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Conformational Complexity in the LH2 Antenna of the Purple Sulfur Bacterium Allochromatium vinosum Revealed by Hole-Burning Spectroscopy Adam Kell, Mahboobe Jassas, Khem Acharya, Kirsty Hacking, Richard J. Cogdell, and Ryszard J Jankowiak J. Phys. Chem. A, Just Accepted Manuscript • Publication Date (Web): 22 May 2017 Downloaded from http://pubs.acs.org on May 25, 2017
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Conformational Complexity in the LH2 Antenna of the Purple Sulfur Bacterium Allochromatium vinosum Revealed by Hole-Burning Spectroscopy Adam Kell,† Mahboobe Jassas,† Khem Acharya,†,‡ Kirsty Hacking,§ Richard J. Cogdell,§ and Ryszard Jankowiak*,†,⊥ †
Department of Chemistry and ⊥Department of Physics, Kansas State University, Manhattan,
Kansas 66506, United States §
Institute of Molecular Cell and Systems Biology, College of Medical, Veterinary and Life
Sciences, University of Glasgow, Glasgow G12 8TA, Scotland
ABSTRACT This work discusses the protein conformational complexity of the B800-850 LH2 complexes from the purple sulfur bacterium Allochromatium vinosum, focusing on the spectral characteristics of the B850 chromophores. Low-temperature B850 absorption and the split B800 band shift blue and red, respectively, at elevated temperatures, revealing isosbestic points. The latter indicates the presence of two (unresolved) conformations of B850 bacteriochlorophylls (BChls), referred to as conformations 1 and 2, and two conformations of B800 BChls, denoted as B800 R and B800 B . The energy differences between average site energies of conformations 1 and
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2, and B800 R and B800 B are similar (~200 cm-1), suggesting weak and strong hydrogen bonds linking two major subpopulations of BChls and the protein scaffolding. Although conformations 1 and 2 of the B850 chromophores, and B800 R and B800 B , exist in the ground state, selective excitation leads to 1 → 2 and B800 R → B800 B phototransformations. Different static inhomogeneous broadening is revealed for the lowest energy exciton states of B850 (fwhm ~ 195 cm-1) and B800 R (fwhm ~ 140 cm-1). To describe the 5 K absorption spectrum and the abovementioned conformations, we employ an exciton model with dichotomous protein conformation disorder. We show that both experimental data and the modeling study support a two-site model with strongly and weakly hydrogen-bonded B850 and B800 BChls, which under illumination undergo conformational changes, most likely caused by proton dynamics.
1. INTRODUCTION Typically, the photosynthetic unit in purple sulfur bacteria consists of two light-harvesting antenna complexes (LH1 and LH2) and a reaction center, where primary charge separation occurs. The characteristic Q y absorption bands of the LH2 family of proteins are produced by two pools of bacteriochlorophyll (BChl) a, referred to as B800 and B850 to indicate the spectral position of the bands. Each LH2 complex consists of multiple repeating heterodimer subunits, each formed from a single α and β polypeptide pair that spans the membrane once and provides a scaffold for the photosynthetic pigments.1,2 In each heterodimer subunit there is a pair of tightly coupled BChl a molecules that gives rise to B850, while B800 is produced by a third, monomeric BChl a. These two pools of BChl a form two independent rings in the LH2 complex. For example, the B850 ring in Rhodoblastus (Rh.) acidophilus (formerly known as Rhodopseudomonas acidophila)1 consists of a nonamer of (BChl a) dimers with strong excitonic
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coupling between nearest neighbors, up to ~300 cm-1,3 while the B800 ring is a nonamer of BChl a monomers with weak nearest neighbor couplings, 20-25 cm-1,4 due to large center-to-center distances (~21 Å). The LH2 complex from Allochromatium (Alc.) vinosum is larger than Rh. acidophilus LH2 and contains 12-13 subunits, as inferred from electron microscopy5 and singlemolecule spectroscopy6 studies. A recent unified analysis of ensemble and single-complex optical data from the LH2 complex of Rh. acidophilus distinguished between static intercomplex and slow conformational intracomplex disorder.7 The same authors argued that single-LH2 complex measurements performed so far may be biased toward complexes with higher Huang-Rhys factors (S).7 It was also demonstrated that the extent of delocalization of a singlet excited state in the B850 ring is strongly time-dependent. When it is “born” it is completely delocalized over the whole ring, then during its lifetime the extent of delocalization is reduced due to the effects of dynamic disorder.7 However, due to strong exciton-phonon interactions the antenna excitations have been proposed to localize (when deformations are allowed in the system), leading to the formation of exciton polarons and/or self-trapped excitons (STE).8-10 It has also been suggested that charge-transfer (CT) states are present in strongly-coupled complexes from purple bacteria,11-13 and efforts were made to detect low-lying dark states in LH2.14 Although such dark states are difficult to detect, by comparing two forms of LH2 from Rhodopseudomonas (Rps.) palustris Ferretti et al.14 demonstrated using 2DES that dark states may play a crucial role in the efficiency of light harvesting, supporting conclusions of previous studies indicating that CT states and/or polaron pairs are present in LH2 antennas.15,16 The presence of CT states in Rh. acidophilus has also been modeled theoretically.17 However, the molecular identity of CT states in LH2 complexes is still an open question.18
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Alc. vinosum, the purple photosynthetic bacterium of interest to this work, has six pairs of pucBA genes that can encode the α and β polypeptides, which form the LH2 complexes.19 It has been established that there are at least three types of LH2 produced by Alc. vinosum (B800-850, B800-840, and B800-820),20 however, no 3D structure has been determined for any of them. Relatively more B800-840 and B800-820 complexes are produced under low-light growth conditions, while relatively more B800-850 LH2 is produced under high-light conditions.20 Additionally, Alc. vinosum displays a spectral splitting of B800 in absorption5,21 not observed for purple non-sulfur bacteria.2,22,23 While the explanation for the B800 splitting is not known, the bands originate from different BChl spectral forms within a single complex.21 A recent singlemolecule study suggested that excitonic interactions between B800 pigments and between B800 and B850 pigments could contribute to the presence of two B800 peaks (B800 R and B800 B ).6 However, the nature of B850 is also not well understood, in particular the very broad emission spectra (suggesting large electron-phonon coupling9), and the role of CT states and/or exciton polarons is still an open question.18 To provide more insight into the B850 chromophores, and the enigmatic split of B800, in LH2 from Alc. vinosum this work uses HB spectroscopy24,25 and simulations while focusing on B800 B → B800 R and B800 → B850 excitation energy transfer (EET). Possible light-induced photoconversion dynamics will be investigated as well. This work concentrates on the nature of the B850 absorption of the LH2 complex and will argue that the data do not support the recent assignment6 that B800 molecules form exciton coupled dimers, which interact with the B850 BChls giving rise to the split 800 nm Q y band. The low-temperature LH2 absorption spectrum is described by a non-Markovian reduced density matrix approach26 employing a dichotomous model of protein conformational disorder. The nature of B800 pigments is discussed in more
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detail in ref 27, where the possibility that BChls contributing to the split B800, i.e., B800 R and B800 B , differ only in the position of the proton in the BChl carbonyl-protein hydrogen bond is discussed, also supporting a two-site model with strongly and weakly hydrogen-bonded B850 and B800 BChls, reflecting sample heterogeneity. 2. MATERIALS AND METHODS 2.1. Isolation and Purification of LH2 Complexes from Alc. vinosum. Alc. vinosum strain D was grown anaerobically in high light (above 60-80 µmol s-1 m-2) at 40 °C (HL40) in media containing either sodium sulfide or sodium thiosulfate as a reduced sulfur source. Cultures were grown in 500 mL flat bottles and harvested by centrifugation at 1,250 × g for 25 min after two days. Cells were lysed by three passes through a French press (950 psi) in the presence of magnesium chloride and DNase1. Lysate was centrifuged for 10 min at 2,000 × g to remove whole cells and cell debris and then the supernatant was spun for 1 hr at 200,000 × g to pellet membranes. Membranes were resuspended in 20 mM Tris-HCl pH 8.0 to an optical density (OD) of 25 at 850 nm. Membranes were solubilized with Dodecyl-β-D-maltoside at 2 % (w/v) at room temperature and were agitated for 90 min. Unsolubilized material was then pelleted by centrifugation for 30 min at 20,000 × g. Solubilized material was then loaded onto sucrose density centrifugation step gradients and centrifuged for 14 hr at 200,000 × g. The discrete LH2 band was then decanted and further purified via anion exchange chromatography using Poros 20 HQ resin on a BioCad 700E perfusion workstation (Applied Biosystems), and then by size exclusion chromatography using a Superdex G200 column (GE Healthcare). Absorption spectroscopy was used to ascertain the best samples, which were then pooled and concentrated to an OD of 50 or 100 and stored at -20 °C. Room temperature absorption spectroscopy was performed with a Shimadzu UV-1700 PharmaSpec.
