Structure of Light-Harvesting Aggregates in Individual Chlorosomes

Article pubs.acs.org/JPCB

Structure of Light-Harvesting Aggregates in Individual Chlorosomes Lisa M. Günther,† Marc Jendrny,† Erik A. Bloemsma,‡ Marcus Tank,§ Gert T. Oostergetel,∥ Donald A. Bryant,§,⊥ Jasper Knoester,*,‡ and Jürgen Köhler*,† †

Experimental Physics IV, University of Bayreuth, D-95440 Bayreuth, Germany Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands § Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, United States ∥ Groningen Biomolecular Sciences and Biotechnology Institute, Nijenborgh 7, 9747 AG Groningen, The Netherlands ⊥ Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717, United States ‡

S Supporting Information *

ABSTRACT: Among all photosynthetic organisms, green bacteria have evolved one of the most efficient light-harvesting antenna, the chlorosome, that contains hundreds of thousands of bacteriochlorophyll molecules, allowing these bacteria to grow photosynthetically by absorbing only a few photons per bacteriochlorophyll molecule per day. In contrast to other photosynthetic lightharvesting antenna systems, for which a protein scaffold imposes the proper positioning of the chromophores with respect to each other, in chlorosomes, this is accomplished solely by self-assembly. This has aroused enormous interest in the structure−function relations of these assemblies, as they can serve as blueprints for artificial light harvesting systems. In spite of these efforts, conclusive structural information is not available yet, reflecting the sample heterogeneity inherent to the natural system. Here we combine mutagenesis, polarization-resolved single-particle fluorescence−excitation spectroscopy, cryoelectron microscopy, and theoretical modeling to study the chlorosomes of the green sulfur bacterium Chlorobaculum tepidum. We demonstrate that only the combination of these techniques yields unambiguous information on the structure of the bacteriochlorophyll aggregates within the chlorosomes. Moreover, we provide a quantitative estimate of the curvature variation of these aggregates that explains ongoing debates concerning the chlorosome structure.

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INTRODUCTION

positioning of the cofactors, which is usually accomplished by exploiting structural constraints imposed by a protein scaffold. Of all known photosynthetic organisms, green sulfur bacteria have been found to survive under the lowest light conditions. Some of these bacteria can grow photosynthetically by absorbing just a few photons per chromophore per day.5−7 The antenna complexes of these bacteria are chlorosomes, which are large ellipsoidal vesicles (lengths of 100−200 nm, widths of 40−60 nm, and heights of 10−40 nm) that may contain up to 250,000 bacteriochlorophyll (BChl) c, d, or e molecules.2,3,8,9 It is known that the monomers of these BChls self-assemble into supramolecular structural elements stabilized by van der Waals forces and hydrogen-bonding interactions. Remarkably, as opposed to most other light-harvesting antenna complexes in Nature, protein scaffolds do not determine the arrangement of the chromophores within chlorosomes.10 Hence, elucidating the design principles of such a natural

The search for technological solutions that will be able to satisfy the growing energy consumption of mankind in a sustainable, long-term, and environmentally clean manner is one of the greatest challenges man faces in the near future. With the evolution of photosynthesis, Nature has demonstrated that biologically produced chromophores can be exploited successfully for efficient solar energy conversion on large scales and under various environmental conditions. However, an order of magnitude estimate reveals that, under optimum conditions, a single molecule would absorb only a few photons per second.1 Therefore, an antenna network composed of many chromophores that harvests photons is used in photosynthesis to perform the light energy concentration step. Nature achieves the high efficiency of light collection and energy transfer by exploiting extremely high densities of chromophores in the antenna systems, which typically correspond to concentrations in the order of 1−3 M.2,3 Such high concentrations of chromophores in solution would result in quenching and the loss of the absorbed energy as heat.4 In natural systems, concentration quenching is avoided by the appropriate © XXXX American Chemical Society

Received: April 12, 2016 Revised: May 30, 2016

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DOI: 10.1021/acs.jpcb.6b03718 J. Phys. Chem. B XXXX, XXX, XXX−XXX

The Journal of Physical Chemistry B

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RESULTS AND DISCUSSION In order to reduce the influence of the BChl homologue heterogeneity on the observable spectral features, we have investigated chlorosomes from the bchR mutant of Cba. tepidum. This mutation affects the composition of only one BChl side chain (see Figure 1a).23 Indeed, this leads to a

