pubs.acs.org/JPCL
Two-Dimensional Electronic Spectroscopy of the D1-D2-cyt b559 Photosystem II Reaction Center Complex Jeffrey A. Myers,† Kristin L. M. Lewis,† Franklin D. Fuller,† Patrick F. Tekavec,† Charles F. Yocum,‡ and Jennifer P. Ogilvie*,† †
Department of Physics and Biophysics, University of Michigan, Ann Arbor, Michigan 48109-1040, and Department of Molecular, Cellular and Developmental Biology, and Department of Chemistry, The University of Michigan, Ann Arbor, Michigan 48109-1048
‡
ABSTRACT We present two-dimensional electronic spectra of isolated D1-D2cyt b559 reaction centers (PSII RC) at 77 K. We decompose the data into twodimensional decay-associated spectra (2D DAS) to reveal the excitation- and detection-dependent spectral signatures associated with energy and charge transfer within the PSII RC. We interpret the 2D DAS in the context of an exciton model and proposed pathways for charge separation. The 2D DAS reveal a high degree of variation in the kinetics as a function of excitation wavelength. We observe rapid ∼50-150 fs energy equilibration throughout the PSII RC and a heterogeneous distribution of time scales associated with charge transfer, ranging from ∼1-3 ps. We observe 6-8 ps energy transfer from states absorbing near 670 nm, as well as ∼40-60 ps processes consistent with secondary charge transfer from a radical pair intermediate or charge transfer from a trap state. SECTION Kinetics, Spectroscopy
he reaction center of photosystem II (PSII) is the heart of oxygenic photosynthesis, taking absorbed light and creating a charge separation capable of splitting water. Although PSII has been extensively studied over the past couple of decades,1-4 the mechanisms and time scales of the ultrafast energy and charge transfer are not fully understood. Consisting of six chlorophylls (Chl) and two pheophytins (Phe), all with partially or fully overlapping absorption bands, the linear absorption spectrum of the PSII D1-D2-cyt-b559 reaction center (PSII RC) is highly congested, making it difficult to assign energy and charge-transfer roles to individual chromophores. In addition, the chromophores are coupled, requiring an excitonic description. A wealth of ultrafast spectroscopic studies have aimed at understanding the PSII RC, and many of the main results are summarized in review papers.1-3 In an effort to track the flow of energy and charge, transient absorption studies have used relatively narrow band excitation at wavelengths spanning the PSII RC Qy absorption5-7 to reveal distinct excitation-dependent kinetics. However, in transient absorption, excitation selectivity comes at the expense of time resolution. Alternatively, Fourier transform two-dimensional electronic spectroscopy (2DES) simultaneously offers high time and frequency resolution for both excitation and detection frequencies,8 providing a detailed picture of the excitation-dependent dynamics and improved discrimination between overlapping signals. 2DES has been shown to provide an unprecedented view of excitonic structure and energy transfer in the FennaMatthews-Olson complex9,10 and other condensed-phase systems.11-14
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In 2DES, three pulses are incident on the sample. In the time domain picture, the first two excitation pulses are separated by a coherence time t1, followed by a third pulse after a waiting time t2. After the third pulse, a signal is emitted and recorded during a time interval specified by t3. Fourier transforming with respect to t1 and t3 provide the respective excitation (ν1) and detection (ν3) frequency axes of the 2D spectrum for a given waiting time t2. The t2 =0 spectrum, or correlation spectrum, provides information about the inherent coupling in the system and also reveals the degree of inhomogenous broadening. There are a number of exciton models for the PSII RC in the literature.15-20 Here, we perform 2DES on the PSII RC at 77K in the Qy spectral region and discuss our data in the context of the model by Novoderezhkin et al.15 In analogy to global analysis methods frequently used in transient absorption spectroscopy, we derive kinetics for each point in our 2D spectrum to reveal the correlated excitation and detection dependence of energy and charge-transfer processes in the PSII RC. Future work will quantitatively compare our 2D data with simulations based on several exciton models, analyzing the 2D line shapes to discern exciton energies, couplings, static disorder, and homogeneous widths for the different exciton states. In this Letter, we restrict our analysis to the overall kinetics, demonstrating that 2DES provides a detailed spectroscopic picture of the intermediate states involved in Received Date: July 16, 2010 Accepted Date: August 27, 2010 Published on Web Date: September 07, 2010
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DOI: 10.1021/jz100972z |J. Phys. Chem. Lett. 2010, 1, 2774–2780