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2.2. Experimental Setup. A Bruker HR125 Fourier transform spectrometer was used to measure low-temperature absorption and HB spectra. In absorption and nonresonant HB the resolution was set to 4 cm-1, while for resonant HB a spectral resolution of 1-2 cm-1 was used. The white light intensity and spectral range were restricted for some measurements by adding colored and gray filters to the beam path before the sample holder. The fluorescence spectra were collected, with a resolution of 0.1 nm by a Princeton Instruments Acton SP-2300 spectrograph equipped with a back0illuminated CCD camera (PI Acton Spec-10, 1340 × 400). The laser source for both nonresonant HB and fluorescence was 496.5 nm, produced from a Coherent Innova 90 argon ion laser. In resonant HB experiments, the tunable wavelengths came from a Coherent CR899-21 Ti:sapphire laser (linewidth of 0.07 cm-1) pumped by a Millenia 10s (Spectra-Physics) diode-pumped, solid-state laser at 532 nm. Power from the laser output was stabilized with a Laser Power Controller (Brockton Electro-Optics Corp.) and precisely set by a continuously adjustable neutral density filter. All experiments were performed inside an Oxford Instruments Optistat CF2 liquid helium cryostat. Sample temperature was read and controlled with a Mercury iTC temperature controller. 3. RESULTS AND DISCUSSION In analogy to previously studied LH2 complexes, the Alc. vinosum absorption bands are also labeled as B850 and B800 even though their low-temperature positions are not exactly at 850 and 800 nm (vide infra). Since B800 in Alc. vinosum is split,5,21 the two resulting bands of B800 BChls are referred to below as B800 R and B800 B (see Figure 1). The experimental 5 K Q y region of the absorption spectrum has maxima of B850, B800 R and B800 B at 11 503, 12 433 and 12 630 cm-1 (869.3, 804.3 and 791.8 nm), respectively. The fwhm of B850 is 360 cm-1, which is similar to that of Ph. molischianum at 20 K (350 cm-1)28 but broader by 100-150 cm-1 than the
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corresponding bands observed for Rb. sphaeroides28 and Rh. acidophilus,28,29 thus suggesting larger heterogeneity. The fwhm of B800 R and B800 B at 5 K are about 170 and 270 cm-1, respectively. 3.1. The Composition of the B850 Absorption Band and its temperature dependence. Figure 1 shows a typical 5 K absorption spectrum and temperature-dependent spectra for the LH2 complex of Alc. vinosum. The B850 peak broadens and shifts blue as a function of temperature, revealing a similar behavior to that previously observed in other LH2 complexes28 (see inset). In contrast, B800 R and B800 B shift slightly to longer wavelengths (see Figure S1 in Supporting Information). The temperature broadening from 5-80 K (~32 cm-1) is similar to that observed for Ph. molischianum (~45 cm-1 from 5-100 K),28 explained via temperature dependence of the site-distribution function (SDF) within the framework of a dichotomous exciton model. The model was originally developed to describe dynamics observed in the fluorescence of single photosynthetic LH2 complexes at room temperature30,31 (see Section 4.2 for more details). Extensive comparative studies of many LH2 samples from Alc. vinosum revealed that the shape and maximum of B850 somewhat varied from batch to batch, even when samples were freshly prepared. Similar changes, e.g., a small blue shift and broadening of B850, were also observed in aged (though refrigerated) samples (data not shown). Comparison of B850 in different samples revealed that B850 BChls exist in two conformations, referred to below as conformations 1 and 2. This is consistent with the presence of an isosbestic point (IP) observed near 11600 cm-1 (862.1 nm). In general, samples studied shortly after preparation contained relatively more contribution from the red-shifted conformation 1. This allows us to extract the approximate shape of the blue-shifted conformation 2, whose band is always somewhat broader.
