Probing Electron-Transfer Times in Photosynthetic Reaction Centers

May 31, 2012 - A brief discussion is presented of transient hole-burned (HB) spectra (and the information that they provide) obtained for isolated rea...
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Probing Electron-Transfer Times in Photosynthetic Reaction Centers by Hole-Burning Spectroscopy Ryszard Jankowiak* Department of Chemistry and Department of Physics, Kansas State University, Manhattan, Kansas 66506, United States S Supporting Information *

ABSTRACT: A brief discussion is presented of transient hole-burned (HB) spectra (and the information that they provide) obtained for isolated reaction centers (RCs) from wildtype (WT) Rhodobacter sphaeroides, RCs containing zinc-bacteriochlorophylls (Zn-BChls), and RCs of Photosystem II (PSII) from spinach and Chlamydomonas reinhardtii. The shape of the spectral density and the strength of electron−phonon coupling in bacterial RCs are discussed. We focus, however, on heterogeneity of isolated PS II RCs from spinach and, in particular, Chlamydomonas reinhardtii, site energies of active (electron acceptor) and inactive pheophytins, the nature of the primary electron donor(s), and the possibility of multiple charge-separation (CS) pathways in the isolated PSII RC. We conclude with comments on current efforts in HB spectroscopy in the area of photosynthesis and future directions in HB spectroscopy.

Reaction Centers (RCs). Photosynthesis, the main source of energy for life on Earth, converts solar radiation into chemical energy via a multistep processes.1 Solar photons are absorbed by membrane-associated antenna complexes, and excitation energy is efficiently transferred to the RC where it is used to drive charge separation (CS). Although the basic mechanisms of photosynthesis have been understood for some time, many questions remain unanswered. In particular, the CS process in the Photosystem II (PSII) RC is not yet fully understood. As energy demand grows around the world, understanding how plants and bacteria harvest and process solar photons continues to be of great interest. Below, we focus on the electron transfer (ET) in various RCs using the high-resolution technique of hole-burning (HB) spectroscopy. We begin with a discussion of the recently obtained transient HB spectra for bacterial RCs (BRCs),2 focusing on the homogeneous (Γhom) and inhomogeneous (Γinh) broadening and phonon line shape function (in Zn-RC and one of its mutants) needed to provide electron− phonon (el−ph) coupling parameters. The role of protein dynamics in guiding electron-transfer pathways in RCs of Rb. sphaeroides is discussed in detail in ref 3. The second part of this Perspective centers on a discussion of the site energies of cofactors, the nature of primary electron donor(s), and CS pathways in isolated PSII RCs. The key spectroscopic markers setting intact PSII RCs apart from destabilized PSII RCs are provided, and future directions of HB spectroscopy in photosynthesis research are briefly addressed. Structural Similarities. The arrangement of cofactors in ZnRC and PSII RC (i.e., D1/D2/Cytb559 complexes) into two symmetrical groups, referred to by subscripts A/B and D1/D2, respectively, is shown in Figure 1.2,4 The structure of Zn-RC (frame A) is based on the very recent 2.0 Å crystal structure of the RC from Rb. sphaeroides PDB ID 3I4D).5 Frame B shows a © 2012 American Chemical Society

Figure 1. (A) Predicted cofactor arrangement in the Zn-RC. Cofactors are shown in different colors: carotenoid (yellow), Zn-BChls (green; Zn atoms shown as blue spheres), and ubiquinones in red. (B) Arrangement of pigments in the active (D1) and inactive (D2) branches of the PSII RC. (Chls, green; carotenes, yellow; pheophytins, purple; plastoquinones, gray; nonheme iron, red; and nitrogen, blue.).

schematic arrangement of the six chlorophylls (Chls) and two pheophytins (Pheos) in PSII RC based on the structure of Received: April 24, 2012 Accepted: May 31, 2012 Published: May 31, 2012 1684

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T. vulcanus (PDB ID 3ARC).4 The PD1 and PD2 Chls are analogous to the PA and PB bacteriochlorophylls (BChls) of the BRC special pair, respectively,6,7 while the ChlD1,D2 and PheoD1,D2 molecules correspond to the monomeric BChlA,B and the BPheoA,B molecules of the BRCs. By analogy with the BRCs, it is believed the PD1/PD2, ChlD1, and PheoD1 molecules participate in primary CS in the PSII RC,8−10 although the nature of the primary electron donor in isolated PSII RCs is still a matter of debate. An obvious difference between the PSII RC and BRC is that the former contains two additional peripheral Chls (i.e., ChlzD1 and ChlzD2, both weakly coupled to the remaining pigments). As argued below, large spectral congestion of pigments in PSII RC and lack of crystals of the isolated RC (i.e., D1/D2/Cytb559) preparations make the description of excitonic structure and excitation energy transfer (EET) dynamics in isolated RCs difficult. What Information Can Be Obtained by HB Spectroscopy? The HB technique (that probes vibrational dynamics) relies on differences observed in the absorption spectrum of a lowtemperature system after narrow-band laser excitation. If a pigment molecule (in resonance with the laser) experiences photochemical reaction, it ceases to absorb at its original wavelength/frequency, and one speaks of photochemical HB (PHB). In the case of nonphotochemical HB (NPHB), the pigment molecule does not undergo a chemical reaction, but its immediate environment experiences some rearrangement (for details, see refs 11 and 12). Both PHB and NPHB result in the formation of persistent holes, meaning that the holes are preserved after the initial excitation is turned off, as long as low temperature is maintained. In either case, the HB spectrum is obtained as the difference between the measured absorption spectrum before and after laser excitation. Generation of transient HB spectra (the focus of this Perspective) requires the presence of a third, relatively long-lived state. That is, the excited state evolves into a triplet state or is converted photochemically to another long-lived (μs to ms range) product (e.g., a charge-separated state), leaving a transient hole in the absorption spectrum with a zero-phonon hole (ZPH) at the frequency of the original excitation (resonant HB) and with shape defined by the strength of el−ph coupling. In this case, the pigment’s ground state is depopulated for the lifetime of the long-lived state, and the spectral hole will be observable only for the duration of this lifetime. The transient holes discussed below are acquired as the difference between the absorption spectra measured while the excitation is on and off (postburn absorption, i.e., after saturation of a persistent hole).