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in the various exciton states is indicated by the colored squares. At t2 = 28 fs, contributions due to pulse overlap effects are largely complete, and a strong cross peak (CP1) is evident below the diagonal. This broad cross peak represents rapid downhill energy transfer between the higher-energy excitons to excitons 1 and 2. A smaller cross peak located symmetrically across the diagonal is also present (CP2). This cross peak is observed even at shorter times, suggesting coupling between excitons 1 and 2 and excitons 6-9. We note that the close spacing of the exciton levels makes it impossible to unambiguously assign excitonic coupling to all of the exciton pairs. The fact that the PD1 and PD2 pigments are common to all pairs with the exception of exciton 8 strengthens this assignment. Figure 2 shows spectra at t2 = 215 fs, 603 fs, 1.5 ps, and 100 ps, with the spectra normalized to the maximum of the t2 = 28 fs spectrum. By t2 = 215 fs, CP1 has grown substantially in magnitude. The t2 = 603 fs spectrum looks similar with slightly broadened features and an overall decrease in amplitude. At t2 = 1.5 ps, the diagonal feature has shortened as the highest-energy exciton states transfer their energy downhill, strengthening the relative amplitude of CP1 compared to the diagonal. By t2 = 100 ps, the spectra show mainly an elongated feature parallel to the excitation axis, indicating that most population resides in the low-energy exciton states. Between 100 ps and our longest recorded delay of t2 = 150 ps, the data do not change significantly. To date, the analysis of 2DES spectra has generally consisted of making comparisons between experimental and simulated 2DES spectra based on modeling. In a system such as the PSII RC, where both energy and charge-transfer processes lead to spectral changes over a broad range of time scales, appropriate models must correctly predict these spectral changes and their associated kinetics. In transient absorption spectroscopy, global-fitting analysis has been beneficial for extracting kinetics and revealing the spectral features displaying characteristic exponential decays via decay-associated spectra.22 These spectral features help identify intermediate species and motivate different kinetic models. Here, we apply a similar analysis to 2D electronic spectra, producing analogous 2D decay-associated spectra (2D DAS) to reveal the excitation and detection frequency dependence of energyand charge-transfer processes in the PSII RC. As seen in Figure 2, the changes in 2D spectra as a function of waiting time are relatively subtle. The 2D DAS provide a convenient way to extract and visualize these subtle changes. While this method does not provide a substitute for full excitonic modeling of a system such as the PSII RC, it makes kinetic processes easier to discern from dominant, slowly varying background signals and will aid in testing our future simulations of the 2D data based on excitonic models. To construct the 2D DAS, we fit each excitation/detection frequency point in our 2D spectra to a sum of four exponential decay processes, where one of these decays was fixed to 2 ns. Each trajectory contained 66 waiting time values spanning t2 = 0-150 ps. To adjust for amplitude noise due to long-term laser drift and the selection of different focal volumes upon translation of the sample between data sets, we invoked the projection-slice theorem,8 using the transient absorption data
Figure 1. Crystal structure of the PSII RC (3BZ1)21 with colors chosen to illustrate the exciton model of Novoderezhkin15 detailed below. Colored squares are to indicate which pigments (shown with the same color scheme above) participate in the exciton state. Exciton 1 is the additional charge-transfer state P-D1PþD2 and is represented by an orange square. The 77 K, t2 =28 fs 2DES spectrum is shown below, with the location of the exciton states shown as bars with the height scaled to represent the relative dipole strengths.
different kinetic processes and the heterogeneous nature of their kinetics. Figure 1 shows the crystal structure of the PSII RC and a 2DES absorptive spectrum at t2 = 28 fs, with the exciton energies of the Novoderezhkin model superimposed as a grid. These energies are derived from Figure 4 of their paper.15 Their model contains nine exciton states, which we label from 1-9 in order of increasing energy as shown. These states include the eight expected states derived from the site energies and appropriate couplings of the eight pigments as well as an additional charge-transfer state involving the two P680 chlorophylls. The participation of the different pigments
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2775
DOI: 10.1021/jz100972z |J. Phys. Chem. Lett. 2010, 1, 2774–2780
pubs.acs.org/JPCL
Figure 2. 2DES spectra of the PSII RC at 77 K for waiting times of t2 = 215 fs, 603 fs, 1.5 ps, and 100 ps. Superimposed as a grid is the exciton model of Novoderezhkin.15
lifetimes given in the middle column, where positive values correspond to exponential decay processes while negative values correspond to exponential growth. The right column shows the distribution of decay rates for each 2D spectral component, indicating a high degree of heterogeneity in the kinetics. 2D DAS Component (I) (