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The latter suggests that heterogeneity is inherent to many natural systems. The result is shown in Figure 2 with red and blue curves representing the experimentally extracted conformations 1 and 2, respectively. An absorption spectrum for a sample with relatively more population of conformation 2 is shown in Figure S2 of the Supporting Information. The width of the pink curve describes the total inhomogeneity of both conformations 1 and 2 of the disordered Frenkel exciton k = 0 state revealed by the measured zero-phonon action (ZPA) spectrum (see Figure 7). In fact, all B850 absorption spectra obtained for various samples can be deconvolved into variable contributions of conformations 1 and 2, with an increasing contribution of conformation 2 in aged samples. It is unlikely that the shift is induced by variable pH values, as samples (with pH ~ 8) were always stored in a frozen state. That is, 1 → 2 transformation occurs in time most likely due to changes in protein conformations, resulting in BChls in conformations 1 and 2 having different site energies. The former may be related to intracomplex disorder, while intercomplex disorder leads to the observed inhomogeneous broadening. The right frame of Figure 2 shows, as an example, difference absorption spectra for temperature changes of 10 K. These data clearly reveal the IP (identical to that observed in Figure 2) supporting the above-mentioned conformational complexity of B850 pigments. There is no clear IP at lower energies. Such behavior is expected for a binary system and is functionally equivalent to a two-site model, suggesting that protein conformational changes do occur. This, in turn, indicates that a standard Gaussian disorder including a dichotomous protein conformational disorder, as described for other LH2 complexes,30,32,33 should be taken into account in order to describe the optical spectra of LH2 complexes from Alc. vinosum. 3.2. Persistent HB Spectra Obtained for Different Excitation Wavelengths. Figure 3A
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shows the 5 K absorption spectrum along with three resonant HB spectra obtained by selective burning into conformation 1 with a burn wavelength (λ B ) of 877.0 nm. Frame B shows the enlarged bleach within the B850 region. The green (curve a), red (curve b) and blue (curve c) HB spectra were obtained with fluences (f = I ∙ t) of 1080, 540 and 180 J cm-2, respectively; where I is laser intensity (~150 mW) and t is burning time in seconds. Note the significantly larger bleach of B800 R , indicating that even very low-energy excitation leads to B800 R → B800 B phototransformation. The shape of the bleach within B850 suggests that laser-induced 1 → 2 phototransformation also occurs, supported by the large (~200 cm-1) anti-hole shift (vide infra). The solid black curve in Figure 4 (left frame) with B850, B800 R and B800 B labeled is the 5 K absorption spectrum in the Q y region. The straight black line at zero is the delta absorption between consecutively measured 5 K spectra probed with low-intensity (~50 µW cm-2) white light to demonstrate that under our experimental conditions sample interrogation by FTIR white light does not induce any spectral changes. See Figure S3 in the Supporting Information for spectral changes induced by high-intensity (~225 µW cm-2) white light over the spectral region of 450-1000 nm. Here we focus on the laser-induced spectral changes by different selective laser frequencies. Curves a-c are the persistent HB spectra obtained with λ B = 496.5, 785.3 and 871.5 nm, respectively. The inset shows that the normalized shapes of all holes and anti-holes within B800 are very similar. The B850 hole depths are very small, however, their shapes (expanded for clarity in the upper left corner of right frame) depend on the excitation frequency. These expanded persistent bleaches (reversible after sample annealing) are arbitrarily shifted and normalized to the bleach of the B800 R band. The shapes of the holes within B850, as well as the large bleaches of B800 R by low-energy excitation, seen in Figures 3 and 4 are highly surprising. Note that curve b (left frame of Figure 4), in addition to the broad hole and anti-hole with
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B800 (whose shapes reflect B800 R and B800 B ), has a sharp resonant hole due to selective excitation within B800 B at 12734 cm-1 (785.3 nm). The zero-phonon hole (ZPH) has a fwhm of 10 cm-1 when fitted with a Lorentzian curve after subtraction of broad changes. The width of the ZPH corresponds to 1.1 ps EET from B800 B to B800 R molecules. The shape of the B800 R bleach clearly indicates that EET is uncorrelated, explaining why the entire B800 R is bleached. The EET time is in perfect agreement with recent transient absorption experiments.21 The bleach within B850 for 12434 cm-1 (804.2 nm) excitation, not shown here, is very similar to curve b. The right frame compares curves c from Figures 3 and 4. The broad bleach at both λ B can be fitted by a Lorentzian with fwhm of 50 cm-1, indicating a very short-lived state. The weak sharp (resolution limited) ZPH is most likely burned at the higher-energy wing of the SDF (see Figure 7). For completeness, we emphasize that all spectra shown in Figures 3 and 4 were probed with low-intensity (50 µW cm-2) white light in a spectral range of 650-1000 nm to avoid white light induced changes. The latter spectral range ensured that carotenoids (Cars) are not excited by the probing FTIR light source in order to eliminate bleaching by very efficient EET from Cars to B850. Car → B850 EET is reflected by significantly deeper B850 holes (Figure 6) than those shown in Figures 3 and 4. In summary, both high- and low-energy laser excitation (as well as high-intensity white light) lead to a phototransformation of B800 R molecules. A detailed analysis of the B800 R → B800 B photoconversion is discussed separately.27 3.3. Fluence-Dependent Nonresonant HB spectra. Figure 5A shows the 5 K Q y region absorption spectrum of Alc. vinosum LH2 and a series of f-dependent HB spectra obtained with λ B = 496.5 nm. HB spectra (from top to bottom) correspond to holes obtained with f = 6, 24, 60, 240, 600, 1680 and 4400 J cm-2, respectively. Again, all spectra were probed with low-intensity FTIR light (50 µW cm-2) in the spectral range of 650-1000 nm to eliminate white light
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illumination of Cars. The B800 R hole is positioned at 12430 cm-1 (804.5 nm) with a corresponding anti-hole peaked at 12620 cm-1 (792.4 nm). The inset shows that for normalized hole depth the shapes of all B800 R holes and anti-holes are the same. The bleaches of the k = 0 state, B850 and B800 R are not correlated, revealing the very complex nature of B850. Figure 5B shows the expanded bleach within B850 from frame A (the corresponding f values are listed for each hole). It is apparent for this λ B that the HB spectra are not conserved. The shapes of B850 HB spectra in Figure 5 suggest that the LH2 samples are very heterogeneous. The so-called B870 (often referred to as the lowest exciton level, k = 0, A symmetry34) with a band position near 11235 cm-1 (890.1 nm), indicated by a dashed arrow, has different relative intensity at low- and high-f. The anticipated structure of the B850 ring (in analogy to other LH2 complexes) renders that the k = 0 state should be forbidden in absorption. It has been proposed that energy disorder mixes the k = 0 and k = ±1 (E 1 ) states and also splits the k = ±1 degeneracy. Interestingly, for very low f, negligible bleach of the k = 0 state is observed. At higher f, two bleaches are resolved near 11235 and 11523 cm-1 (890.1 and 867.8 nm), tentatively assigned to the k = 0 and k = ±1 states (likely mixed with a dark state(s) present within B850), respectively. At large f, the hole minimum assigned to k = ±1 shifts to ~11480 cm-1 (871.1 nm) due to the relatively larger bleaching of k = 0 (see a well-developed shoulder near 11235 cm-1 [890.1 nm] with an accompanying anti-hole near 11745 cm-1 [851.4 nm]). 3.4. Q y Region Absorption and HB Spectra Obtained for Fresh and Partly Damaged Samples Probed with Higher-Intensity White Light. 6A shows the Q y region HB spectra probed with a larger white light intensity of 225 µW cm-2 in a broader spectral range (450-1000 nm covering the absorption region of Cars) for different OD samples, i.e., samples 1 (OD ~ 0.073) and 2 (OD ~ 0.36). Curve 2 in frame B is normalized to the B800 R intensity of sample 1,