in the low-temperature limit as the integrated area of the spectral density)11 for phonons and/or pseudolocalized phonon frequencies. The width of the ZPH in resonant PHB spectra depends on the lifetime of the excited state and “pure” dephasing and/or ET time. ZPHs can be fitted with a Lorentzian profile as they reflect the homogeneous line width (i.e., Γhom = 1/2 ZPH width11,12). The ET time is obtained from Γhom using eq 1 Γhom(cm−1) = (1/2πcT1 + 1/2πcτET) + 1/πcT2* ≈ 1/2πcτET

(1)

where T1 is the fluorescence lifetime; T2* is the “pure” dephasing time, which (at T = 5 K) is very large in comparison to T1; c is the velocity of light in (cm s−1); and τET is the ET time.11,12 Equation 1 provides τET because the latter is ≪T1. A general schematic for numerical simulations of HB spectra in excitonic systems is shown in Figure 2.13 Disorder is

Figure 2. Modeling of HB spectra. Adopted from ref 13.

accounted for by ensemble averaging over pigment site energies chosen randomly from a Gaussian distribution characteristic of each pigment within the complex. Each randomly generated set of (“pre-burn”) site energies corresponds to a realization of disorder for one complex in an ensemble. For each complex, the Hamiltonian generated by the preburn site energies and coupling constants is diagonalized, and a line shape is calculated using either an assumed (static) single-site spectrum (convolution method)14,15 or a more advanced approach including delocalization and energy-transfer effects.13,16−18 The excitonic line shapes from all states are then summed to produce the preburn absorption spectrum. After the preburn spectrum is generated, a decision is made whether to “burn” the selected complex; this corresponds to a random event depending on whether or not the given complex is excited by the laser and whether a particular excitation event leads to HB. The calculated line shapes depend in both cases on the temperature (T), el−ph coupling strength S, and phonon spectral density or “one-phonon profile” (equivalent to spectral density up to a temperature-dependent weight on the one-phonon profile).11,13 Note that the one-phonon profile at low temperatures can be accessed by HB experiments. If a given complex is selected for burning, a single pigment is selected randomly with probability proportional to its contribution to the lowest-energy excitonic state, and its preburn site energy is altered, mimicking the HB event. Finally, the postburn absorption spectrum is calculated using the new “postburn” site energies as input parameters; the difference between the

Generation of transient HB spectra (the focus of this Perspective) requires the presence of a third, relatively long-lived state. Selected information provided by HB spectroscopy (relevant to data discussed below) includes11 (1) lifetimes of the zeropoint level of S1(Qy) states due to EET and/or ET, as determined by the widths of ZPH; (2) Γinh values, typically ∼50−200 cm−1, derived from the ZPH action spectrum, that is, the envelope of ZPHs burned at different wavelengths under constant irradiation dose conditions; and (3) the el−ph (protein) coupling parameter (Huang−Rhys factor, S, defined 1685

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postburn and preburn absorption spectra produces the HB spectrum. Both resonant and nonresonant (transient and/or persistent) holes can be calculated.11,13,19

The width of the ZPH in resonant PHB spectra depends on the lifetime of the excited state and “pure” dephasing and/or ET time. For situations in which excitonic effects are not of interest, the more rigorous calculations just described are not necessary, and a simple time-dependent expression for the HB spectrum can be obtained by assuming a single (static) line shape for an isolated state. The expression briefly discussed below is then valid as long as hole-refilling (from either photoproduct or thermal effects) can be neglected. In this case, the photochemical (transient) HB spectra of P870 in BRC can be also defined by ΔA = A t (Ω, t ) − A t = 0(Ω)

(2)

where At=0(Ω) is the preburn absorption spectrum and A t (Ω , t ) =

∫ dω LA(ω − Ω)N(ω)e−PσϕL (ω −ω)t A

B

(3)

is the postburn absorption spectrum. ωB is the burn/ excitation frequency, σ and ϕ are the integrated absorption cross sections of the molecule aligned with the transition dipole parallel to the laser polarization and HB quantum yield, respectively. P is the photon flux, t is the burn time, N(ω) is the preburn site distribution function (SDF), describing the probabilities of encountering different zero-phonon transition frequencies, and LA(ω) is the single site absorption profile. In the Franck−Condon approximation for an absorptive transition linearly coupled to a harmonic bath, the single-site absorption profile can be expanded in terms of R phonon profiles.2 In this approach, a static absorptive line shape function can be used to describe the phonon line shape function more accurately than that in the “mean phonon approximation”.20,21 For the calculations presented in Figure 3A/B, the contributions of R phonon transitions to the absorption spectrum (both creation and annihilation) were obtained by numerical convolution of the one-phonon profile with itself (R − 1 times) and with the ZPL.2 To account for lifetime broadening of the transition, the line shape function (the single-site absorption profile, LA(ω)) was convolved with a Lorentzian line shape function before evaluating At(Ω,t). For details, see refs 2 and 22. Bacterial RCs. We continue with ET in a novel Zn-RC of Rb. sphaeroides, containing six Zn-BChls and a “Zn-β-RC” mutant in which the Zn-BChl in the BPhe-binding site on the A side (HA) has the Zn pentacoordinated.23−25 In wild-type (WT) BRCs, the energetics of the charge-separated intermediates, that is, P+BA− and P+HA− (where P is the initial electron donor, the so-called “special pair” of BChls, i.e. PA/PB), and ET rates are well-understood.26 Due to strong coupling between the PA and PB molecules (in WT Rb. sphaeroides), the respective lowest excitonic state of this dimer is shifted red to 865 nm (at T = 298 K in comparison with the BA/BB and HA/HB absorption bands) and is referred to as the P870 band. The P870 band in Rb. sphaeroides is located at 897 nm at T = 5 K.27 The wellunderstood redox potentials of each step in the A branch result in a series of energetically favorable downhill reactions, and the 11,20

