Article pubs.acs.org/JPCB
Energy Transfer Pathways in Light-Harvesting Complexes of Purple Bacteria as Revealed by Global Kinetic Analysis of Two-Dimensional Transient Spectra Evgeny E. Ostroumov,† Rachel M. Mulvaney,‡ Jessica M. Anna,† Richard J. Cogdell,‡ and Gregory D. Scholes*,† †
Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario, M5S 3H6, Canada Glasgow Biomedical Research Centre, IBLS, University of Glasgow, 126 Place, Glasgow G12 8TA, Scotland, U.K.
‡
S Supporting Information *
ABSTRACT: Excited state dynamics in LH2 complexes of two purple bacterial species were studied by broad-band two-dimensional electronic spectroscopy. The optical response was measured in the 500−600 nm spectral region on the 0−400 fs time scale. Global target analysis of two-dimensional (2D) transient spectra revealed the main energy transfer pathways between carotenoid S2, 1Bu− and S1 states and bacteriochlorophyll Qx state. Global analysis ascertained the evolutionary and vibration-associated spectra, which also indicated the presence of a higher-lying vibrational level in the carotenoid S1 state. The estimation of the spectral overlap between the 1Bu− state and the Qx state indicated a significant contribution of the 1Bu− state to the overall S2-to-Qx excitation energy transfer.
1. INTRODUCTION Studies of electronic energy transfer mechanisms are influencing how researchers are designing assemblies of chromophores for harvesting light.1 Bioinspiration has been the backbone of much of this work.2−14 In that respect, studies of the light harvesting complexes of photosynthetic organisms have been important.15,16 An especially detailed body of work has been carried out on the major light harvesting complex LH2 from purple bacteria (see Figure 1A).17−22 These complexes contain two types of chromophores: bacteriochlorophylls (BChl) and carotenoids (Car). The two lowest singlet states of BChls, Qx and Qy, absorb in visible (590 nm)23 and near-IR (800/850 nm)24 spectral regions (Figure 1B). The transition into the higher lying Car 1Bu+ (S2) excited state in 400−500 nm region has a strong dipole transition moment, while the transition to the lowest Car 2Ag− (S1) excited state is symmetry forbidden and therefore Car S1 is often referred to as a “dark” state.25−28 Carotenoids are incredible molecules. In photosynthetic light harvesting complexes, they play a role in dissipating excess excitations (nonphotochemical quenching),29−32 they function to photoprotect the proteins by quenching chlorophyll triplet and singlet oxygen species,33−37 they contribute to light harvesting,38−47 and in some cases are used for heat transduction.48 Recently we studied the light harvesting by carotenoids in LH2 complexes of two purple bacterial species and concluded that the excitation was transferred from the Car S2 state to the BChl Qx state via a dark Car state lying below S2.49 The dark carotenoid states are a subject of extensive and contentious research, both theoretical and experimental.50−62 © XXXX American Chemical Society
The experimental observations of the intermediate dark states are not broadly accepted because no well-resolved absorption (or bleach) nor fluorescence features have been reported. The presence of the dark states was deduced from the excited state absorption signals occurring on a time scale close to the temporal resolution of the experiments.54,56,57,63 In the recent work we took advantage of the combined high frequency and time resolution gained in Fourier transform two-dimensional electronic spectroscopy (2DES).64,65 Using 2DES, the intermediate excited X state was directly observed via its ground state bleach (GSB) signal in both LH2 complexes and in isolated carotenoids.49 It was shown that the signature frequencies of carotenoid ground state vibrational modes are excited via both Car S2 and X states, confirming that the X feature is due to a carotenoid excited state, most likely the 1Bu− state, predicted by Tavan and Schulten.50 Here we report 2D electronic spectroscopic studies of exceptionally fast electronic energy transfer (EET) from the S2 and X electronic states of Car molecules to the Qx state of the BChl molecules in LH2 complexes of purple bacteria Rhodopseudomonas acidophila and Rhodobacter sphaeroides. The focus of this report is how to analyze the 2D spectroscopic data to resolve quantitative information about the EET kinetics and the Special Issue: Rienk van Grondelle Festschrift Received: March 27, 2013 Revised: June 11, 2013
A
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Figure 1. Molecular structure of the LH2 complex of Rps. acidophila and distances between neighboring carotenoid-bacteriochlorophyll pair (A). Absorption spectrum and electronic level structure of LH2 complexes of Rps. acidophila (B). Red color indicates carotenoid; green color indicates bacteriochlorophyll.