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with all HB spectra scaled accordingly. Both samples are form the same batch, the only difference is that sample 1 was freshly prepared before the experiment and studied immediately after the addition of glass-forming solvent, while sample 2 (higher OD sample) was used in two prior experiments and kept in a freezer. The fwhm of B850 increased in sample 2, as shown by spectra 1 and 2 in frame B. A somewhat broader and blue shifted B850 suggests larger disorder, in agreement with a much stronger bleach of k = 0 near 11250 cm-1 (888.9 nm) observed for sample 2 (see frame A). However, based on data shown in Figures 1 and 2 it also appears that sample 2 has a different distribution of conformations 1 and 2. Recall that the absorption spectra shown in Figure 6B were probed with total white light f = 0.2 J cm-2, which also led to partial white light-induced photobleaching. Persistent HB spectra (red and black curves correspond to samples 1 and 2, respectively) in frame A (λ B = 496.5 nm) were obtained with f = 1, 7 and 300 J cm-2 for curves a/d, b/e and c/f, respectively. These spectra were not corrected for white light bleaching, which accounts for about 50% of the difference in hole depth observed for curves a and d when scaled to account for the difference in sample OD. All B850 persistent holes in Figure 6A are positioned at 11500 cm-1 (869.6 nm) and again the B850 hole shapes reveal complex, sample-dependent behavior with the bleach likely contributed to by photochemical HB (PHB) and protein conformational changes. B800 R holes are located at 12415 (black) and 12417 (red), i.e., 805.3 and 805.2 nm, respectively. The small differences are due to a small shift and different distribution of peak intensities in the absorption spectra of samples 1 and 2 (frame B). B800 anti-hole peaks are located at 12600 (black) and 12616 cm-1 (red), i.e., 793.7 and 792.6 nm, respectively. Interestingly, at the early stage of burning the black curves, in contrast to the red curves in frame A and data shown in Figures 3 and 4, reveal less anti-hole near 12600-12616 cm-1 (793.7-792.6 nm). These data could indicate more initial bleach
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of B800 B as shown by the blue curve (d – a) in the inset of frame B. The latter, however, is most likely an artifact due to normalization at B800 R . Since the integrated bleach of B800 R is always larger than the corresponding anti-hole within B800 B , the more likely interpretation of differences exposed in Figure 6A is that sample 2 (curves d-f) has less efficient B800 R → B800 B photoconversion than the direct nonresonant bleach of B800 R pigments. Note that a similar antihole (though still less intense than that observed in the red curves) is clearly visible at higher f in curve f. The less efficient photoconversion is also supported by data in Figure 4. Thus, we do not believe the bleach in the blue curve (d – a) in Figure 6B (indicated by an asterisk) corresponds to the upper exciton component of B850 BChls (see also Section 4.2). 3.5. SDFs for B850 and B800 R . HB spectroscopy can be used to reveal inhomogeneous broadening parameters, i.e., the position and width of the SDF. This can be obtained via ZPA spectroscopy.9,24,25,35,36 In this technique HB spectra are measured for fixed, low-irradiation doses at various λ B across the inhomogeneously broadened absorption profile. The ZPA spectrum is then obtained as a plot of resonant hole depth versus λ B and thus provides a convenient means to directly measure the extent of static disorder. The ZPA spectrum obtained for the k = 0 state (Γ inh ~ 195 cm-1) and B800 R (Γ inh ~ 140 cm-1) are shown in Figure 7. The sharp peaks are the inverted ZPHs, while the broader Gaussian curves reflect inhomogeneous broadening. The solid line shows the bulk absorption spectrum for comparison. Note that the distribution widths for excitonic state energies are narrower than the fwhm for distributions of pigment site energies due to delocalization and exchange narrowing. 3.6. Fluorescence Spectra. Although absorption and HB spectra showed in Figure 6 reveal clear differences, the shape of corresponding fluorescence spectra for these two samples are nearly identical, as emission always occurs from the k = 0 state (see Figure 8). The red and black
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spectra (curves 1′ and 2′) correspond to fluorescence spectra obtained for samples 1 and 2, respectively, whose absorption spectra are shown in Figure 6B. The fact that the measured fluorescence intensity of sample 1 is significantly smaller than that of sample 2, suggests that sample 1 is somewhat less disordered and/or has a larger contribution from a nearly dark quasidegenerate state(s) overlapping (and mixing) with the k = ±1 states, in agreement with HB spectra shown in Figure 6A. The latter could lead to more efficient energy dissipation via a triplet state. Spectra 1′ and 2′ have similar fwhm (310 and 335 cm-1, respectively), however, the maxima of these spectra are significantly red-shifted in comparison to the expected position of the k = 0 level emission, whose spectral position is indicated in Figures 2, 4 and 7. This, besides a subpopulation of LH2 complexes with relatively weak electron-phonon (el-ph) coupling (revealed via sharp resonant holes burned within the k = 0 state) there must be complexes with large el-ph coupling to account for the broad and significantly red-shifted emission band. Such a broad emission observed in other LH2 complexes was first explained by exciton self-trapping, i.e., trapping the exciton on a limited spatial region of the B850 ring.7,9,18,37-3941-43 In fact, the large fwhm of Alc. vinosum emission spectra, in comparison with the fwhm observed for Rb. sphaeroides (310 cm-1)9 and Rh. acidophilus (245 cm-1),40 suggest that Alc. vinosum has a similar, if not slightly larger, contribution from STE formation. 4. MODELING OF ABSORPTION SPECTRA 4.1. Ph. molischianum-Based Structural Model for Alc. vinosum LH2. The X-ray structure of Alc. vinosum is not known as of yet. Since its protein sequence is very similar to that observed in Ph. molischianum, which has C 8 -symmetry,2 it is reasonable to assume that the major building block (i.e., the B850 dimer and B800 monomer) might have similar structures. However, it has been proposed recently that Alc. vinosum may possess C 12 -symmetry. The latter
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was indicated by the number of peaks observed in fluorescence excitation spectra of single complexes at 1.7 K.6 Electron microscopy measurements also suggested that this complex may have 12- or 13-fold symmetry,5 while Cleary et al. has shown that LH2 antenna networks are only stable for distinct symmetries, i.e., C 8 , C 9 , C 10 and C 12 .41 Therefore, in our qualitative modeling study we also adopt C 12 -symmetry. The resulting model structure of Alc. vinosum is shown in Figure 9. That is, the initial atomic coordinates of a single αβ polypeptide heterodimer are taken from the LH2 structure of Ph. molischianum (PDB ID: 1LGH).2 To create an arbitrary LH2 ring subunit coordinates are translated away from the origin (the center of the resulting ring) such that the “outer” most BChl atom (C8 of the B800 BChl) is the desired radius away. The value used in this work is 46 Å, which is in agreement with approximate LH2 particle size (9.2 nm) as determined by electron microscopy measurements.5 A 2D rotational operator (describing rotation about the z-axis) is applied to the input coordinates in order to generate N duplicate copies equidistant about the ring. As stated above, in this work N = 12. Note that each of the twelve subunits is identical to the subunit structure of Ph. molischianum and only the distance of neighboring subunits changes with ring size. Thus, as far as structure-based parameters are concerned intrasubunit (i.e., αβ heterodimer) coupling constants are the same as for Ph. molischianum while intersubunit coupling differ. 4.2. Dichotomous Model. As mentioned in the introduction, the splitting of the B800 band and possible B800-B850 interactions have been recently modeled in ref 6. The authors argued, based on the fits of their fluorescence excitation spectra, that it is essential to take into account a dimerization of the B800 BChl a molecules in a twelve member ring structure. Since our absorption spectra are significantly different than the fluorescence excitation spectra reported in ref 6, we provide an alternative model, also based on the LH2 complex from Ph. molischianum,