Figure 3. (A) Schematic view of Zn-BChls with five histidine (His) residues that ligate the BChl Mg2+ in the Zn-β-RC and one leucine (Leu) residue that is close to HB. The letters L and M refer to the protein chains to which the residue belongs. (B) Black and red curves show experimental and calculated absorption spectra of P870 for Zn-βRC, respectively. (C) Calculated (red) and experimental (black) curves show the P+QA− transient spectra of Zn-β-RC. Solid arrows refer to laser burn frequencies (ωB). The inset shows a magnified view of the experimental (black) ZPH obtained with ωB = 11 127 cm−1 (λB = 898.7 nm) for Zn-β-RC and its Lorentzian fit (blue). T = 5 K. Reprinted with permission from ref 2. Copyright 2012, American Chemical Society.

times for ET from P* → HA → QA → QB at room temperature are 3 ps, 200 ps, and 200 μs, respectively.26 The yield of the P+QA− formation depends on the competition between forward ET and the recombination process. A low-temperature absorption spectrum of Zn-β-RC in the Qy region (black curve) is shown in Figure 3B. The peak of the PA,B Qy band (Qy transition of PA,B Zn-BChls) at T = 5 K appears at 884 nm, but to be consistent with literature data (see ref 2), we refer to this band as P870. Recently, the average (weakly frequency-dependent) low-temperature ET rates of the Zn-RC and Zn-β-RC (measured from ZPHs in resonant transient HB spectra) were both ∼1 ps (vide infra). Efficient ET in the Zn-RC was ascribed to the coordination state of the Zn-BChl bound to the HA site. In the WT-RC and Zn-RC, the PA,B and B A,B sites (Mg- and Zn-BChl, respectively) are both pentacoordinated.23,24 In the WT-RC, the HA site binds BPhe, which does not contain a metal ion and possesses a more positive redox potential than BA-BChl. It was suggested that in the Zn-RC, the Zn-BChl bound to the HA site is not penta- but tetracoordinated because a fifth coordinating ligand 1686

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absorption and multiple PHB spectra of P870 for Zn-RC and Zn-β-RC are summarized in Table 1 in the Supporting Information (SI). The presence of strong el−ph coupling in BRCs21,30,31 reveals that P870 possesses a significant chargetransfer (CT) character with the lowest-energy absorption band largely homogeneously broadened. It has been suggested that previously obtained parameters for el−ph coupling in BRCs (via transient HB spectra) need to be re-examined as their values have been underestimated.2 It has been shown recently that in Rb. sphaeroides after dynamic stabilization of the state P + H A −, the environment is optimized to avoid rapid recombination of the charge-separated state, and forward ET to the quinone with very high yield on the hundreds of picoseconds time scale takes place. Thus, by employing protein dynamics, the RC is able to optimize multiple reactions, on very different time scales involving the same reaction intermediate.3 Photosystem II RCs. Historically, the excited state of the PSII RC has been named P680*, with its cationic state referred to as P680+. Below, however, we use the terminology given in Figure 1B with the primary electron donor (depending on CS pathway) being either PD1 and/or ChlD1, while destabilized and intact PSII RCs will be referred to as RC680 and RC684, respectively.32,33 Recent data obtained in various laboratories for the isolated PSII RCs,8,9,32−37 in particular, those recently obtained from spinach35,37 and C. reinhardtii,32,33 continue shedding light on this important biological system. We do not provide a comprehensive analysis of experimental/theoretical efforts describing the excitonic structure of PSII RCs; rather, we place recent findings (e.g., the unusual shapes of transient HB spectra obtained for C. reinhardtii) into a broader context of PSII (CP47-RC-CP43) data.32 For example, it has been shown based on a recent crystal structure of PSII with a 1.9 Å resolution (PDB ID 3ARC) that two vinyl groups of the PD1 and PD2 are oriented differently4 (i.e., due to a dimer formation, a vinyl group from PD1 coordinates to the magnesium in PD2, making this six-coordinated), in agreement with recent calculations using the dispersion-corrected density functional theory of the PD1/PD2 Chl a dimer.38 It has been also suggested that structural asymmetry in the PD1/PD2 dimer may lead to ET along the PD1 branch.38 This is consistent with the midpoint potential (Em) of PD1, which is lower than that of PD2, favoring preferential localization of the charge of the cationic state on PD1.39 The latter suggests that PD1 is the most likely primary electron donor in intact PSII RC. Although our understanding of the PSII RC steadily improves, a satisfactory global understanding of its electronic structure and dynamics has yet to be achieved. The key uncertainty in modeling RC optical spectra (independent of the level of the excitonic theory involved) is that different sets of optical spectra are interpreted in the context of different pigment site energies (i.e., different transition energies induced by the protein environment), which cannot be reliably calculated at the present time due to the very complex protein environment and insufficient structural resolution. Instead, the search for realistic site energies is guided by experimental constraints and aided by fitting algorithms.8,10,33 Active and Inactive Pheophytins. There is general agreement that PheoD1 is the primary electron acceptor in the PSII RC; however, the assignment of its site energy is not agreed upon. While most researchers assigned the Qy states of PheoD1 and PheoD2 (inactive Pheo a) bands at ∼678−684 nm and ∼668− 672 nm, respectively (ref 33 and references therein), recent modeling of the electronic structure of the RC reversed their