2.2. 2D Electronic Spectroscopy. The 2DES experimental setup is described in detail elsewhere.79 Briefly, a regenerative amplifier (Spitfire, Spectra-Physics) seeded by Ti:sapphire oscillator generates 150 fs pulses at 800 nm. A home-built NOPA converts the 1.5 W of 800 nm pulses into 10 mW broadband pulses (30) with varied starting values of the system parameters.
scheme with kinetic rates as global variables. The oscillatory contribution was modeled by a set of frequency modes which were generated by simulating the Fourier spectrum with Gaussian functions and wavelength-dependent phase factors. The phase factor of each oscillation mode was fitted for each excitation/detection wavelength separately (independently) within the global analysis (see Figure S2). We found this analysis algorithm with combined global variables and wavelengthdependent phases to be less time-expensive and a more efficient fitting process as compared to implementation of a 2D function for describing the wavelength-dependent oscillation phases. The calculated oscillations were transformed into the timedomain and treated as “coherent” species in addition to incoherent population species. As a result, 2D species-associated spectra were obtained for both exponentially decaying population species (species-associated spectra, 2DSAS) and coherent oscillatory modes (vibration-associated spectra, 2DVAS). Several kinetic schemes were tested in the analysis of the incoherent dynamics. The evolutionary kinetic scheme produced the most meaningful and detailed spectra (2D evolutionary-associated spectra, 2DEAS). For discussion of the kinetic scheme selection procedure see Ostroumov et al.49 The analysis was applied to the absorptive data, as well as to absorptive data combined with rephasing and nonrephasing data. In the latter case, the parameters were kept global for all three data sets. The resulting 2DEAS and fit quality for the absorptive data did not differ significantly, indicating that dispersive
3. RESULTS AND DISCUSSION 3.1. Two-Dimensional Spectra. 2D spectra of the absorptive, rephasing and nonrephasing signals from Rps. acidophila and Rba. sphaeroides at selected population times are shown in Figures 3 and 4. These spectra reveal signals from at least four excited states: S2, X, S1 and Qx. In recent work it was shown that the X state most probably corresponds to the excited electronic 1Bu− state of carotenoids,49 predicted theoretically by C
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Figure 4. 2D spectra of Rba. sphaeroides taken at selected t2 delays (indicated in each panel): absorptive (A−C), rephasing (D−F), and nonrephasing (G−I).
Tavan and Schulten.50,51 The oscillation frequencies of the X feature allow it to be identified unambiguously as a carotenoid state, and the ratio of the amplitudes of these frequency modes (Fourier spectrum) indicates that the X feature belongs to an excited state different from the lower lying S1 state (see discussion below). In the presented data, the S2, X, and Qx states are observed via their GSB and stimulated emission (SE) signals (positive peaks), while the S1 state is observed only via its excited-state absorption (ESA) transition to a higher lying state SN (negative peaks).25,87−91 In the spectra, taken at various t2 delays, shown in Figures 3 and 4, a substantial decrease of the amplitude of GSB and SE signals from S2, X and Qx can be observed as a function of t2, while the amplitude of the negative ESA signal, attributed to S1 state, increases as t2 increases. This observation is in agreement with the literature, since it is known that the higher lying states (S2, X, Qx) decay on the ∼100 fs time-scale populating the lower energy S1 state,38,91−96 which has lifetimes of 1.2 and 3.3 ps in LH2 complexes of Rba. sphaeroides and Rps. acidophila, respectively.72 In the current work, the time-range of the experiments was limited to t2 = 400 fs; for this reason no contribution from the vibrationally relaxed S1 state is present in the data. We rather detect the decay of the signals from high lying states and rise of the signals from the vibrationally excited S1 state. The t2 time traces taken at points lying along the diagonal of the absorptive 2D spectra are shown in Figure 5A,B. The decay of the positive
amplitude can be readily observed in these time traces; however, the magnitude and rate of this incoherent decay of the excited states can only be estimated qualitatively owing to presence of strong coherent oscillations. The oscillations originate from the carotenoid ground state vibrational modes as follows from the Fourier spectra of the oscillatory contribution. Indeed the three main frequencies observed in the high-frequency region (Figure 5C,D) are signatures of the carotenoid ground state.25,49,93,97 In Figure 5C,D, the Fourier transformed data taken from traces of the diagonal peaks corresponding to S2 and X (red and blue lines) reveal almost identical profiles, confirming that X is a carotenoid state. In contrast, the Fourier spectrum sampled at the Qx peak consists mostly of the 1600 cm−1 mode. Since BChl does not have significant ground state vibrational modes in the high frequency range except for a weak 1340 cm−1 mode,98,99 the 1600 cm−1 mode observed at the Qx peak coordinate is likely due to the carotenoid as well. This assignment becomes obvious from the 2D maps of Fourier transformed data in Figure S5. These 2D maps were obtained by extraction of the exponential contribution from the data and integration over a 1000−1700 cm−1 frequency range. The four peaks in the 2D Fourier maps are due to excitation of the carotenoid ground state vibrational modes via the S2 and X states at 530 and 560 nm, respectively. 3.2. Global Analysis. The features observed in the 2D spectra (Figures 3,4) and t2 time traces (Figure 5) qualitatively agree with the spectral and temporal properties of the LH2 complexes known from transient absorption and time-resolved D
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Figure 5. t2 time traces of absorptive signal taken at selected excitation and detection coordinates of 2D data of Rps. acidophila (A) and Rba. sphaeroides (B): 530/530 nm (S2, blue line), 560/560 nm (X, red line), 580/580 nm (Qx, green line). Corresponding Fourier spectra after extraction of exponential contribution (C, D). The three indicated modes correspond to CH3 rocking mode (∼1000 cm−1), C−C stretching mode (∼1200 cm−1) and CC stretching mode (∼1600 cm−1). The spectrum in (C) was obtained using padding in order to better resolve the frequency of each mode by reducing their bandwidths. No padding was applied in (D).