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which in the first approximation should provide a good description of the conformational complexity of LH2 from Alc. vinosum. Figure 10 shows a proposed theoretical description of the 5 K B800-850 absorption spectrum (curve a). Bands labeled as 1 (solid purple) and 2 (solid red) are the experimentally extracted conformations mentioned previously (see Figure 2) while 1′ and 2′ (dashed lines) within B850 correspond to the calculated conformations using a dichotomous model.30,32,33 The dotted (narrow) blue and pink curves represent the calculated contributions from B800 R and B800 B BChls, respectively. Curve a′ is the sum of all conformations, showing a qualitative agreement with the experimentally measured absorption spectrum. The main difference between the experimental and calculated spectra is at the low-energy wing of B850, as in our simulations only (diagonal) static disorder of structurally perfect C 12 rings is taken into account. Apparently, in real samples there will also be a (off-diagonal) contribution to the static disorder from structurally disordered rings, as reflected by a variable amount of the k = 0 level observed experimentally. It is well known that disorder-induced mixing relaxes the strict selection rules for perfect rings and leads to a redistribution of dipole strength among other states.34 In structurally disordered rings the wave functions shrink and the excitons localize on smaller parts of the aggregate,10 allowing the the k = 0 state to become optically allowed. The presence of sample-dependent k = 0 state absorption is manifested in data shown in Figures 2, 4 and 7. Table 1 summarizes the parameters used in our modeling study. Fits are obtained for site energies for the B850 and B800 pigments of 11990 and 12580 cm-1 (834.0 and 794.9 nm), respectively, with corresponding inhomogeneous broadening (fwhm) of 390 and 145 cm-1. Note that these fwhm values for inhomogeneous broadening are not the same as measured
16
experimentally for low-energy exciton states. The values reported in Table 1 are adjusted while fitting optical spectra in order to ensure that the static broadening for calculated exciton states (taking exchange narrowing into account) is consistent with the measured ZPA spectra. For simplicity B850 BChls bound to the α and β proteins have the same average site energies, although some studies indicate they may differ.42,43 In order to account for protein conformations, site energy shifts (ΔE) and conformation 2 (higher-energy conformation) probabilities (p 2 ) are included as well. Lognormal spectral densities are used to describe el-ph coupling.44 The lognormal parameters are approximated by fitting experimentally-determined Gaussian-Lorentzian curves for B85045 and B80046 BChls from delta fluorescence line narrowing (ΔFLN) and HB spectroscopies, respectively. Interestingly, the ΔFLN spectra (λ ex = 869 nm) for the low-energy B870 region displays a prominent phonon mode at 18 cm-1, which was described previously by a single Gaussian contribution.45 The same Gaussian parameters for the 18 cm-1 mode are used in the present work (see Table 2). BChl a intramolecular vibrational modes measured by HB47 are incorporated into the line shape with S vib = 0.3 and 0.37 for B850 and B800, respectively. Note that the coupling constant between nearest neighbor B850 BChls calculated from the structure of Section 4.1 is ~200 cm-1 (μ = 6.1 D). It has previously been reported that this value could be as large as 300 cm-1.3,43 Using the same spectral densities as Table 2 with nondegenerate B850 site energies and a coupling constant of 300 cm-1, the experimental absorption and extracted conformations can still be well fit with only small changes to the parameters describing static disorder, i.e., Γ inh, , ΔE and p 2 (data not shown). This demonstrates the robustness of the dichotomous model even though the exact structure, and hence structure-based parameters, is not known.
17
5. FURTHER DISCUSSION 5.1. Fluorescence Excitation Spectra. Figure 11 shows the 1.2 K fluorescence excitation spectrum (curve a) from ref 6 obtained for an ensemble of LH2 complexes from Alc. vinosum embedded in a glycerol matrix using a high excitation intensity I = 100 W cm-2. Note a significantly smaller B800 R /B800 B ratio (r ~ 0.74) in comparison with intact absorption spectra of Alc. vinosum shown in this work (Figure 1) for which r ~ 1.2. This is not surprising as the large excitation intensity used in ref 6 (vide supra) led to B800 R → B800 B phototransformation. To illustrate this point, the pink dots in Figure 11 show data for 5 K fluorescence excitation obtained for a purposefully phototransformed sample in glycerol, with curve b the corresponding absorption (r ~ 0.9). This comparison clearly indicates that such a fluorescence excitation spectrum should not be used to reveal the electronic structure of LH2 complexes. In particular, the fluorescence excitation spectrum obtained for Alc. vinosum in a PVA matrix and described theoretically had an even smaller r ~ 0.65.6 Thus, the suggested dimerization of B800 chromophores and excitonic coupling of B800 and B850 pigments as the origin of the B800 splitting, based on modeling of photoconverted fluorescence spectra, is most likely incorrect. Moreover, none of the persistent HB spectra support such a model. In addition, comparison of curve b and the dotted fluorescence excitation spectrum (for low OD sample) shows that in excitonically coupled LH2 complexes, where B800 R → B850 EET is present, the difference in relative B800 intensity between absorption and fluorescence excitation spectra must be taken into account (unless sample OD is extremely low). Therefore, we conclude that fluorescence excitation spectra obtained for Alc. vinosum with large excitation intensities (~100 W cm-2) should not be used to model absorption spectra of LH2 complexes, and out modeling results in Figure 10 provide a more feasible description of the electronic structure of Alc. vinosum LH2
18