(His residue) is absent in the HA site. Thus, it was thought the tetracoordinated Zn-BChl would have a more positive redox potential and be more similar to BPhe than to a pentacoordinated Zn-BChl in the BA site. This difference in coordination state between the cofactors in the BA and HA sites of the Zn-RC was proposed to enable efficient ET, avoiding the deficiency observed in the β-RC mutant (an analogous (Mg)BChl-containing mutant of Rb. sphaeroides26 wherein both BA and HA sites of the Mg-BChls are pentacoordinated). The hypothesis that the coordination state of the HA cofactor tunes the Zn-RC ET rate was tested recently2 by studying the Zn-βRC, in which one amino acid (leucine, Leu) at position 214 of the M protein (M)214 in the Zn-RC HA site was modified so that a His was present in place of the usual Leu; the predicted mutant’s structure is illustrated in frame A of Figure 3, which shows the Zn-β-RC, wherein both BA and HA sites are pentacoordinated.23,24 By analogy with the original β mutant of the WT-RC of Rb. sphaeroides (β-RC), the predicted effect of this change would be a 2-fold reduction to the ET time (PA → BA; vide infra) compared to the unmodified Zn-RC.26 However, recent HB studies2 demonstrated that both Zn-RC and Zn-βRC have similar ET times, suggesting that the difference in coordination state of the HA cofactor is not responsible for preservation of efficient ET in the Zn-RC. In BRC, the primary donor is PA, and the primary ET step is from PA to accessory BChl a (BA). HB probes this primary step; it does not probe the second step that involves BPheo (HA) or whatever is residing in the HA site. Frame B shows the experimental (black curve) and calculated (red) P870 absorption band (at ∼884 nm at T = 5 K; 11 312 cm−1) for Zn-β-RC and the corresponding ZPH action spectrum (with Γinh of ∼130 cm−1). The sharp blue spikes shown in frame B are the inverted ZPH spectra for the Zn-β-RC. (A slightly smaller Γinh of ∼110 cm−1 was obtained for Zn-RC.2 Black spectra a, b, and c in Figure 3C correspond to three transient (P+QA−) spectra of Zn-β-RC obtained for different burning wavelengths (see the arrows). Again, the calculated THB spectra are shown in red. The sharp peaks in the THB spectra in Figure 3C correspond to the ZPHs, which can be fitted with a Lorentzian profile reflecting a homogeneous line width. The primary ET times are obtained using eq 1. An example of ZPH (black) and its Lorentzian fit (blue) is shown as an insert in Figure 3C. In summary, the average ET time in both Zn-RC and Zn-β-RC is ∼1 ps,2 which is similar to a rate previously measured in the WT Rb. sphaeroides RC.28 Thus, the coordination state of HA in the Zn-RC does not tune the ET rate.2 It was also demonstrated in ref 2 that the bleach near 12 346 cm−1 (810 nm), not shown in Figure 3C for brevity, does not represent the P+ component (upper excitonic component of the PA/PB dimer), which must be hidden below the band of accessory BChlA/B, in agreement with the original suggestion by S. Boxer et al.29 Experimentally determined Γinh and a more accurate phonon line shape function (no excitonic features included, just a static line shape) used in calculations allowed a simultaneous fit of absorption (Figure 3B) and transient HB spectra (Figure 3C), providing the HR factors (S), as well as ωSP, often referred to as the special pair marker mode in BRCs.2 Note that a very good agreement exists between experimental curves and their fits in Figure 3B,C. Total HR factors/reorganization energies (=∑i Siωi; in cm−1) for Zn-RC and Zn-β-RC are 4.6/238 and 3.6/ 188, respectively. The weaker el−ph coupling in Zn-β-RC is consistent with more pronounced ZPH in experimental PHB spectra. The parameters obtained from simultaneous fit of 1687

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site energies,40,41 suggesting that the mean site energies of PheoD1 and PheoD2 are ∼672 and ∼678 nm, respectively. In this case, PheoD2, not PheoD1, would strongly contribute to the lowest-energy state. However, studying a large number of isolated RCs from spinach and C. reinhardtii (at different levels of intactness), as well as the C. reinhardtii mutant (D2-L209H) in which PheoD1 has been genetically replaced with Chl a, we have shown recently that the Qx/Qy region site energies of PheoD1 and PheoD2 are ∼545/680 and ∼541.5/670 nm, respectively, in good agreement with previous assignments for the spinach RC.32,33 Frames A and B of Figure 4 show the

the Chl a that replaced PheoD1; all remaining site energies in the WT and mutant RCs remained the same.33 Such a blue shift is justified as mutation of PheoD1 (to Chl a) could break the H-bond between 13′-keto group of PheoD1 and glutamine 130 present in WT RC,42 although changes in the pKA value in the mutated site cannot be excluded. The weaker and blueshifted Q x band of the PheoD2 in the mutant (curve b′ in Figure 4B) and selective bleach of the Qx band of PheoD1 (reflected by curve c′) prove that PheoD1 was replaced with Chl a. In turn, a significant bleach near 684 nm (curve c) in the Qy region shows that PheoD1 strongly contributes to the lowest-energy exciton state. Therefore, its site energy must be red-shifted in comparison with that of PheoD2.33 Yet, a very different conclusion was reached using a chemical modification approach43,44 along with detergent extraction and pigment reconstitution, where it was suggested that site energies of both Pheos have their Qy transition at 676−680 nm.43,44 While chemical modification is suitable for stable isolated BRCs,45,46 it is not clear to what degree it is applicable to the unstable isolated RCs of PSII. Thus, the site-directed mutagenesis approach used in ref 33 should be more reliable. The kinetics of primary CS were not substantially altered in the D1-L210H RC mutant (PheoD2 replaced with Chl a)47 but were strongly modified in the D2L209H (with PheoD1 replaced).33 This indicates that mutation in the inactive D2 branch does not perturb the energetics of the primary electron donor/acceptor pair, which is also consistent with the blue-shifted site energy of PheoD233 as this pigment does not appear to play a major role in the ET process. The blue-shifted Qy transition of PheoD2 is also consistent with the room-temperature absorption spectra of WT-RC and the L210H RC mutant reported in ref 47. Thus, in agreement with our modeling studies, it is unlikely that PheoD2 contributes to the lowest-energy exciton state, as suggested in refs 40 and 41. This leads to a question tackled next. Do the Typically Studied D1/D2/Cytb559 Complexes Provide a Representative Model System for the Intact RC within the PSII (i.e., CP43-D1/D2/Cytb559-CP47) Complex? It has been suggested48,49 that isolated RCs from spinach contain a sampledependent ratio of destabilized (RC680) and intact fractions of RCs, that is, RC684.32−34,49,50 A much larger contribution from RC684 has been recently observed for isolated RCs from C. reinhardtii, where several fractions (in different proportions) referred to as RC680 (no QA), native RC684 (no QA), and native RC684 with QA were observed.32 (One observation that needs to be confirmed indicated that small quantities of isolated RCs prepared from thylakoids membranes contained a larger contribution from intact RC684.) Figure 5 shows normalized 5 K absorption spectra of a typical spinach RC sample (frame A; assigned mostly to RC680 preparation) and the RC from C. reinhardtii (C), which possess a large contribution from RC684 (∼60%); importantly, destabilized RCs from C. reinhardtii have absorption spectra similar to that of the spinach RC.32 Insets in frames A and C show the Qx band of both Pheos with band maxima near 543.0 and 544.2 nm, respectively. The red-shifted maximum of the Qx band in frame C is consistent with the redshifted Qx band observed in intact PSII51,52 (see the SI), suggesting that data shown in frames C and D represent a more intact RC sample than those obtained from spinach. The shape of the transient hole spectrum (Figure 5D), whose hole depth is by a factor of ∼2 larger than that in spinach RC, is also consistent with (PD1+QA−−PD1QA) spectra obtained for the PSII RC core.8,48 The bleach near 673 nm and the increase of absorption observed at >690 nm are signatures of the