Figure 6. 2DVAS of Rps. acidophila for the absorptive signal (left column), rephasing signal (center column), and nonrephasing signal (right column). The 2DVAS-1−3 are due to the 1070 cm−1, 1225 cm−1, and 1602 cm−1 modes, respectively. White lines indicate excitation/emission wavelengths.
fluorescence spectroscopic studies. In order to assess qualitatively the energy transfer pathways and spectral signatures of excited species, a global target analysis was applied. The analysis
was applied to absorptive, rephasing and nonrephasing signals. Owing to the dispersive contribution in rephasing and nonrephasing signals, however, these spectra cannot be easily E
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interpreted. Nevertheless, the dispersive signal is connected to the absorptive signal by Kramers−Kronig relations. The rephasing and nonrephasing signals, therefore, have t2 evolution identical to the absorptive signal and, therefore, can be described by the same kinetic model. The analysis resulted in 2D spectra associated with population (evolutionary) species (2DEAS) and with vibrational species (2DVAS). The 2DVAS correspond to the ground state vibrational modes, and they do not carry information on dynamics of the excited states (see Feynman diagrams in Figure S6). Therefore 2DEAS and 2DVAS can be discussed separately. 3.2.1. Vibration-Associated Spectra. Although ground state vibrations do not reflect dynamics in excited states, they provide significant information on the electronic structure of the chromophores. In Figure 6, the 2DVAS of Rps. acidophila are shown for the absorptive, rephasing, and nonrephasing signals (2DVAS of Rba. sphaeroides are shown in Figure S7). These 2DVAS correspond to three different vibrational modes having frequencies of 1070 cm−1, 1225 cm−1, and 1602 cm−1. Values of these frequencies were obtained from global analysis, where they were treated as global variables. The rephasing and nonrephasing 2DVAS reveal a particular arrangement of the peaks: off-diagonal for rephasing, and diagonal for nonrephasing. This arrangement reflects pathways, which can be illustrated by Feynman diagrams (Figure S6). As suggested by the vertical elongation of the absorptive and nonrephasing peaks in Figure 6, all three modes are excited at 530 nm, 560 nm, and 580 nm. The lower frequency modes reveal higher amplitudes at 530 and 560 nm excitation, while the 1600 cm−1 mode shows substantial amplitude at 580 nm excitation. Since the frequencies of the modes are much larger than the energy associated with the room temperature (kT = 207 cm−1), the corresponding vibrational levels of the ground state are not populated prior to excitation and these three excitation wavelengths indicate transitions from the ground S0,0 state. Here and further in this work we will use a second digit/symbol after the comma to emphasize a vibrational level of the given electronic state, e.g., S0,0 designates vibrationally relaxed ground state, S1,N designates the Nth vibrational level of the S1 state, etc. The two high-energy transitions in Figure 6 are due to the excitation of the carotenoid states S2 (530 nm) and X (560 nm). The lower energy transition coincides with the BChl Qx state (∼580 nm). Our preliminary experiments on isolated carotenoids find that their Fourier transformed 2D spectra in the high-frequency range (1550−1650 cm−1) have a similar shape to the 2DVAS-3 in Figure 6, but the three transition energies (see Figure S8) are spectrally shifted owing to the different dielectric environment and conformation of carotenoids in solution compared to the LH2 protein. Therefore the high frequency oscillatory signals at 580 nm excitation in LH2 proteins are not due to resonant excitation of the BChl Qx state, but rather indicate the presence of a lower lying carotenoid state. We can speculate that this state might be a high lying vibrational level of the S1 state (S1,N), lying above the well-known first vibrational level of S1 state (S1,1 or hot-S1 state) as shown in Figure 7. The interpretation of the origin of signals observed in 2DVAS can be performed using the theory of resonance Raman scattering. Indeed, since the Fourier spectra are dominated by the ground state vibrational modes of carotenoids (see Figure 5CmD), only stimulated Raman pathways contribute to the 2DVAS (i.e., no vibrational modes in the excited states are excited; see Figure 7). The amplitude of the off-resonance
Figure 7. Schematic presentation of (A) peaks from nonrephasing 2DVAS-1−3 from Figure 6, and (B) corresponding transitions. Colors of the arrows in B correspond to colors of diagonal peaks in A. Note that amplitudes of X and S1,N peaks in Figure 6 depend on the frequency of the 2DVAS (see text) and are overlapped.