complexes. Nevertheless, we hasten to add that the X-ray structure is needed to confirm or reject the validity of the model discussed above. 5.2. Sample Heterogeneity. Figure 12 shows spectra c and f replotted from Figure 6A (for easy comparison) to illustrate a drastic difference between the nonresonant HB spectra obtained for samples 1 and 2. The green spectrum is curve c normalized with curve f at the B850 bleach (i.e., multiplied by a factor of four). The comparison of normalized spectra reveals that samples represented by absorption spectra 1 and 2 in Figure 6B possess not only different amounts of disordered LH2 rings (see differences near the k = 0 state), but also different bleaching of B800 R with modified B800 R → B800 B phototconversion (see also discussion of data shown in Figure 6B). These data illustrate that not only sample variability, whose origin likely arises due to differing apoproteins encoded by multiple pucBA genes, but also the intensity and spectral range of probing light used in experiments noticeably affect the absorption and HB spectra. For example, this work shows that more bleach in B850 is observed due to very efficient Car → B850 EET, simply induced via probing light expanded to the Car absorption region. Similar spectral changes within B850 are also induced by a narrow (nonresonant) laser excitation of Cars at 496.5 nm as illustrated in Figures 4-6. The k = 0 state, B850 and B800 R hole shapes shown in these figures suggest that B850 BChl a molecules exist in two conformational state and that samples must be mixtures of LH2 complexes with different degrees of disorder and delocalization. The nature of B800 pigments is also complex as they can easily undergo lightinduced phototransformation, however, this issue is beyond the scope of this work and is discussed elsewhere.27 5.3. On the Origin of Nonresonant and Resonant HB Spectra. The data shown in Figures 2, 7B and S2 suggest that LH2 samples of Alc. vinosum are a mixture of differently disordered
19
complexes and the presence of at least two major conformational states, significantly complicating the interpretation of experimental HB spectra. However, excitation- and fdependent hole shapes can provide insight into the excitonic structure of complex biological systems (for this work the composition of B850).25 Thus, this section begins by discussing the shapes of HB spectra bleached within B850. It is well known that the k = 0 state is the lowest energy excitonic state which becomes optically allowed in spatially and energetically disordered LH2 complexes.34 As a result, this state should act as a trap and contribute significantly to observed bleaching. However, as shown for λ B = 496.5 nm (nonresonant) excitation in Figures 4, 5B and 6A, there is a major bleach near the maximum of B850 suggesting that there must be another (nearly dark) state (e.g., a CT state) strongly mixing with the strongly allowed k = ±1 transitions. Such a scenario is consistent with the recent model by Ferretti et al.14 developed for Rps. Palustris in which the dimeric LH2 ring was assumed to be coupled to a single CT state. Resonant excitations used in this work provide additional support for the presence of a dark state; namely, as shown in Figures 3 and 4, burning at 877.0 and 871.5 nm reveals a very broad resonant hole with fwhm ~ 50 cm-1. This broad hole corresponds to a short-lived state (~200 fs) and cannot reflect fast relaxation from the k = ±1 to k = 0 states, as in this case bleaching near the B850 maximum would be negligible. However, it is very likely that the lifetime of the dark state is much longer, i.e., a 70 ps lifetime was assigned to the dark state observed for Rps. palustris.14 Thus, the dark (or nearly dark) state could be mixed with the excited states and this mixed exciton-CT state will look similar to the usual exciton states, in agreement with the model by Ferretti et al.14 This is why the major bleach occurs near the B850 maximum coincident with the intense k = ±1 states. Note that the B850 bleach is not conservative, especially when highintensity (225 μW cm-2) white light in a broad spectral range (450-1000 nm) is used in
20
absorption measurements (see Figure 6A). The latter could suggest that the non-conservative nature of the B850 bleach is related to a mixed exciton-CT state, where the CT state may perhaps be formed between BChl and Car. This conclusion supports the 2D electronic spectroscopy data obtained for the M-LH2 mutant of Rps. palustris14 and confirms that fluorescence quenching in LH2 complexes may indeed occur via CT states. In the case of Alc. vinosum the CT state must be blue-shifted compared to the lowest energy k = 0 exciton states. Finally, we conclude that although the CT state is largely homogeneously broadened, inhomogeneous broadening is also present as demonstrated by HB spectra shown in the right frame of Figure 4. 5.4. On the Origin of Light-Induced Transformational Changes. Note that holes can be nonphotochemical (NPHB) or photochemical (PHB) in nature, while also revealing persistent and/or transient bleaching. In both NPHB and PHB, however, the product is reached via a tunneling process. PHB means that light affects the chromophore itself, whereas NPHB affects the matrix surrounding the chromophore.24,25 Regarding HB spectra shown in Figures 3 and 4, it is likely that due to a very low fluorescence quantum yield (~10%) typically observed in isolated LH2 complexes48 that a large fraction of the absorbed energy could be quenched (vide supra) by mixed exciton-CT states, and/or dissipated by some radiationless process(es) resulting in the excitation of nuclear motions of the protein matrix.49 The latter could lead to a phototransformation of B800 R chromophores. Another possibility is that laser excitation near 871-877 nm may induce protein conformational changes affecting the B850 BChls (e.g., changing the strength of hydrogen bonds in a subpopulation of BChls) and driving B800 R → B800 B . Both processes could lead to the observed spectral changes characterized by a different strength of hydrogen bonds between amino acids and BChls in conformations 1 and 2 (within B850), B800 R BChls, and B800 B BChls. Note that high-intensity probing white light also leads
21
to the same phototransformation of B800 R chromophores (see Supporting Information). Thus, we propose that conformation 1 → 2 phototransformation of B850 BChls, due to conformational changes of the protein environment, can also induce spectral changes of the B800 BChls in the excited state. PHB spectra, the B800 B → B800 R EET time, as well as the rate of B800 R → B800 B phototransformation likely reflects proton dynamics. 5.5. Dark (or Nearly Dark) State within B850. BChls within B850 strongly absorb via short-lived states, i.e., the k = ±1 levels, which can quickly relax to the lowest energy k = 0 level in disordered aggregates from which emission occurs. Thus, as discussed above, one should not expect a strong bleach at the B850 maximum during the HB process, in contrast to the observed shapes of HB spectra presented in this work. Therefore, it is suggested (vide supra) that there must be a dark (or nearly dark) state resonant with the k = ±1 states. Since exciton states mix with the CT state(s), the resulting transition dipole moment will be non-zero and can be directly excited from the ground state. This is why for nonresonant HB (λ B = 496.5 nm) the main bleach occurs near the B850 maximum (see curve a in Figure 4 and HB spectra in Figure 5B). Interestingly, the broad holes for λ B = 877.0 and 871.5 nm (blue and red spectra in the right frame of Figure 4, respectively) can be well-fit with a Lorentzian curve with fwhm ~ 50 cm-1. The latter corresponds to a ~200 fs decay and is assigned to the fast decay time from the mixed exciton-CT state to the superradiant lowest energy k = 0 level. The broad bleach for the k = 0 state is consistent with the shape of the SDF revealed by ZPA spectroscopy in Figure 7. The very weak and narrow (resolution limited) ZPH at λ B = 877.0 nm and its absence for λ B = 871.5 nm in Figure 4 (right frame) indicates that this narrow spike was burned resonantly at the blue edge of the k = 0 state SDF. 5.6. Is B850 Contributed to by Disordered and Nearly Perfect B850 Rings? We have
22