Figure 4. (A) Predicted arrangement of PD1, PD2, ChlD1, ChlD2, and PheoD1 replaced with Chl a, and PheoD2 pigments in the D2-L209H RC mutant and their ligands. (B) 5 K absorption of isolated WT-RC (curve a) and D2-L209H RC mutant (curve b) in the Qy and Qx regions (T = 5K). The left inset shows the corresponding Qx region absorption of Pheos. Curves c (c = b − a) and d are the experimental and calculated absorption difference spectra. Adopted from ref 33.

predicted cofactor arrangement in the D2-L209H RC mutant and a comparison of absorption spectra for the WT (curve a) and D2-L209H RC (curve b).33 The areas of spectra a and b were scaled based on the normalized oscillator strength of the cofactors. The red curve c in the right inset is the difference between spectra b and a.33

The key uncertainty in modeling RC optical spectra (independent of the level of the excitonic theory involved) is that different sets of optical spectra are interpreted in the context of different pigment site energies. The blue curve (d) was obtained using excitonic calculations, assuming a blue shift (∼125 cm−1) of only the site energy of 1688

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good agreement with the total absorption shape of RC684, with long-wavelength excited resonant holes matching well the lowenergy absorption of RC684,32 we suggest that the spectrum shown in frame E of Figure 5 represents intact RC. These findings raise the important question of whether conclusions reached over the years based on data obtained in the frequency- and time-domain spectroscopies for isolated RCs, in particular, those obtained for the extensively studied spinach RCs (i.e., dominated by RC680 with the main transient bleach near 680 nm), can be used to describe the intact RC within the PSII system in which the main triplet bleach is near 684/685 nm.8,32 Because isolated RCs can be a mixture of destabilized RC680 and intact RC684, the question is whether the recently observed red-shifted bleach (∼682 nm) in the evolution associated difference spectra (EADS) for narrow femtosecond excitation at 685 nm (fwhm ∼5 nm) in the spinach RC37 (with a major bleach near 680 nm) is caused by a photoselection within a distribution of RC680 complexes or is due to a selective excitation of RC680 and a small contribution of intact RC684. This is important as the data for 680, 682, and 685 nm excitation in ref 37 were not included in the target analysis of EADS and microscopic decay rates, and only six data sets, that is, 660, 665, 670, and 675 nm (5 nm fwhm); 662 nm (8 nm fwhm); and 675 nm (12 nm fwhm) have been linked. Of course, an assumption of photoselection for long-wavelength excitations (due to a large static disorder, Γinh ≈ 160 cm−1, present) is justified as a laser at 685 nm (fwhm ≈ 115 cm−1) should reveal a weak photoselection. The question, however, is what subpopulation of RCs is photoselected at 680 and/or 685 nm excitations in ref 37. Is this a red-shifted subpopulation of RC680 due to large Γinh, or do these excitations select different proportions of RC680 and RC684? If spinach RCs contain a small contribution from RC684, the analysis of data obtained for 685 nm excitation could provide more insight into CS events in intact RCs. However, due to a very broad excitation bandwidth, the excitation at 685 nm must excite a mixture of both RC680 and RC684. This is illustrated in Figure 6A, which shows two femtosecond transient Δ-absorption (77 K) spectra of spinach RC (a and b), adopted from ref 37. These spectra

Figure 5. Frames A and C show the normalized absorption spectra of spinach and C. reinhardtii RCs, respectively. The corresponding insets show the Qx bands of both Pheos. The transient HB spectra of the above RCs λB of 665.0 nm (with a laser intensity of ∼100 mW/cm2) are shown in frames B and D. Frames E and F show absorption spectra of the RC684 and RC680, respectively.32 All spectra were measured at T = 5 K. Reprinted with permission from ref 32. Copyright 2012, American Chemical Society.

PD1+QA− formation (specifically PD1+). Frames E and F show the extracted absorption spectra of pure RC684 and RC680, respectively.32 An absorption spectrum for RC684 from frame E (red curve) is compared with an absorption spectrum for the PSII from spinach51 in Figure S1 in the SI; both spectra are normalized at the Qx band of pheophytins. Integrated areas of these spectra suggest that some of the PSII lack antennas. Therefore, the extracted RC absorption spectrum given in ref 51, obtained by subtracting CP43 and CP47 contributions (scaled based on the number of pigments and their oscillator strengths) from the PSII, may not represent an intact PSII RC absorption. Additionally, PSII complexes seem to be slightly contaminated with CP29 and/or LHCII; this could explain why the spectrum shown in frame E of Figure 5 significantly differs from that reported in refs 51 and 53 (see Figure S2 in the SI). Because the shapes of inverted transient spectra are also in

Figure 6. (A) EADS adopted from ref 37. Spectra a and b (Qy region) were obtained for an isolated RC from spinach at 77 K for 680 and 685 nm excitation. Spectrum a′ is scaled curve a. Spectrum c = b − a′, with a minimum near 684 nm, is very similar to the nonresonant transient hole in RC684 from C. reinhardtii32 and to the shoulder near 684 nm in the transient holes obtained for the spinach RC (see frame B). (B) Spectra d−f are the triplet bottleneck (transient) HB spectra (λB = 665.0 nm; T = 5 K) obtained for three different D1/D2/Cytb559 preparations from spinach. Modified from refs 32. (C) Resonant transient HB spectra for C. reinhardtii; spectra a−d were obtained with λB of 682.0, 684.0, 686.0, and 688.0 nm, respectively. The inset corresponds to the Lorentzian fit (black curve) of the ZPH of curve b. 1689