Raman signal will be negligible compared to the resonance conditions, therefore the resonance transitions will appear as isolated peaks in the 2DVAS. According to the theory of resonance Raman scattering, there are two mechanisms of intensity enhancement.100,101 In the Franck−Condon scattering mechanism, the enhancement is due to displacement of the potential energy surface of the excited state relative to the ground state along the vibrational normal coordinate Q under transition of the molecule from its ground to excited state. According to the second mechanism, the resonant vibronic level of a weakly allowed excited state ‘e’ borrows intensity from a higher lying excited state ‘s’, which has a strong transition dipole moment, due to Herzberg−Teller vibronic coupling. The S2 state has a strong transition dipole moment and moderate displacement; therefore, the 2DVAS signal observed at 530 nm (S0−S2 transition) is due to Franck−Condon scattering.102 The transition to the X state (1Bu−) is generally forbidden due to pseudoparity selection rules; however, it can gain a weak transition dipole moment owing to environmental effects (deviation from the ideal geometry predicted for a gas phase environment). Even more important may be intensity enhancement, because of coupling to the S2 state, which is close in energy (≤1000 cm−1) and has similar symmetry (1Bu+). Therefore under resonance excitation of the X state, the enhancement of the resonance Raman signal will be mostly caused by Herzberg− Teller vibronic coupling. In that case, the selection rules are relaxed if the symmetry of the coupling vibrational mode is the same as the product of symmetries of the weakly allowed resonant state and strongly allowed higher lying excited state.103 Since S2 (1Bu+) and X (1Bu−) states have the same symmetry (except for pseudoparity index), the signals observed in 2DVAS at 560 nm excitation are likely due to coupling via totally symmetric ag modes. The Fourier spectra taken at S2 and X peaks of 2D spectra reveal an almost identical profile (see Figure 5CD), therefore we can conclude that the Franck−Condon factors are similar for transitions resonant with S2 and X states, i.e., that minima of their potential energy surfaces have similar displacements relative to the minimum of the ground state potential energy surface. This Herzberg−Teller enhancement mechanism should be responsible also for the signals observed in 2DVAS at 580 nm excitation, where the excitation is in resonance with a vibrational level of S1 state. However, because the S1 and S2 electronic states have different symmetries, the coupling between them can only take place via nontotally symmetric bu modes.104,105 The Fourier spectra taken at 580 nm on the diagonal of 2D spectra reveal a different profile from Fourier spectra taken at the S2 and X peaks (Figure 5C,D), indicating a substantially different displacement of the resonant state (S1,N) as compared to displacements of the F
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2DEAS of absorptive signals are discussed here. The absorptive 2DEAS of Rps. acidophila together with EET pathways and kinetic rates are shown in Figure 8.
