Excitonic structure and charge separation in the Heliobacterial Reaction Center probed by multispectral multidimensional spectroscopy
Photochemical reaction centers are the engines that drive photosynthesis. The reaction center from heliobacteria (HbRC) has been proposed to most closely resemble the common ancestor of photosynthetic reaction centers, motivating a detailed understanding of its structure-function relationship. The recent elucidation of the HbRC crystal structure motivates advanced spectroscopic studies of its excitonic structure and charge separation mechanism. We perform multispectral two-dimensional electronic spectroscopy of the HbRC and corresponding numerical simulations, resolving the electronic structure and testing and refining recent excitonic models.Through extensive examination of the kinetic data by lifetime density analysis and global target analysis, we reveal that charge separation proceeds via a single pathway in which the distinct A 0 chlorophyll a pigment is the primary electron acceptor. In addition, we find strong delocalization of the initial excited state and charge separation intermediate. Our findings have general implications for the understanding of photosynthetic charge separation mechanisms, and how they might be tuned to achieve different functional goals.
Introduction<!>Results<!>Discussion<!>Methods
<p>Photosynthesis drives life on Earth by converting solar energy into chemical energy 1,2 . In photosynthesis, light is absorbed by the antenna pigments, which transfer their excitation energy to the reaction center (RC) 3,4 . The primary energy conversion step, charge separation (CS), takes place in the RC 5 , driving the subsequent chemical reactions for synthesizing high-energy chemical compounds. All RCs are believed to have evolved from a common homodimeric ancestor RC 6,7 . Until recently, the crystal structures of the existing homodimeric RCs were unknown 8 , hindering mechanistic understanding of these systems.</p><p>Recently, the crystal structure of the heliobacterial RC (HbRC) from Heliobacterium modesticaldum, was characterized by Gisriel and coworkers 8 . The HbRC has been proposed to be the RC most similar to the common ancestor of all photosynthetic RCs 6,7 . Furthermore, it is the simplest known RC and structural analog to the photosystem I RC 9 (PSI RC), and possesses three chemically distinct pigments.</p><p>The HbRC binds 54 BChl g, four BChl g', two 8 1 -hydroxychlorophyll a F (8 1 -OH Chl a), two carotenoids (4,4'-diaponeurosporene), and one [4Fe-4S] cluster 8 . The majority of the BChl g pigments serve as an antenna to direct excitation to the homodimeric core which serves as the electron transfer domain, depicted in Fig. 1. Throughout this paper we adopt the language commonly used in discussing RCs, where we reserve the term RC for the electron transfer domain, and the other pigments are collectively referred to as the "antenna". The RC consists of two BChl g' pigments (P 800 ), two BChl g (Acc), two Chl a (A 0 ) and one [4Fe-4S] cluster 8 .</p><p>A structure-based excitonic model of the full HbRC was recently proposed by Kimura and coworkers 10 . They report that the HbRC pigments form 58 delocalized excitonic states with 0-0 transition energies spanning 771 to 812 nm and two excitonic states with dominant absorption peaks centered at 667 nm. Until now, this model has not been tested by advanced experimental techniques.</p><p>The diverse pigment composition of the HbRC in comparison to other RCs makes it particularly appealing for spectroscopic studies of its primary CS processes. Energy transfer and CS in the HbRC have been studied by several groups [11][12][13][14][15][16][17][18][19][20][21] , with the early work reviewed by Neerken et al 17 . In light of previous proposals of the CS mechanism and the new structure, Gisriel et al. proposed two possible charge-separation mechanisms: one in which Acc acts as the initial electron acceptor, and another in which Acc acts as the initial electron donor 8 . Two-dimensional electronic spectroscopy (2DES) has proven to be a powerful tool to study photoexcited dynamics in a variety of systems including light-harvesting complexes [22][23][24] , photosynthetic RCs 25,26 and solar cells 27,28 . A 2DES spectrum is a frequency-frequency map that directly highlights correlations between photoexcited and photodetected states via cross-peaks induced by excitonic interactions. 