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Recently, Novoderezhkin et al.10 have commented that the absence of sharp ZPHs (and enhancement of phonon side bands) can be produced by selective coupling between CT states and vibrationally excited exciton states, rather than by strong local Huang−Rhys factors for each site. In this regard, it should be emphasized that HB experimentally probes the line shape of the absorption spectrum and the distribution of these line shapes across an absorption band. Whether the broad line shapes (and absence of ZPHs) observed in HB measurements are interpreted in terms of strong el−ph coupling or in terms of displacement between the nuclear coordinates of CT and exciton states is essentially unimportant from a HB perspective as these measurements probe the final result (the actual absorption spectrum). A detailed agreement between the two descriptions could be confirmed by incorporating the line shape modeling of Novoderezhkin et al. into the excitonic HB calculations outlined in ref 19 and described briefly above, allowing for direct comparison of these calculations with experimental HB data. Chl Site Energies and CS Pathways. Various assignments of the site energies of PD1, PD2, ChlD1, and ChlD2, in the absence of disorder, have been proposed; for references, see the SI. Regarding site energies, no consensus exists, though accurate site energies, as well as coupling constants, the strength of el−ph coupling, and contribution from CT states are critical for description of the RC excitonic structure and CS mechanism(s). It has been suggested recently that both PD1 and ChlD1 can serve as primary electron donors.32,37 In contrast, Renger et al.40 argued that ChlD1 is the major electron donor, both in isolated RCs and the PSII (CP47-RC-CP43). In this case, no distinction between destabilized and intact RC complexes has been made; in theoretical modeling of isolated RC spectra, the site energy of ChlD1 was arbitrarily blue-shifted by ∼4 nm. The latter shift, in fact, supports our analysis that isolated RCs from spinach are mostly destabilized (i.e., RC680). However, our modeling data (to be published elsewhere) suggest that both PheoD1 and ChlD1 strongly contribute to the lowest-energy exciton state and not only ChlD1, as suggested in ref 40. Although resonant HB spectra alone, reported by Acharya et al. in ref 32, cannot distinguish if CS times correspond to one or another CS path, the authors suggested that the ZPHs observed in the 680−685 nm region (corresponding to CS times of ∼1.4−4.4 ps) characterize the ChlD1 pathway, while the observation of the CT state(s) in RC684 (in the 686− 695 nm range) and the absence of ZPHs at λB > 685 nm was considered evidence of the PD1 pathway, for which CS could be ≤1 ps. Thus, on the basis of our recent findings,32 the PD1 pathway, if present in the spinach RC,37 operates only in a very small subpopulation of intact RCs (i.e., RC684) but not in destabilized RC680. That is, we believe that the typically observed bleach near 680 nm (via a triplet-bottleneck state localized on ChlD1) is characteristic of the ChlD1 CS pathway in destabilized (due to weakened PD1−PD2 coupling) RC680. This is consistent with the observed ∼4 nm blue-shifted minimum of the transient hole40 mentioned above. Thus, we conclude that the small and variable transient contribution near 684 nm, which is not accompanied by the 673 nm bleach, originates from the triplet-bottleneck hole in intact RC684 that lacks QA. Consequently, we believe that the major electron donor in intact RCs (i.e., RC684) is PD1, as in the BRCs, though contribution from the ChlD1 path, in particular, at physiological temperature, at proper realization of energetic disorder, is likely present. Thus, the coexistence of both mechanisms might be

can be compared with typical nonresonant transient HB spectra (also for spinach RC) shown in frame B of Figure 6. Curves a and b (scaled at the low-energy wing) are the EADS obtained at 680 and 685 nm, respectively, and correspond to the component with a common lifetime of 20 ps. (Similar spectra were obtained for the 3 ps component37.) It is of interest to reveal the composition of curve b (685 nm excitation); spectrum a′ is the same as curve a but scaled to match the highenergy side of curve b. Curve c = b − a′ has a minimum near 684 nm and is very similar to the 684 nm shoulder observed in 5 K transient HB spectra (also for spinach RC) shown in frame B, assigned to a contribution from the RC684. Note that the latter contribution in spectra d−f (frame B) varies from sample to sample, demonstrating that spinach RCs contain a variable mixture of RC680 and RC684. The 684 nm shoulder is not resolved in EADS at 77 K due to thermal broadening and possibly a smaller contribution from RC684. We suggest that the red-shifted EADS obtained at 685 nm excitation (see curve c in frame A) reveals relatively more contribution from a minor subpopulation of intact RC684 that is also excited at 680 nm and lower-wavelength excitations (e.g., 665 nm) but in different proportions. The estimated ratio of RC680/RC684 in spinach RC from ref 37 for 680 and 685 nm excitations is ∼5 and ∼1, respectively. Thus, the target analysis (reported in ref 37) of data obtained for the excitation wavelength of 660−675 nm favors CS events taking place in destabilized RC680. Figure 6C shows resonant (transient) HB spectra (0.5 cm−1 resolution) for the most intact RC sample studied so far in our laboratory.32 These holes are obtained with λB = 682.0 (green curve a), 684.0 (red curve b), 686.0 (brown curve c), and 688.0 nm (blue spectrum d). Curve c reveals an extremely weak ZPH, as indicated by the brown arrow at 686.0 nm. Note that all spectra exhibit bleaching near 673 nm (revealed for the first time in the resonant transient HB spectra of isolated PSII RCs) and in the 684−686 nm region. The ZPH widths, which varied from 2.4 to 7.6 cm−1 at 682 nm, depending on illumination dose, are believed to reflect a distribution of CS time (τCS). The ZPH widths mentioned above correspond to τCS in C. reinhardtii (at 682−684 nm) in the range of 1.4−4.4 ps, in agreement with previous data obtained for primary CS in spinach RCs.9,49,54,55 The distribution of τCS is consistent with data obtained by 2-D electronic spectroscopy for spinach RCs at 77 K.54 In the latter work, however, no clear response was observed at 684/685 nm, suggesting that this particular sample contained mostly RC680. Interestingly, the ZPHs were nearly absent at λB = 686 nm and entirely absent at 688.0 nm and longer wavelengths, suggesting that excited states of cofactors in intact RC684 are strongly coupled with CT state(s) lying in the long-wavelength region.32 This assignment is in agreement with E. Krausz et al.,51 who showed that excitation wavelengths as long as 695.0 nm (T = 1.7 K) can induce CS in PSII and thus QA− formation.51 However, E. Krausz et al.51 argued that the CT state in the PSII extends far beyond 700 nm (700−730 nm), a notion that requires further confirmation, as the long absorption tail in the PSII is strongly contributed to by antennas and no such absorption was revealed in our intact isolated RC684 complexes.32 In addition, a very small shift of the 673 and 684 nm holes for λB of 686, 688, 690, and 695 nm is consistent with the presence of a weakly absorbing (and predominantly homogeneously broadened) CT state(s) lying near 688−695 nm. Thus, the absence of ZPHs for λB ≥ 686 nm supports the assignment that the lowest-energy CT state is largely homogeneously broadened.56 1690

