probe spectroscopy in the region between 1600 and 1800 cm(-1) was used to investigate electron transfer, radical pair relaxation, and protein relaxation at room temperature in the Rhodobacter sphaeroides reaction center (RC). Wild-type RCs both with and without the quinone electron acceptor Q(A), were excited at 600 nm (nonselective excitation), 800 nm (direct excitation of the monomeric bacteriochlorophyll (BChl) cofactors), and 860 nm (direct excitation of the dimer of primary donor (P) BChls (P(L)/P(M))). The region between 1600 and 1800 cm(-1) encompasses absorption changes associated with carbonyl (C=O) stretch vibrational modes of the cofactors and protein. After photoexcitation of the RC the primary electron donor P excited singlet state (P*) decayed on a timescale of 3.7 ps to the state P(+)B(L)(-) (where B(L) is the accessory BChl electron acceptor). This is the first report of the mid-IR absorption spectrum of P(+)B(L)(-); the difference spectrum indicates that the 9-keto C=O stretch of B(L) is located around 1670-1680 cm(-1). After subsequent electron transfer to the bacteriopheophytin H(L) in approximately 1 ps, the state P(+)H(L)(-) was formed. A sequential analysis and simultaneous target analysis of the data showed a relaxation of the P(+)H(L)(-) radical pair on the approximately 20 ps timescale, accompanied by a change in the relative ratio of the P(L)(+) and P(M)(+) bands and by a minor change in the band amplitude at 1640 cm(-1) that may be tentatively ascribed to the response of an amide C=O to the radical pair formation. We conclude that the drop in free energy associated with the relaxation of P(+)H(L)(-) is due to an increased localization of the electron hole on the P(L) half of the dimer and a further consequence is a reduction in the electrical field causing the Stark shift of one or more amide C=O oscillators.

FIGURE 1

Two representative time traces measured using Rb. sphaeroides R26 RCs lacking Q_A, excited at 600 nm, and probed at 1685 cm⁻¹ and 1715 cm⁻¹. The solid lines through the data points are the result of a global fit using a sequential model with time constants of: τ₁ = 120 fs, τ₂ = 3.8 ps, τ₃ = 16 ps, τ₄ = 4 ns, and τ₅= infinite. IRF width = 150 fs FWHM. The timescale is linear up to 3 ps and logarithmic thereafter.

FIGURE 2

EADS of R26 RCs lacking Q_A, excited at 600 nm, resulting from a global analysis using a sequential model with increasing lifetimes. The measurements were carried out over a 1780–1600 cm⁻¹ window with a spectral resolution of 6 cm⁻¹.

FIGURE 3

R26 RCs lacking Q_A excited at 805 nm and probed using a spectral resolution of 3 cm⁻¹. The EADS are the result of a global analysis of the data using a sequential model with increasing lifetimes of 2.6 ps, 7.2 ps, 1.7 ns, and 4 ns.

FIGURE 4

EADS of R26 RCs lacking Q_A calculated from measurements carried out over a broad spectral range between 2280–1600 cm⁻¹. The spectra between 2153–2075 cm⁻¹ are not shown due to the high IR absorption by D₂O in this region. The lifetimes shown are the result of global analysis of the data obtained in 1780–1600 cm⁻¹ region (Fig. 2). Excitation was at 600 nm in each case.

FIGURE 5

R26 RCs with Q_A excited at 600 nm and probed in the region 1775–1590 cm⁻¹. Displayed are EADS resulting from a global analysis using a sequential model with increasing lifetimes of 0.1 ps, 4.4 ps, 222 ps, and a nondecaying component.

FIGURE 6

Scheme for energy transfer and electron transfer in R26 and AM260W RCs. Green arrows (plus associated time constants) refer to forward processes, red arrows to back reactions. Forward electron transfer from the state in RCs with Q_A led to the state, whereas in Q_A-deficient RCs it led to the triplet state of P (³P) and recombination to the ground state.

FIGURE 7

SADS derived from data obtained with R26 RCs lacking Q_A that had been excited at 805 nm. The SADS were produced from a target analysis of all data sets using the kinetic scheme outlined in Fig. 6.

FIGURE 8

Comparison of the P* and spectra resulting from target analysis of R26 RCs. (A) P* state spectra resulting from excitation at 805 nm (black) and 860 nm (red). (B) spectra resulting from excitation at 600 nm (black) and 860 nm (red).

FIGURE 9

Overlaid SADS of R26 RCs. The green spectrum is that shown in Fig. 7 (green), obtained after excitation of RCs not containing Q_A with 805 nm. The black spectrum is the average of the remaining five SADS.

FIGURE 10

Comparison of fs mid-IR spectra and FTIR spectra of radical pair states. (A) Comparison of EADS of the state measured for the samples with (black) and without quinone Q_A (red) excited with 600 nm light and summed FTIR spectra of P⁺ (,–49) and (blue) (43,44) (note that the FTIR spectra were not normalized). (B) Comparison of the EADS of the state (black line) formed after 600 nm excitation and summed FTIR spectra of P⁺ (,–49) and (blue line) (–,,–49).

FIGURE 11

The average of all the (black) and (red) spectra, and difference between them (green). An increase or decrease of an intensity of the indicated bands was marked with arrows directed up or down, respectively.