2DES also reveals the evolution of excited state populations, enabling the testing of kinetic models and the identification of spectroscopic fingerprints of reaction intermediates. Here were have applied 2DES to the HbRC, revealing excitonic correlations between Acc, A 0 and P 800 and refining the proposed excitonic structure. We find that CS in the HbRC proceeds via a two-step process in which A 0 acts as the primary electron acceptor. Moreover, our results suggest that the hole is initially delocalized over Acc and P 800 immediately after the primary CS event, but later becomes localized to P 800 . The dashed lines in the 2DES spectrum and the grey lines in the absorption spectrum indicate the various peak positions discussed in the main text. c) The experimental and simulated slice spectra under excitation at 668 nm at T = 0 ps extracted from the 2DES absorptive spectra. The site energies of BChl g and BChl g' are set to be equal according to Kimura's excitonic model. d) The experimental and simulated slice spectra under excitation at 668 nm at T = 0 ps extracted from the 2DES absorptive spectra. Modifying the site energy of accessory BChl g to be 13150 cm -1 yields considerably better agreement with the experimental data. The presence of distinct BChl Q y peaks under excitation at 668 nm reveals excitonic delocalization between A 0 and the BChl pigments Acc and P 800 . The stick bars reveal the participation ratios of the RC pigments in each excitonic state as given in the respective exciton models. Fig. 2 | 2DES probes the photoexcited dynamics in the HbRC at 77K. a) 2DES absorptive spectra at 0.05 ps, 0.5 ps, 10 ps and 946 ps. Alongside the 2D absorptive spectra are the experimental absorption spectrum and the simulated stick spectrum (red lines) from of Kimura et al 10 . The dashed lines in the 2DES and the grey lines in the absorption spectrum indicate the various peak positions discussed in the main text. b) The slice spectra under excitation at 668 nm (solid) and 690 nm (dashed) at various T extracted from the 2DES absorptive spectra. The derivative lineshapes at 573, 578 nm and the blue-shifted A 0 peak at 666 nm (inset) are induced by the Stark effect due to the presence of an adjacent charge. These features are fingerprints for the charge-separated state.</p><!><p>To probe the HbRC excitonic structure and accentuate CS processes originating from RC excitation, we excited the ~590-720 nm region, spanning the Q y A 0 Chl and vibronic shoulder of Q y BChl g transitions.</p><p>The multispectral probes in our recently developed 2DES instrument 29 access both Q x and Q y of Chls and BChls, providing dynamical information about energy transfer and CS processes. We also performed complementary 2DES studies with excitation of the BChl g Q y region.</p><p>Fig. 1b displays 2DES absorptive spectra of the HbRC at 77K, in which the sample was treated with sodium dithionite to reduce the terminal [4Fe-4S] cluster (F x ). This blocks forward electron transfer from A 0 to F x , resulting in charge recombination of the P 800 A 0 state in ~15 ns 12 . The 2D absorptive spectrum at T = 0 ps provides insight into the HbRC's excitonic structure and tests the current structure-based excitonic model 10 . The diagonal peak at 668 nm is consistent with photoexcitation of the highest energy RC excitons assigned to two states primarily involving A 0 with some participation from Acc. 10 The presence of cross peaks in the region λ ex = 668 nm and λ det = 770-800 nm (denoted CP1) is key to understanding the pigment composition of the excitonic states in the crowded BChl Q y region. The model of Kimura and coworkers 10 predicts roughly three states in the 760-800 nm region that involve delocalization over A 0 and could therefore produce CP1 in the T = 0 2D spectrum. These states, as well as the other RC excitons from the Kimura model, are indicated by solid red lines in the linear absorption spectrum shown in Fig. 1b (right panel). To test the Kimura exciton model, we simulated the 2DES absorptive spectrum in the CP1 region.</p><p>The simulations employ a commonly modified Redfield approach, where the optical response functions are obtained within the adiabatic limit during electronic coherence periods: diagonal system-bath fluctuations are included exactly using the cumulant expansion, truncated at the second order, and off-diagonal fluctuations are included within Markovian second-order perturbation theory 30,31 . The resulting slice spectrum upon excitation of A 0 is shown in Fig. 1c. The two cross peaks in the BChl Q y band provide firm evidence for excitonic delocalization, but are red shifted relative to the experimental data, indicating that refinement of the excitonic parameters is needed to capture the tuning of pigment site energies by the protein environment. We find better agreement between experiment and theory by shifting the site energy of Acc from 12739 cm -1 (785 nm) to 13150 cm -1 (760 nm (see Fig. 1d). We also note that not all the excitonic peaks involving A 0 pigments appear in the simulated 2DES, due to low oscillator strength and overlapping contributions of the ground-state bleach (GSB), stimulated emission and excited state absorption (ESA) signals. Further simulation details are given in the Section S6 of Supplementary Information (SI).