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responsible for the efficient CS in PSII RC at physiological temperatures as room-temperature protein motions induce a large conformational protein disorder, as originally suggested by R. van Grondelle’s group.57 It appears that conformational protein dynamics modulate pigment−pigment and pigment−protein interactions, leading not only to efficient excitation energy transfer but also to efficient and fast ET times, utilizing different CS pathways. Figure 7 summarizes the most likely CS pathways in both RC680 and RC684 samples with characteristic transient spectra.

HB and single-complex RC studies should be combined with time-domain spectroscopies, in particular, 2D-electronic spectroscopy. Future Directions. The theoretical description of nonresonant and resonant transient holes is restrictive in terms of possible site energies of RC cofactors and should be combined with the simultaneous fit of other linear and nonlinear spectra. Resonant (persistent) holes burned into the lowest-energy state of RCs (and their modeling) should provide information on interaction of low-energy pigments with the protein environment and spectral diffusion, that is, information on protein motion in conformational phase space, as the mechanism of NPHB is associated with a hierarchy of configurational relaxation events triggered by electronic excitation.11 NPHB (including hole growth kinetics measurements), along with single photosynthetic complex spectroscopy and spectral diffusion experiments, could also provide valuable information on the distributions of the barriers on the protein energy landscapes (see refs 59 and 60). Higher-resolution structural data and improved theories for calculating pigment site energies are needed as the composition of various excitonic states is crucial for understanding how these systems work. Advanced descriptions of energy-transfer broadening and coupling to phonons and vibrational modes, as well as experimentally determined spectral density profiles, are desired to further tune pigment site energies and calculate EET dynamics. For example, it remains to be established if spectral densities are the same for individual cofactors of RCs. HB and single-complex RC studies should be combined with time-domain spectroscopies, in particular, 2D-electronic spectroscopy54 using the same samples, to shed more insight into the excitonic structure and mechanisms of CS, in particular, in PSII RC complexes, by simultaneous fitting of both linear and nonlinear spectra. It might be necessary to explore whether the isolated PSII RC complexes are less disrupted using a different buffer system than the one traditionally used in RC sample preparation. The exposure of samples to room temperature and ambient light during isolation must be also minimized to provide more intact complexes for spectroscopic studies. If possible, RCs should be reconstituted into phospholipid bilayer membranes to provide a more in-vivo-like environment. It would be also of interest to study single photosynthetic crystals; then, polarization experiments could be performed on likely narrowed excitonic bands, providing more insight into their excitonic structure. Regarding Zn-RC and Zn-β-RC complexes, it remains to be shown whether these complexes partly lose the HB cofactor during the isolation procedure, as suggested by the absorption spectra in the Qx region.2 Crystals and their X-ray structures, when available, will help to resolve this problem and will help to better interpret changes observed in both the Qx and Qy spectral regions. Finally, regarding BRCs (with less congested spectral overlap of cofactors), we suggest that two-laser experiments (with one laser saturating the PA+QA− state and the second selectively exciting the BB) could reveal more insight on branch B electron transfer.

Figure 7. Summary of CS events in the isolated RCs from C. reinhardtii based on HB spectroscopy data. Frames A and B illustrate possible PD1 and ChlD1 paths of CS (with possible CT states formed) in isolated RC684 (intact) and RC680 (destabilized) complexes. Characteristic transient spectra (with labeled band minima) observed in C. reinhardtii for both types of RCs (i.e., RC680 and RC684) are shown for clarity; see text for details.

Concluding Remarks. The key spectroscopic markers setting RC684 apart from RC680 (both of which lack QA) are (i) the Qx band of PheoD1 in the absorption spectrum is at 545 nm and (ii) a triplet-bottleneck hole in the Q y region (with no response in the Qx region of pheophytins) must be near 684 nm. The shapes of nonresonant transient HB spectra obtained for more intact PSII RC (i.e., RC684 with QA present), with bleaching near 673 and 685 nm, are very similar to P+QA− − PQA absorbance difference spectra measured in PSII complexes from Synechocystis PCC 6803.58 We suggested that in the RC684 complexes, both PD1 and ChlD1 may serve as primary electron donors, leading to two different CS pathways. Resonant HB spectra cannot distinguish the CS times corresponding to different paths, but it is likely that the ZPHs observed in the 680−685 nm region (corresponding to CS times of ∼1.4−4.4 ps) reveal the ChlD1 pathway; conversely, the observation of the CT state(s) in RC684 (in the 686−695 nm range) and the absence of ZPHs at λB > 685 nm likely stem from the PD1 pathway, for which CS could be ≤1 ps. We showed that the average ET time for ZnRC and Zn-β-RC is ∼1 ps, with the ET rate in the Zn-RC in good agreement with recent room-temperature, time-domain data.25 Experimentally determined Γinh decreased the number of variables in theoretical fits of the absorption and frequency-dependent shapes of resonant THB spectra, leading to more reliable HR factors for both low-frequency phonons and a pseudolocalized phonon, ωSP. We anticipate that HB spectroscopy will continue to provide insight into the electronic structure of complex biological systems as HB spectra offer important constraints and parameters for excitonic calculations.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information on RC684 and PSII (i.e., CP43-D1/ D2/Cytb559-CP47) spectra. This material is available free of charge via the Internet at http://pubs.acs.org. 1691