S2 and X states. This agrees with the data on linear polyenes, where the emission spectrum from the S1 state was observed to show a maximum at the third or fourth vibrational level, while the maximum of S2 fluorescence has maximum of the intensity at the zeroth or first vibrational level.106,107 It is known that the energy of the S1 state is 12800 cm−1 and 13400 cm−1 in isolated rhodopin glucoside and spheroidene, respectively.108,109 Therefore, the high frequency peaks observed in the isolated rhodopin glucoside at 560 nm (Figure S8C) and in isolated spheroidene at 580 nm (Figure S8F) correspond to an energy gap of ∼5060 cm−1 and ∼3840 cm−1. These energy gaps are equal to four and three quanta of a ∼1250 cm−1 mode, respectively, which is known to be one of the vibrational modes of the carotenoid S1 state.110−112 Thus our observations are in agreement with expected positions of the vibrational levels of the S1 state. The presence of the high vibrational levels of the S1 state provides an additional explanation for the anomalous conjugation length dependence of the S2-to-S1 relaxation rate, an effect that has been controversially discussed in the literature for ́ and Sundström25). the past two decades (for review, see Polivka Several experimental studies have shown that the lifetime of the Car S2 state in different carotenoids/polyenes does not follow the energy gap law,113−115 revealing a minimum at conjugation length N = 9. Two hypotheses have been discussed to explain this abnormal dependence. Frank et al. suggested that a higher density of accepting vibrational modes causes a decrease of the S2 lifetime.114,116 Another proposed mechanism is the presence of an intermediate state, which would mediate the S2-to-S1 energy transfer (for review see Polivka and Sundstrom59). The results of the current study, as well as the previous work,49 suggest that both mechanisms can be active. In this case, the complete energy level ladder in carotenoids would be S2→X→S1,N→S1,1→S1→S0, where the X state is the 1Bu− state, and the S1,1 (hot-S1) state is the well-known feature from transient absorption studies.90,117−119 This energy level ladder is very similar to the scheme proposed by Maiuri et al.,57 where the Sx state was equivalent to X state in current work. Another feature in the 2DVAS is the change in the relative amplitude of the peaks emitted at 560 and 580 nm wavelengths (the systematic red-shift of the emission wavelength in 2DVAS-1, 2DVAS-2, and 2DVAS-3 in Figure 6). Because the two signals emitting at 560 and 580 nm overlap and appear as a single peak, the redistribution of the amplitudes in different 2DVAS appears as a visual “shift” of the emission of this peak to longer wavelengths for modes of higher frequency (1070 cm−1 at 565 nm, 1225 cm−1 at 570 nm, 1602 cm−1 at 580 nm). The increase of the relative amplitude of the lower energy peak (580 nm transition) for higher frequency modes (1600 cm−1) can be due to Franck−Condon factors larger for the 580 nm transition and smaller for the 560 nm transition, when the 1600 cm−1 frequency mode is involved in the transition. An additional factor, which can cause an actual red-shift of the signal’s emission wavelength, is a change (increase) of the Franck−Condon factors during the evolution (relaxation) of the system after excitation (i.e., Franck−Condon factors of the transitions corresponding to the third and fourth field-matter interaction in the four-wave mixing process). This effect has been indicated previously to explain the delay of the maximum of the response signal in Fourier-transform spectroscopy of both LH2 complexes and isolated carotenoids.120 3.2.2. Evolution-Associated Spectra of Rps. acidophila. Although the absorptive, rephasing, and nonrephasing signals were analyzed together within single global kinetics model, only
Figure 8. 2DEAS of Rps. acidophila of absorptive real signal (left column). Diagonal features are designated by a single symbol, offdiagonal features are marked by two symbols (indicating the locally excited state, and the emitting state, respectively). Energy transfer processes corresponding to each 2DEAS are shown on the right. The rates of the evolutionary kinetic scheme are shown in blue. In kinetic schemes carotenoid excited states are indicated by red bars, BChl excited states are indicated by green bars, ground states are indicated by black bars, EET pathways are indicated by solid black arrows, and SE and ESA transitions observed in 2DEAS are shown by downward and upward dashed blue arrows, respectively. The GSB signals from S2, X, and Qx states are not indicated in the schemes.