</p><p>While both the RC and the antenna excitons are simultaneously excited in this experiment, the spectral signatures are separated along the excitation and detection wavelengths. This enables investigation of the CS mechanism(s) initiated by direct excitation of the RC or excitation of the antenna. The 2DES spectra in Fig. 2 show that by 0.5 ps the 668-nm diagonal peak has largely decayed, accompanied by the rise of two cross peaks at 794 nm and 814 nm. We interpret these signatures as redistribution of energy within the RC and equilibration with the antenna. The contribution of antenna excitons to these cross peaks is evident in the similarity of the cross-peak structure at an excitation wavelength of 690 nm, which corresponds to predominantly antenna excitation. At 946 ps, the 2DES absorptive spectrum has become excitation wavelength independent, suggesting that excitons have been converted to the final charge-separated state (CSS)-P800 + A 0 -, as reported in previous studies 11, [14][15][16][17][18]32,33 . In this 2D spectrum, a derivative lineshape near 575 nm (Q x band of BChl g) shown in Fig. 2b is induced by the Stark effect due to the presence of an adjacent charge 13 , providing definitive fingerprints for CS. The CS is also evident in the evolution of the GSB peak of the 668-nm RC exciton, which shifts to λ det = 666 nm in accordance with previous studies 9, 13 , consistent with the formation of A 0 -. Two other GSB peaks at λ det = 794 and 807 nm decay with A 0 and can thereby be assigned to the upper and the low excitonic peaks of P 800 +12,13</p><p>.</p><p>To make full use of the rich information from 2DES, we used a combination of lifetime density analysis (LDA) 34,35 to obtain an overview of the kinetics, followed by global-target analysis 36 to test specific kinetic models. LDA fits the data using exponential functions with a continuous distribution of time constants. As no prior knowledge is required for this analysis, this approach is well suited to systems like the HbRC, in which multiple relaxation pathways are expected. Lifetime density maps (LDMs) that reveal the kinetics following RC and antenna excitation are shown in Supplementary Fig. 3 and  To test the proposed CS mechanism and extract spectroscopic signatures of the intermediates, we turned to global-target analysis. This is often used in kinetic analysis of transient absorption spectroscopy 36 and has been applied in 2DES studies [37][38][39][40][41] . This approach fits the data using a trial kinetic model and produces a set of species-associated spectra (SAS) and rate constants. Guided by the LDA analysis and the revised Kimura model, we considered multiple kinetic models before arriving at the one presented in Fig. 3a.</p><p>Further justification of our choice is given in Section S4 of the SI, where we discuss a subset of alternative models, including ones that incorporate additional and alternative CS pathways. The Fig. 3a model provides the best fit to the 2D data and produces SAS with spectroscopic characteristics consistent with the target model. In this model, two RC excitons and two antenna excitons are excited by our broadband pulses, after which downhill energy transfer precedes rapid CS via a single intermediate CSS. Species S0, with GSB peaks at λ det = 668 nm and 780 nm, and an ESA peak at λ det = 810 nm, is assigned to the delocalized RC exciton (i.e., (A 0 Acc)*). This assignment is consistent with the presence of GSB peaks in the T = 0 2DES spectrum and is further supported by the large coupling strength of 200-300 cm -1 between A 0 and Acc 10,13 in the Kimura model. Our proposed shift of Acc's site energy suggests a stronger degree of participation from P 800 in this RC exciton compared to the Kimura model.</p><p>After photoexcitation, S0 transitions to S1 with a time constant of 0.09 ps. S1 exhibits GSB peaks at λ det = 668 nm and 790 nm. S1 was not observed with excitation at 690 nm (see Supplementary Fig. 6), leading us to infer that S1 is also an RC excitonic state. Consistent with the Kimura model, we attribute S1 to a lower energy RC exciton involving some degree of delocalization over all RC pigments (P 800 AccA 0 )* 10,42 .</p><p>S1 transitions to S4 with a time constant of 0.89 ps. S4 exhibits three GSB peaks at λ det = 666 nm, 794 nm and 814 nm and a Stark lineshape near λ det = 575 nm. As discussed above, the presence of the Stark lineshape and the blue-shifted A 0 peak are fingerprints of a CSS involving A 0 and at least one BChl g pigment. Compared to P 800 + A 0 -, S4 exhibits a larger splitting between the two GSB peaks in the BChl Q y band, suggesting the hole wavefunction in S4 may be more delocalized in this state than in P 800 + A 0 -. Given the coupling strength of ~200 cm -1 between Acc and P 800 10,42 , we speculate that the hole wavefunction is delocalized over both of them in S4. One of the most striking features in this fit is that the CSS intermediate S4 is present in both charge-separation pathways, whether initiated by the antenna exciton or the RC exciton.