observed that the fwhm of B850 increased in sample 2, as shown by spectra 1 and 2 in Figure 6B. A broader B850 suggests larger disorder, consistent with a much stronger bleach of the k = 0 band near 11230 cm-1 (890.5 nm) observed for sample 2. We also found that the relative intensity of B850, in comparison with the intensity of B800, varies from sample to sample; likely due to apoprotein compositional variability (vide supra). It is likely that the larger intensity of B850 (curve 1 in Figure 6B), when normalized to B800 (in which the excitons are more localized), could be caused by larger exciton delocalization indicating more contribution from lessdisordered B850 C 12 rings. It has been shown that when excitons are more delocalized in the B850 ring, the corresponding transitions dipoles should be stronger than those of B800 BChls.33 The changes observed in absorption and HB spectra are not surprising as Alc. vinosum revealed the potential for a remarkable degree of plasticity in its LH2 structure.20 Thus, in summary, data presented in this work are consistent with a mixture of heterogeneous rings. Single-molecule spectroscopy data also showed that individual LH2 complexes from Rps. palustris 2.1.6 are quite heterogeneous, with alterations in the type of α and β apoproteins within a single LH2 ring.50 Since light intensity conditions/fluctuations under which the cells are grown can also lead to heterogeneity,51 the distributions of BChl a site energies do not have to be symmetrical in heterogeneous rings, leading to changes in the exciton band structure which in turn can alter the absorption and HB spectra. 5.7. STE Emission from B850. The fluorescence spectra of a typical LH2 sample studied in this work is shown in Figure 13 (curve a). The calculated fluorescence (curve b) is obtained as the convolution of the SDF from Figure 7 (fwhm = 195 cm-1) with the single-site fluorescence spectrum (see Table 2 for parameters used in calculating the single-site line shape). The HuangRhys factor S = 1.06 is taken from ΔFLN measurements.45 Curve b represents Frenkel exciton
23
emission, which clearly is insufficient to describe the LH2 fluorescence. It has been shown that STEs emit at longer wavelengths in various LH2 antennas and are more localized than the k = 0 state.9,10 We suggest that the above emissions likely originate from different B850 antenna rings, though it has been argued that they can also originate from the same rings as well.9 The latter seems to be supported by the recent work of Pajusalu et al.,7 who provided deeper insight into the fluorescence spectra of LH2 complexes. For example, the study of LH2 complexes from Rh. acidophilus on both the individual complex and bulk ensemble level revealed that: i) the fluorescence spectra of exciton-like complexes are generally narrower than the STE-like spectra; ii) only the exciton-like spectra reveal narrow k = 0 zero-phonon lines at the origin of the absorption of fluorescence spectra; and iii) the Stokes shift between the absorption and fluorescence spectra increases with increasing exciton-phonon coupling.7 These conclusions are in agreement with the fluorescence spectra reported in this work. 6. CONCLUSIONS This work has demonstrated that B850 and B800 exist in two major conformations (1/2 and B800 R /B800 B , respectively), even at the low temperatures employed. Since the proteins are known to behave non-exponentially, the corresponding states at 5 K, in the absence of lightinduced transformation, most likely become immobile due to an extremely long transition time. Therefore, these states become static inhomogeneous configurations. Thus, the thermodynamic relations discussed in refs 28 and 32 do not hold for Alc. vinosum LH2 proteins studied in this work. However, light-induced transformations take place via high-intensity (≥ 100 J cm-2) probing white light, wavelength-independent selective laser excitation, and an increase in temperature, revealing a dichotomous behavior. Interestingly, even relatively low-intensity white light (≥ 100 µW cm-2) in the spectral region covering Car absorption can lead to very efficient
24
Car → B850 EET that modifies the bleach within B850. Thus, probing light intensity has to be considered in all frequency- and time-domain experiments, especially while using selective resonant excitations within the Q y absorption region. The best fits of the LH2 absorption spectrum, using Redfield theory with a conformational energy difference ΔE = ±100 cm-1 for both B850 and B800 chromophores, is obtained assuming the average B850 and B800 BChl site energies are 11990 and 12580 cm-1 (834.0 and 794.9 nm), respectively. That is, the site energy difference between BChls in conformations 1 and 2 for B850 and between B800 R and B800 B is very similar (~200 cm-1), suggesting weak and strong hydrogen bonds linking subpopulations of BChls with the protein scaffolding. We also suggest that the broad B850, in contrast to B800, is not split due to significantly larger (by a factor of ~3) inhomogeneous broadening. In contrast to ref 6, none of our data support the previous model of dimerized B800 BChl molecules. This work supports a two-site model with strongly and weakly hydrogen-bonded B850 and B800 BChls (independent of each other) which under illumination undergo conformational changes, likely caused by proton dynamics.
25
FIGURES
Figure 1. Left: Typical 5 K absorption spectrum of the LH2 complex from Alc. vinosum. Right: B850 from the intact (freshly prepared) sample measured in the temperature range of 5-80 K. The inset shows temperature dependence of the peak position and bandwidth (fwhm) of B850. IP indicates the energy of the isosbestic point (11600 cm-1; 862.1 nm).
Figure 2. Left: B850 of the intact LH2 complex and its contribution from conformations 1 and 2. k = 0 indicates the maximum of the SDF (pink curve) for the disordered Frenkel exciton k = 0 state, obtained from the ZPA spectrum. Right: Spectra a-d show temperature-induced difference spectra of B850 plotted in 10 K increments, i.e., ΔT = 10 K; IP = isosbestic point.
26
B800 R
A
0.02
a
0.002
0.01 0
λ B = 877.0 nm
0
10000
12000
T=5K 14000
Wavenumber (cm-1)
0.001
Δ Absorbance
0.1
B850
b Δ Absorbance
Absorbance
0.2
c 0
-0.01
-0.001
-0.02
-0.002
B 11000
11500
12000
Wavenumber (cm-1)
Figure 3. Frame A: 5 K Absorption spectrum (black curve) and three f-dependent HB spectra obtained with λ B = 877.0 nm. Frame B: Enlarged bleach within B850. Curves a-c are obtained with f = 1080, 540 and 180 J cm-2, respectively.
Figure 4. Frame A: 5 K B800-850 absorption and persistent HB spectra obtained with λ B of 496.5 nm (curve a), 785.3 nm (curve b), and 871.5 nm (curve c). The inset shows normalized holes and anti-holes bleached within the B800 region. Frame B: 5 K B850 absorption and expanded curve c from left frame (red curve) compared with the HB spectrum obtained with λ B = 877.0 nm (blue curve) from Figure 4.
27
Figure 5. Frame A: Absorption and f-dependent nonresonant HB (λ B = 496.5 nm) spectra. The inset shows normalized B800 holes. Frame B: B850 region of the nonresonant HB spectra. The corresponding f values are listed for each hole.
Figure 6. Frame A: Q y region HB spectra (λ B = 496.5 nm) probed with a white light intensity of 225 µW cm-2 in the 450-1000 nm range for samples 1 (curves a-c) and 2 (curves d-f). Persistent holes were obtained with f = 1, 7 and 300 J cm-2 for curves a/d, b/e and c/f, respectively. Frame B: Q y region absorption spectra of LH2 complexes for different OD samples 1 and 2 normalized at the maximum of B800 R . Frame C: Normalized B800 R bleach for curves a and d compared to the difference curve (d – a).
28
Figure 7. 5 K absorption and inverted ZPA spectra for k =0 and B800 R with corresponding Gaussian curves representing the SDFs.
Fluorescence (arb. units)
0.003
Alc. vinosum T=5 K
0.002
1' 0.001
0 10000
2'
11500 10500 11000 Wavenumber (cm-1)
Figure 8. Spectra 1′ and 2′ are normalized fluorescence spectra of samples 1 and 2, respectively. The fluorescence band maximum is near 11110 cm-1 (900.1 nm). λ ex = 496.5 nm. Nearly identical fluorescence spectra were obtained for 665 nm excitation (data not shown).
29
Figure 9. Ph. molischianum based structure for Alc. vinosum assuming C 12 -symmetry. The B800 and B850 rings of BChls are shown in brown and green, respectively.
Figure 10. Curves a (green) and a′ (dashed black) are the experimental and calculated (using the dichotomous model) 5 K absorption spectra of LH2 from Alc. vinosum. Curves labeled as 1 (solid purple) and 2 (solid red) correspond to experimentally extracted conformations 1 and 2, respectively. 1′ and 2′ (both dashed lines) correspond to the calculated contributions from B850 BChls. The dotted blue and pink spectra represent the calculated contributions from B800 R and B800 B BChls, respectively.
30
Fluorescence Intensity (a.u.)