The 2DEAS-1 decays with a rate constant of 48 ps−1. The positive signals at 530 nm excitation are due to S2 GSB/SE (diagonal peak) and SE from X state (cross-peak), which is populated by EET from S2 state. The weak diagonal shoulder at 560 nm is due to X GSB. The positive diagonal signal at 585 nm excitation is due to Qx state GSB. The cross peak at 585 nm excitation and 560 nm detection was previously speculated to arise from GSB due to mixed excitation of Qx and X states.49 However, the thorough analysis of coherent contributions described above revealed a weak signal at ∼580 nm in 2DVAS arising from the carotenoid, likely from S1,N state (for detailed G
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Supplemental Material in ref 49). Two sources of instability are (i) insufficient signal-to-noise ratio, and (ii) short t2 timerange of the measurement. Optimization of the fitting algorithm is an additional factor that can affect the sensitivity of the analysis. 3.2.3. Evolution-Associated Spectra of Rba. sphaeroides. The absorptive 2DEAS of Rba. sphaeroides shown in Figure 9
discussion see Figure 6 and corresponding text). Therefore another possible origin of the Qx/X cross-peak is a carotenoid GSB signal indicating the common ground state between the X state and S1,N state. Whether the Qx/X cross-peak is produced by one of these two mechanisms or by a contribution from both cannot be answered at this time. In summary, signals at all three excitation coordinates (530 nm, 560 nm, 585 nm) and the 560 nm detection wavelength represent decay of the population of the X state. We can conclude that the S2 state has an even faster relaxation rate and is not resolved in the analysis, because the time scale of S2 state dynamics coincides with the pulse overlap time-range (∼15 fs), which is not included in the global analysis. The 2DEAS-2 decays with a rate constant of 16 ps−1. A significant feature of this 2DEAS is the positive signals observed in the 530−590 nm excitation range at a single detection wavelength of 590 nm. These positive signals are due to SE from Qx state populated by EET from the S2 state (530 nm excitation), X state (560 nm excitation), and by direct excitation at 590 nm. The diagonal signal at 530 nm is due to GSB from the S0−S2 transition. One expects to see the GSB from the S0-X transition as well; however, it is not visible owing to overlap with negative signals. The negative signals are most probably due to ESA from the vibrationally excited S1 state (S1,N). This S1 ESA signal is present at both 530 and 560 nm excitation wavelength, therefore we conclude that both the S2 and X states transfer excitation energy to the S1 state. The 2DEAS-3 decays with a rate constant of 8 ps−1. The three positive diagonal signals are due to GSB from S2, X, and Qx states. These GSB peaks suggest energy transfer pathways on a time scale of ∼125 fs. Despite the presence of the negative S1 ESA signal below the diagonal, the 8 ps−1 EET pathway is not due to the population of the S1,1 state. Because the dipole transition moment of the S1−SN transition is stronger than the dipole transition moment of the S0−X transition, the S1 ESA obscures the GSB signal from the X state, as is evident from 2DEAS-2 and 4. We speculate that the 8 ps−1 component represents population of the BChl Qy state (populated by EET from S2 and X states and internal conversion from Qx state), which is known to occur on the 100−300 fs time scale in LH2 complexes from different organisms.72,92 In this case the S1 ESA signal is observed because the temporal resolution within the evolutionary kinetic scheme is not perfect, and a small amount of the slower S1,1 ESA signal mixes into the 2DEAS-3. The 2DEAS-4 decays with a rate constant of 2.6 ps−1. It reveals GSB signals from S2 and Qx states as well as three S1 ESA peaks at 530 nm, 560 and 590 nm excitation wavelengths. Therefore the 2DEAS-4 can be assigned to the S1,1 state populated from all three S2, X, and Qx states. The weak X/Qx peak might be due to the same GSB signals as the Qx/X cross-peak in 2DEAS-1. In 2DEAS-4 and 2DEAS-2 the negative S1 ESA signal is spectrally overlapped with the GSB from X state and obscures it, as discussed above. The proposed kinetic pathways are summarized in the right column of Figure 8. Several EET processes can contribute to a single 2DEAS. The evolutionary kinetic schemes allows the pathways to be conveniently observed, resolving their spectral signatures and arranging them according to their relaxation rates. The actual kinetic scheme accounting for these EET processes has been applied in the global analysis, however, due to multiple intermingled pathways the model appeared to be unstable, revealing species-associated spectra with strong mixing of actual spectral profiles of the excited states (for details see
Figure 9. 2DEAS of Rba. sphaeroides of absorptive real signal (left column). Diagonal features are designated by a single symbol of locally excited state, off-diagonal features are marked by two symbols (indicating the locally excited state, and the emitting state, respectively). Energy transfer processes corresponding to each 2DEAS are shown on the right. The rates of the evolutionary kinetic scheme are shown in blue. In kinetic schemes carotenoid excited states are indicated by red bars, BChl excited states are indicated by green bars, ground states are indicated by black bars, EET pathways are indicated by solid black arrows, and SE and ESA transitions observed in 2DEAS are shown by downward and upward dashed blue arrows, respectively. The GSB signals from S2, X, and Qx states are not indicated in the schemes.