</p><p>To support this claim, we compared the fits of the pump-probe under excitation at 666 nm and 690 nm using one to three CS pathways (Supplementary Fig. 5-6) and the fits of the 2DES using two to three CS pathways (Supplementary Fig. 7-10). All of these fits show the presence of an intermediate CSS with the characteristics of S4, providing strong evidence that A 0 is the primary electron acceptor in the initial CS step, independent of the initial excitation. The intermediate CSS S4 relaxes to S5 with a time constant of 19 ps. S5 exhibits a derivative lineshape at 575 nm, a blue-shifted A 0 peak at 666 nm and two P 800 + peaks at 794 and 807 nm as described earlier, leading to its assignment as P 800 + A 0 -.</p><p>We considered several models for the antenna relaxation, finding that a minimum of two distinct antenna excitons, S2 and S3, were needed. While S2 and S3 can be generated via direct photoexcitation of the vibronic shoulder of the BChl Q y transitions, a small fraction of S0 is also transferred to the antenna, populating S2 and subsequently S3. We include a back-energy-transfer step from S3 to S2, inspired by a recent study 43 that suggests the low-energy exciton is located far from the RC. The spectroscopic characteristics of S2 and S3 are consistent with their assignment as antenna excitons: both lack amplitude at 668 nm indicative of A 0 excitation. S2 exhibits GSB signals at λ det = 570-590 nm and 790-810 nm and transitions to the CSS S4 with a time constant of 2.2 ps. Due to the transfer of S0 to S2, we attribute S2 to an antenna exciton involving pigments near to the RC. In contrast, S3 exhibits GSB signals that are redshifted relative to S2 and undergoes relatively slow backwards energy transfer to S2, leading us to assign S3 to low-energy antenna exciton(s) 16,18 . We note that an RC exciton involving P 800 and/or Acc only may be present between S2 and S4 and act as the reactant for the initial charge separation step. However, this component cannot be isolated from S2 probably due to their similar spectral profiles and fast exchange rate.</p><!><p>The energy transfer and CS processes of the HbRC have been studied since the 1980s [11][12][13][14][15][16][17][18]20,21,44,45 . Lin and coworkers 11 reported that excitonic equilibrium within the antenna is complete within ~0.8 ps, while CS takes place at ~25 ps at room temperature. They further claimed that energy transfer is the rate-limiting step and reported the upper limit of the intrinsic CS to be 1 ps -1 . At cryogenic temperatures, Chiou et al. 18 reported that all excitations are funneled into the lowest energy exciton prior to CS, which takes place within 55-70 ps. However, the Vos 20 and Amesz groups 16,17 observed fast CS occurring on the time scale of a few picoseconds upon photoexcitation of both antenna and A 0 , suggesting that excitonic equilibrium need not be established prior to fast CS.</p><p>Another debate concerns the difference in CS mechanisms upon excitation of the RC and antenna domains [16][17][18] . Initially, a one-step CS mechanism (i.e., hole transfer from A 0 to P 800 or electron transfer from P 800 to A 0 ) was used to describe CS for both RC and antenna excitation 16,17,20,44 . However, the one-step mechanism cannot explain the multiple time scales observed for CS, or 'uphill' CS from the lowest energy antenna exciton 16,18,21 . To resolve these issues, Chiou et al 18 proposed a two-step mechanism in which A 0 is the primary acceptor. Without knowledge of the location of A 0 and its coupling to other pigments, the feasibility of this mechanism, and the relationship to CS mechanisms in other RCs, were unclear.</p><p>Upon resolution of the HbRC structure, Gisriel et al. 8 proposed two different two-step CS mechanisms for the HbRC. In the first, Acc acts as the primary electron acceptor, similar to the dominant mechanism proposed for the purple bacterial RC 37,46 . In the second, Acc acts as the primary electron donor, with A 0 playing the role of primary electron acceptor in analogy to PSI 1 and the dominant mechanism in both PSI 35,[47][48][49] and PSII 35,49,50 . The LDA and global target analysis of our 2DES data strongly favor the latter mechanism. Regardless of initial excitation, RC and antenna excitons both transition to a common intermediate CSS (S4), in which A 0 is the primary electron acceptor and the hole wavefunction is delocalized over Acc and P 800 . We cannot entirely rule out additional CS intermediates obscured by spectral congestion in the BChl Q y region and overlapping time scales of energy equilibration and primary CS.</p><p>However, our extensive consideration of other target models consistently found that any intermediates exhibiting the hallmark of a CSS also involved A 0 .