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(9) Novoderezhkin, V. I.; Romero, E.; Dekker, J. P.; van Grondelle, R. Multiple Charge-Separation Pathways in Photosystem II: Modeling of Transient Absorption Kinetics. ChemPhysChem 2011, 12, 681−688. (10) Novoderezhkin, V. I.; Dekker, J. P.; van Grondelle, R. Mixing of Exciton and Charge-Transfer States in Photosystem II Reaction Centers: Modeling of Stark Spectra with Modified Redfield Theory. Biophys. J. 2007, 93, 1293−1311. (11) Jankowiak, R.; Reppert, M.; Zazubovich, V.; Pieper, J.; Reinot, T. Site Selective and Single Complex Laser-Based Spectroscopies: A Window on Excited State Electronic Structure, Excitation Energy Transfer, and Electron−Phonon Coupling of Selected Photosynthetic Complexes. Chem. Rev. 2011, 111, 4546−4598. (12) Jankowiak, R.; Hayes, J. M.; Small, G. J. Spectral Hole-Burning Spectroscopy in Amorphous Molecular Solids and Proteins. Chem. Rev. 1993, 93, 1471−1502. (13) Reppert, M. Modeling of Resonant Hole-Burning Spectra in Excitonically Coupled Systems: The Effects of Energy-Transfer Broadening. J. Phys. Chem. Lett. 2011, 2, 2716−2721. (14) Reppert, M.; Acharya, K.; Neupane, B.; Jankowiak, R. Lowest Electronic States of the CP47 Antenna Protein Complex of Photosystem II: Simulation of Optical Spectra and Revised Structural Assignments. J. Phys. Chem. B 2010, 114, 11884−11898. (15) Reppert, M.; Zazubovich, V.; Dang, N. C.; Seibert, M.; Jankowiak, R. Low-Energy Chlorophyll States in the CP43 Antenna Protein Complex: Simulation of Various Optical Spectra. II. J. Phys. Chem. B 2008, 112, 9934−9947. (16) Renger, T.; Trostmann, I.; Theiss., C.; Madjet, M. E.; Richter, M.; Paulsen, H.; Eichler, H. J.; Knorr, A.; Renger, G. Refinement of a Structural Model of a Pigment−Protein Complex by Accurate Optical Line Shape Theory and Experiments. J. Phys. Chem. B 2007, 111, 10487−10501. (17) Zhang, W. M.; Meier, T.; Chernyak, V.; Mukamel, S. ExcitonMigration and Three-Pulse Femtosecond Optical Spectroscopy of Photosynthetic Antenna Complexes. J. Chem. Phys. 1998, 108, 7763− 7774. (18) Ishizaki, A.; Fleming, G. R. On the Adequacy of the Redfield Equation and Related Approaches to the Study of Quantum Dynamics in Electronic Energy Transfer. J. Chem. Phys. 2009, 130, 234110/1− 234110/8. (19) Reppert, M.; Naibo, V.; Jankowiak, R. Analytical Formulas for Low-Fluence Non-Line-Narrowed Hole-Burned Spectra in an Excitonically Coupled Dimer. J. Chem. Phys. 2009, 131, 234104/1− 234104/10. (20) Hayes, J. M.; Lyle, P. A.; Small, G. J. A Theory for the Temperature Dependence of Hole-Burning Spectra. J. Phys. Chem. 1994, 98, 7337−7341. (21) Small, G. J. On the Validity of the Standard Model for Primary Charge Separation in the Bacterial Reaction Center. Chem. Phys. 1995, 197, 239−257. (22) Acharya, K.; Neupane, B.; Reppert, M.; Feng, X.; Jankowiak, R. On the Unusual Temperature-Dependent Emission of the CP47 Antenna Protein Complex of Photosystem II. J. Phys. Chem. Lett. 2010, 1, 2310−2315. (23) Yeates, T. O.; Komiya, H.; Chirino, A.; Rees, D. C.; Allen, J. P.; Feher, G. Structure of the Reaction Center from Rhodobacter Sphaeroides R-26 and 2.4.1: Symmetry Relations and Sequence Comparisons between Different Species. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 7993−7997. (24) Allen, J. P.; Feher, G.; Yeates, T. O.; Rees, D. C.; Deisenhofer, J.; Michel, H.; Huber, R. Structural Homology of Reaction Centers from Rhodobacter Sphaeroides and Rhodopseudomonas Viridis as Determined by X-ray Diffraction. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 8589−8593. (25) Lin, S.; Jaschke, P. R.; Wang, H.; Paddock, M.; Tufts, A.; Allen, J. P.; Rosell, F. I; Mauk, A. G.; Woodbury, N. W.; Beatty, J. T. Electron Transfer in the Rhodobacter Sphaeroides Reaction Center Assembled with Zinc Bacteriochlorophyll. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 8537−8542.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The author declares no competing financial interest. Biography Professor Ryszard Jankowiak (http://www.k-state.edu/chem/people/ faculty/jankowiak.html) joined the Department of Chemistry at Kansas State University (KSU), Manhattan, KS, in 2005 after 20 years of research at Ames Laboratory (U.S. Department of Energy) and the Department of Chemistry at Iowa State University, Ames, IA. He is also an Ancillary Professor in the Department of Physics at KSU. His current research interests include excitonic structure and excitation energy-transfer and electron-transfer processes in various natural and artificial photosynthetic complexes.

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ACKNOWLEDGMENTS This work was supported by the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Science, U.S. Department of Energy (Grant EC9987), and the NSF ARRA Grant (CHE-090795). I would like to thank all coauthors and collaborators who contributed to the papers discussed in this work: Prof. V. Zazubovich (Concordia University, Montreal, Canada), M. Reppert (MIT, Cambridge, MA), Dr. Michael Seibert (NREL, Golden, CO), Dr. Rafael Picorel (CSIC, Zaragoza, Spain); and Prof. Tom Beatty (University of British Columbia, Vancouver, Canada; and his research group). In particular, the author acknowledges Dr. K. Acharya (KSU) for help with figures and references. M. Reppert and V. Zazubovich are also acknowledged for the critical reading of this Article.

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REFERENCES

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