reveal features similar to the features discussed above. The 2DEAS-1 shows positive diagonal signals due to GSB from S2, X, and Qx states, as well S2/X cross-peaks. These cross-peaks are due to the GSB via a common ground state and SE from the X state. Therefore this component represents the fastest step of energy redistribution between the S2 and X states, with a small contribution from the Qx state. The relaxation rate of this H
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spectrum is 30 ps−1, which is slower than the corresponding rate of the Rps. acidophila. The 2DEAS-2 reveals two diagonal GSB signals from S2 and Qx states as well as negative S1 ESA signals. The S1 ESA signal is observed at 530, 560 and 580 nm excitation wavelengths, implying population of the S1 state from S2, X and Qx states (compare with 2DEAS-2 of Rps. acidophila in Figure 8). The weak positive cross-peak at 580 nm detection wavelength is due to Qx SE signal, indicating S2-to-Qx EET. Thus the two main EET processes revealed in 2DEAS-2 are (i) population of vibrational level of the S1 state from S2, X, and Qx states (see discussion of 2DEAS-2 of Rps. acidophila, Figure 8), and (ii) population of the Qx state from the S2 state. The relaxation rate 5.7 ps−1 is much smaller than the rate of 2DEAS-2 in Rps. acidophila. The decrease of the rate values in Rba. sphaeroides can be caused by the different carotenoid structure (e.g., shorter conjugation length) and/or by different structure of the LH2 protein in these two organisms. The 2DEAS-3 shows strong GSB signal from the S2 state, weaker diagonal signals from X and Qx states and a positive crosspeak. Observation of this cross-peak in 2DEAS-3 can be explained by two processes: (i) the X-to-Qx EET in Rba. sphaeroides has a slower rate compared to the similar EET process in Rps. acidophila (see 2DEAS-2 in Figure 9), (ii) both the diagonal peak and cross-peak indicate a bleach of Qx state due to relaxation to a lower lying BChl state (i.e., Qy state). The latter hypothesis was used to explain X and Qx GSB signals in 2DEAS-3 of Rps. acidophila in Figure 8. The relaxation rate of the current 2DEAS is 2.8 ps−1, confirming the slower dynamics in LH2 complexes of Rps. sphaeroides. The two negative signals indicate mixing of the S1 ESA signals into this component because the model cannot separate their rate constants. The 2DEAS-4 is dominated by S1 ESA signal observed at 510− 570 nm excitation wavelengths, with a weak shoulder at 580 nm excitation. The 1.7 ps−1 relaxation rate suggests assignment of this 2DEAS to the S1,1 (hot-S1) state, which according to literature decays on a time-scale of 700 fs in the case of spheroidene in solution117 (as compared to 320 fs for rhodopin glucoside in LH2 protein72). Thus the S1,1 state is populated from S2, X, and Qx states. 3.3. Spectral Overlap. The evolutionary kinetic scheme reveals the main energy transfer pathways and characteristic relaxation times of the associated states, allowing for one to follow the dynamics of the excitation. However, the efficiency of particular energy transfer pathways cannot be determined from these results because the exact values of the rates between different states are not known, only their effective product (“characteristic relaxation times”). Nevertheless, a rough estimate of the contribution of the intermediate X state to the overall Car-to-BChl energy transfer process can be obtained by calculating the Förster-type spectral overlaps. The overall rate associated with the Car-to-BChl energy transfer is the summation of all the rates associated with the different pathways contributing to the overall energy transfer process. Equation 1 gives the expression for the rate constant associated with a given pathway for which electronic energy is transferred between two weakly coupled states.121−123
k=
4π 4 2 |V | J h2c
having units of cm, the above expression can be written as k = 1.18|V|2J.122 As can be seen from the above equation, the rate of electronic energy transfer depends on two terms, the spectral overlap, and the electronic coupling. In this manuscript we focus on how the spectral overlap influences the rate constant. To determine the electronic coupling term for the system being studied would require challenging quantum chemical calculations of the X state due to the fact that the ideal dipole approximation cannot be used to appropriately describe the transition density in the current system.121 There are two reasons for this. The first being that the carotenoids and BChl molecules are tightly packed in the LH2 protein matrix. Owing to the small spatial separation, the chromophores interact in such a way that the transition density is no longer approximated by the transition dipole moments.121 The second arises from the fact that the intermediate Car X state is an electronically forbidden transition, having a negligible transition dipole moment.121 Theories based on the ideal dipole approximation therefore do not provide reasonable descriptions of the transition density.121,124 Previous computational studies of the LH2 complex have used the transition density cube method to overcome these limitations and have found that the electronic couplings between the Car S2 and BChl states121 and Car S1 and BChl states124 are similar. Therefore the variation in rates associated with the energy transfer pathways between Car states (S2 and S1) and BChl states were mainly due to the variation in the spectral overlap values. Because both Car X and S1 states are formally dark states, and because X (1Bu−) and S2 (1Bu+) states have similar symmetry, one expects the couplings between the X state and BChl states to be similar to couplings between S2 and/or S1 states and BChl states. Therefore a study of the spectral overlaps can give a rough estimate of the rate of EET from the X state to the Qx state. The spectral overlap, J, was calculated between the relevant states, the Car S2 and BChl Qx and the Car X and BChl Qx, according to eq 2, where AA is the area-normalized absorption spectrum of the acceptor molecule and FD is the area-normalized fluorescence spectrum of the donor molecule:122,123,125 J=
∫0
∞
FD(v)AA (v) dv
(2)
The absorption spectra associated with the S2, Qx and X states are plotted in Figure 10 as solid lines. The absorption spectra associated with the S2 and Qx transitions were obtained by fitting the linear absorbance spectrum to Gaussian functions. Since the absorption spectrum of the intermediate X state is unknown, we simulated the spectrum as a Gaussian function with maximum at 560 nm (determined from the 2D electronic spectra) having a fwhm equivalent to that of the Car S2 0−0 transition. We note here that an increase in the spectral width of the simulated X absorption would lead to an increase in the X−Qx spectral overlap. The fluorescence spectrum associated with the Car S2 state was calculated according to the previous work of Krueger et al.,126 and the resulting spectrum is plotted as a dashed blue line in Figure 10. The fluorescence spectrum associated with the X state was calculated according to the Kennard−Stepanov relationship127,128 and is plotted as a dashed green line in Figure 10. From these absorption and emission spectra, the spectral overlaps for the S2−Qx and X−Qx pathways were calculated to have the same value of J = 2.49 × 10−4 cm. In Figure 10 the area normalized spectral overlaps for the two scenarios are shown. Under assumption of equal coupling strengths, the equivalent spectral overlaps imply that the rate constants for energy transfer are approximately the same for the two pathways.