</p><p>The mechanism we have proposed makes good chemical and biological sense. We have argued that the ancestral homodimeric RC used quinones as terminal electron acceptors to couple light-driven electron transfer to proton pumping across the membrane, driving ATP production. Inherent inefficiencies of quinone reduction in this RC drove the evolution of type I and type II RCs 9 . While type II RCs became heterodimeric and specialized the two branches, type I RCs added the F X cluster to catalyze semiquinone dismutation; the subsequent switch to reducing ferredoxin necessitated a more reducing cluster as well as an upstream donor (A 0 ) with much lower reduction potential. This explains why all type I RCs use a version of Chl a in the A 0 site due to the considerably lower reduction potentials of chlorins, including A 0 12 , compared to bacteriochlorins 51,52 . The use of Chl a as A 0 places serious constraints on the CS mechanism.</p><p>It would be extremely difficult for Acc -(BChl) to act as an electron donor to A 0 (Chl), rendering the "purple bacterial RC model" very unlikely. This means that the excited state of BChl g, which has a significantly lower potential than the ground state, must be used as the primary donor to A 0 . Our data are most consistent with a model in which the primary donor is actually an excited state delocalized across several BChl g (P 800</p><p>and Acc). This mechanism may protect the HbRC from wasting energy through charge recombination reactions: delocalization of the hole moves cation density away from A 0 in the initial CSS, which should serve to slow charge recombination of the initial radical ion pair. This may be especially important for anoxygenic type I RCs, in which the redox gradient from BChl to Chl would be uphill if not for the use of the excited state. (In contrast, the redox gradient from (B)Chl to (B)Pheo is downhill in type II RCs.) The subsequent step of localizing the hole to the P 800 BChl dimer would be a natural consequence of its higher reduction potential, enabling the special pair to stabilize the hole and place it near the external side for reduction by the external donor (cytochrome c).</p><p>In conclusion, multispectral 2DES has provided insight into the excitonic structure and CS mechanism of the HbRC. Our extensive kinetic analysis revealed A 0 as the primary electron acceptor independent of whether the initial excitation occurs in the RC or antenna domain. We find RC excitonic delocalization over Acc and A 0 while the intermediate CSS is delocalized over P 800 , Acc and A 0 . The use of delocalization across the special pair and accessory pigments could well be a general strategy used by other photochemical RCs 53 . The HbRC presents an especially tractable case, due to the chemically different pigment in the A 0 site. Our study highlights the different ways in which nature has made use of the basic RC architecture that emerged over 3 billion years ago.</p><!><p>Sample Preparation: The isolation of the HbRC is described in previous studies 13 . The HbRC sample was stored in a buffer solution that contains 5.38 µM HbRC, 100 mM glycine buffer (pH = 10), 0.02% ndodecyl-β-D-maltoside. Before ultrafast spectroscopic measurements, the HbRC solution was treated with 20 mM dithionite to reduce the terminal [4Fe-4S] cluster (F x ). This blocks forward electron transfer from A 0 to F x , resulting in charge recombination of the P 800 A 0 state in ~15 ns 12 . The 1:1 mixture of the HbRC solution and glycerol was loaded into a sealed quartz sample cell with an optical path length of 380 μm.</p><p>The OD values at 786 nm and at 668 nm of the samples were 1.1 and 0.13, respectively. The transient measurements were performed at 74 K using a liquid nitrogen cryostat from Oxford Instrument.</p><p>Spectroscopic Measurements: 2DES spectra were measured by using a pump-probe geometry 2DES setup as described previously (see Section S5 of SI for further details). 29 . The pump pulse was compressed to 12 fs. A two-phase cycling scheme was used to remove scattering and background signals. A shutter added in the probe arm removed residual scattering from the pump. In the experiments, the pulse energy of pump pulses was ~40 nJ and the beam waists (1/e 2 ) for both pump and probe were ~200 μm. 2D experiments were performed under the magic-angle condition and at least three times to ensure reproducibility. The data was analyzed using home-written MatLab scripts. The chirp of the probe pulse was corrected using a third-order polynomial function as described previously 54 . The lifetime density analysis was performed using the OPTIMUS software 34 . The global-target analysis was implemented using CarpetView3D (Light Conversion). Fluence dependence studies were also conducted to avoid the exciton-exciton, exciton-charge, and charge-charge annihilation (Supplementary Fig. 12).</p>