B800R
B800B
b
a 800
810
790
780
Wavelength (nm)
Figure 11. Curve a is the 1.2 K fluorescence excitation spectrum obtained for Alc. vinosum with high excitation intensity (I = 100 W cm-2).6 The dots represent the 5 K fluorescence excitation spectrum for purposely photoconverted Alc. vinosum LH2, with curve b the corresponding 5 K absorption spectrum. All spectra were obtained for an ensemble of LH2 proteins embedded in a glycerol matrix. λ B = 496.5 nm
0
B800R
ΔAbsorbance
k= 0 c f B850 band
c×4 11000
12000
13000
Wavenumber (cm-1)
Figure 12. Curves c and f are replotted from Figure 7A. The green spectrum is curve c normalized with curve f at the B850 bleach.
31
Figure 13. Curve a represents a typical LH2 fluorescence spectrum obtained with λ ex = 496.5 nm. The Frenkel exciton emission (curve b) corresponds to the k = 0 level fluorescence that was obtained by convolution of the SDF from Figure 7 (curve b′) with the single-site fluorescence spectrum.
32
TABLES Table 1. Disorder Parameters Describing a Dichotomous Model for Alc. vinosum LH2 B850 α
B850 β
B800
Site Energy (cm-1)
11990
11990
12580
Γ inh (cm-1)
390
390
145
p2
0.58
0.58
0.54
ΔE (cm-1)
100
100
100
Table 2. Electron-Phonon Coupling Parameters Defining Lognormal Spectral Densities ω c (cm-1)
σ
S
ω G (cm-1)
Γ G (cm-1)
S
S tot
B850a
85
0.7
2.35
18
18
0.14
2.49
B800
30
0.4
0.4
0.4
a
For B850 the total spectral density is the sum of lognormal and Gaussian components
ASSOCIATED CONTENT Supporting Information. Absorption changes induced by high-intensity white light over a broad spectral range and temperature dependence of the B800 bands. (PDF) This information is available free of charge via the Internet at http://pubs.acs.org AUTHOR INFORMATION Corresponding Author
33
*E-mail:
[email protected]. Phone: 785-532-6785 Present Addresses ‡
Orchid Science College, Bharatpur-10, Chitwan, Nepal
Author Contributions LH2 samples were prepared by K.H. and R.J.C. Experiments were performed by A.K., K.A. and M.J. Calculations were performed by A.K. and M.J. The manuscript was written by R.J. and A.K. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Award Number DE-SC-0006678 (to R.J.). R.J.C. and K.H. acknowledge support from the Photosynthetic Antenna Research Center (PARC), an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Award Number DE-SC-0001035. R.J. acknowledges useful discussions with Drs. Darius Abramavicius and Olga Rancova (Vilnius University, Lithuania). REFERENCES (1) McDermott, G.; Prince, S. M.; Freer, A. A.; Hawthornthwaite-Lawless, A. M.; Papiz, M. Z.; Cogdell, R. J.; Isaacs, N. W. Crystal Structure of an Integral Membrane Light-Harvesting Complex from Photosynthetic Bacteria. Nature 1995, 374, 517–521.
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(2) Koepke, J.; Hu, X.; Muenke, C.; Schulten, K.; Michel, H. The Crystal Structure of the Light-Harvesting Complex II (B800–850) from Rhodospirillum molischianum. Structure 1996, 4, 581–597. (3) Sauer, K.; Cogdell, R. J.; Prince, S. M.; Freer, A. A.; Isaacs, N. W.; Scheer, H. StructureBased Calculation of the Optical Spectra of the LH2 Bacteriochlorophyll-Protein Complex from Rhodopseudomonas acidophila. Photochem. Photobiol. 1996, 64, 564–576. (4) Sundström, V.; Pullerits, T. Photosynthetic Light-Harvesting: Reconciling Dynamics and Structure of Purple Bacterial LH2 Reveals Function of Photosynthetic Unit. J. Phys. Chem. B 1999, 103, 2327–2346. (5) Kereiche, S.; Bourinet, L.; Keefstra, W.; Arteni, A. A.; Verbavatz, J. M.; Boekema, E. J.; Robert, B.; Gall, A. The Peripheral Light-Harvesting Complexes from Purple Sulphuric Bacteria have Different ‘Ring’ Sizes. FEBS Lett. 2008, 582, 3650–3656. (6) Löhner, A.; Carey, A.-M.; Hacking, K.; Picken, N.; Kelly, S.; Cogdell, R.; Köhler, J. The Origin of the Split B800 Absorption Peak in the LH2 Complexes from Allochromatium vinosum. Photosynth. Res. 2015, 123, 23–31. (7) Pajusalu, M.; Kunz, R.; Rätsep, M.; Timpmann, K.; Köhler, J.; Freiberg, A. Unified Analysis of Ensemble and Single-Complex Optical Spectral Data from Light-Harvesting Complex-2 Chromoproteins for Gaining Deeper Insight into Bacterial Photosynthesis. Phys. Rev. E 2015, 92, 052709.
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(8) Timpmann, K.; Ellervee, A.; Pullerits, T.; Ruus, R; Sundström, V.; Freiberg, A. ShortRange Exciton Couplings in LH2 Photosynthetic Antenna Proteins Studied by High Hydrostatic Pressure Absorption Spectroscopy. J. Phys. Chem. B 2001, 105, 8436–8444. (9) Freiberg, A.; Rätsep, M.; Timpmann, K.; Trinkunas, G.; Woodbury, N. W. Self-Trapped Excitons in LH2 Antenna Complexes Between 5 K and Ambient Temperature. J. Phys. Chem. B 2003, 107, 11510–11519. (10) Freiberg, A.; Trinkunas, G. Unraveling the Hidden Nature of Antenna Excitons. In Photosynthesis in silico, Laisk, A., Nedbal, L., Govindjee, Eds.; Advances in Photosynthesis and Respiration, Volume 29; Govindjee, Ed.; Springer: Dordrecht, Netherlands, 2009; pp 55–82. (11) Beekman, L. M. P.; Steffen, M.; van Stokkum, I. H. M.; Olsen, J. D.; Hunter, C. N.; Boxer, S. G.; van Grondelle, R. Characterization of the Light-Harvesting Antennas of Photosynthetic Purple Bacteria by Stark Spectroscopy. 1. LH1 Antenna Complex and the B820 Subunit from Rhodospirillum rubrum. J. Phys. Chem. B 1997, 101, 7284–7292. (12) Beekman, L. M. P.; Frese, R. N.; Fowler, G. J. S.; Picorel, R.; Cogdell, R. J.; van Stokkum, I. H. M.; Hunter, C. N.; van Grondelle, R. Characterization of the Light-Harvesting Antennas of Photosynthetic Purple Bacteria by Stark Spectroscopy. 2. LH2 Complexes: Influence of the Protein Environment. J. Phys. Chem. B 1997, 101, 7293–7301. (13) Somsen, O. J. G.; Chernyak, V.; Frese, R. N.; van Grondelle, R.; Mukamel, S. Excitonic Interactions and Stark Spectroscopy of Light Harvesting Systems. J. Phys. Chem. B 1998, 102, 8893–8908.
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