(1)
In the above equation, V is the electronic coupling between the two states, and J is the Förster spectral overlap. For a rate constant, k, having units of ps−1, V having units of cm−1, and J I
dx.doi.org/10.1021/jp403028x | J. Phys. Chem. B XXXX, XXX, XXX−XXX
The Journal of Physical Chemistry B
Article
Figure 11. Electronic level scheme and corresponding characteristic populating times (inverse of corresponding rate constant kij−1) of LH2 complexes from Rba. sphaeroides and Rps. acidophila.
Figure 10. The area-normalized spectral overlaps before integration, FD(λ)AA(λ) (gray area), between (A) Car S2/BChl Qx states and (B) Car X/BChl Qx states within LH2 protein. The blue, green, and orange solid lines represent the absorption spectra of the Car S2, X, and BChl Qx states, respectively. Dashed blue and green lines represent the emission spectra of the Car S2 and X states.
By resolving 2D spectra of the high-frequency ground state vibrational modes, we observed three low-lying carotenoid excited states: S2, X (1Bu‑) and, presumably, S1,N. The S1,N represents a high vibrational level of the S1 state (N > 2), which has not been reported previously in the literature. 2DES showed strong advantages in revealing excited states with weak transition dipole moments, while the global target analysis allowed assignment of the spectral features and uncovering the underlying dynamics of excited states.
The results of 2D spectroscopy and estimation of the spectral overlap indicate that the X state acts as a sink accepting energy from the S2 state and efficiently redirecting it to the Qx state, therefore sensitizing BChl. This qualitative analysis gives insight into the excited state dynamics reported in previous studies of LH2 complexes. Krueger et al. performed calculations of the Car S2−BChl energy transfer pathways using the transition density cube method, which allowed accounting for the electronic coupling with a high precision.121 These calculations, which did not include the intermediate X state, revealed the efficiency of the direct S2−Qx energy transfer to be not more than 21% in LH2 protein from Rps. acidophila.121 This value is significantly lower than the values obtained experimentally from stationary fluorescence and time-resolved spectroscopies (50−60%).72,126 The disagreement between theory and experiment indicates the importance of the intermediate Car X state in the Car-to-BChl energy transfer, suggesting that up to 50% of the total S2-to-Qx EET efficiency is due to the X state. The magnitude of the contribution of the X state in the light harvesting process will vary from organism to organism, e.g., depending on the molecular and electronic structure of the particular carotenoid.
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ASSOCIATED CONTENT
S Supporting Information *
Figures S1−S8, Movies S1−S2. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Natural Sciences and Engineering Research Council of Canada, DARPA (QuBE) and the United States Air Force Office of Scientific Research (FA9550-10-1-0260) to G.D.S., by research scholarship from the BBSRC to R.M.M. as well as by the U.S. Department of Energy (DOE), Office of Basic Energy Sciences, as part of the Photosynthetic Antenna Research Center (PARC) Energy Frontier Research Center, DE-SC0001035 to R.J.C. Authors thank T. Polivka for comments and stimulating discussions.
4. CONCLUSIONS Studies of excitation dynamics in multichromophore complexes present a challenging task due to the high density of electronic states and strongly intermingled energy transfer pathways. Applying global analysis to 2D electronic spectra, we resolved energy transfer pathways between four excited states of LH2 proteins occurring on an ultrafast