system could greatly impact the strategies that are considered for the development of novel organic solar cells.11−13 The detailed supramolecular organization of the BChls in chlorosomes, which dictates the nature of the electronically excited states that determine their light-harvesting performance, is the subject of a still ongoing debate, and previous studies have led to various models for the structure, including tubular, lamellar, and rolled lamellar formations.10,14−21 This diversity is a direct consequence of the large degree of structural heterogeneity of the chlorosomes; i.e., variations occur in the size of the aggregates and mixtures of the various types of BChl molecules, which prevent structural determination with atomic resolution using X-ray crystallography. The most detailed structural model that has been proposed to date has emerged from combining genetic modification of the BChl homologue distribution, cryo-EM imaging, molecular modeling, and solidstate nuclear magnetic resonance (NMR).22 Owing to the more uniform pigment content of a triple mutant (bchQRU) from the species Chlorobaculum (Cba.) tepidum, in which the compositions of three of the BChl side chains were controlled,23 it was possible to grow bacteria with chlorosomes that were structurally more homogeneous than those of the wild type (WT). This yielded a detailed microscopic picture for the mutant, which in turn has been translated into a structural model for the WT chlorosomes, featuring syn−anti stacked dimeric [E,M]-BChl c molecules arranged in multilayer tubular superstructures. Additional information about the supramolecular arrangement of the molecular building blocks is accessible from optical spectroscopy.2,24 This is because the properties of the electronically excited states, such as their energetic positions, their oscillator strengths, or the mutual orientations of their transition-dipole moments, depend crucially on the structure of such aggregates.25−27 In particular, circular dichroism (CD) spectroscopy has proven to be a powerful tool to obtain information about the excitonic couplings and the chirality of such structures.28,29 However, the inherent sample heterogeneity greatly affects the application of these methodologies, because decisive features in the optical spectra are often obscured due to ensemble averaging. This can be mitigated by studying individual chlorosomes.20,21,30,31 The recently reported polarization-resolved, fluorescence−excitation spectroscopy on individual chlorosomes provides a unique view on polarization-dependent features of the spectra over a broad excitation range at low temperatures.32,33 On the one hand, a strong modulation of the fluorescence−excitation spectra as a function of the polarization of the incident radiation was found, which suggested a strong spatial organization of the individual BChl molecules. On the other hand, these studies also confirmed the large degree of structural disorder in the WT chlorosomes and the measurements did not support a detailed structural model. In this study, we combine mutagenesis, single-particle spectroscopy, cryo-EM imaging, and theoretical modeling to elucidate the supramolecular arrangement of the BChl molecules within the chlorosome, and to obtain a quantitative estimate of the curvature variation of these aggregates. Remarkably, the comparison of the obtained information with predictions based on previous model structures reveals that it is impossible to pinpoint the structure of the chlorosomes based on data from just single-particle spectroscopy or cryo-EM imaging; only their combination leads to an unambiguous assignment of the chromophore assembly.

Figure 1. (a) Molecular structure of BChl. The table shows the possible side groups R1−R3 for natural BChl c, and BChl d, as well as for two mutants. (b) Stack of 250 fluorescence−excitation spectra for one particular chlorosome as a function of the polarization of the excitation light in a two-dimensional representation. The spectra have been recorded at 1.5 K. (c) Fluorescence−excitation spectrum of panel b averaged over all polarizations (black), together with its decomposition into four Gaussians (colored). (d) Fitted polarization-resolved fluorescence excitation spectra according to eq 1.

decrease of the full width at half-maximum (fwhm) of the low temperature (room temperature) Qy absorption band from 900 cm−1 (990 cm−1) for the WT to 540 cm−1 (730 cm−1) for the mutant (Supporting Information, Figure S1), reflecting a significant reduction of the sample heterogeneity. These mutants show a similar photosynthetic growth rate as the WT, albeit at a slightly reduced light-harvesting performance,23 which is likely due to the decrease of the absorption bandwidth. Before going into detail, it is worthwhile to clarify once more the hierarchies in the structural arrangements of the BChl molecules to avoid confusion. First, the BChl monomers form supramolecular aggregates, which form the internal structure of chlorosomes. Hence, a single chlorosome refers to a small subensemble typically believed to consist of several tens of molecular aggregates, each consisting of thousands of BChl molecules, and we distinguish three organizational levels: monomers - aggregates - chlorosomes. Figure 1b shows the low-temperature, polarization-resolved, fluorescence−excitation spectrum of an individual chlorosome. The pattern displays 200 consecutively recorded fluorescence−excitation spectra, for B


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Figure 2. (a) Distributions of the spectral peak positions of the polarization-averaged spectra (gray) and of the four fitted Gaussians 1−4 (colored). (b) Distributions of the spectral widths (fwhm) of the polarization-averaged spectra (gray) and of the four fitted Gaussians 1−4 (colored). (c) Energetic separations between bands i and j. (d) Ratios of the oscillator strengths Oi/Oj of the fitted Gaussians i and j. (e) Relative phase angle between the fitted Gaussians i and j. The circles in the histograms indicate the parameters for the particular chlorosome whose spectra are displayed in Figure 1. (f) Correlation between the energetic separations ΔE12 and ΔE34 obtained for individual chlorosomes.

observed in each of the 66 recorded spectra from individual chlorosomes of the mutant. In order to analyze the fluorescence-excitation spectra and the underlying spectral contributions more quantitatively, we developed a fit routine based on the following general expression for the polarization-resolved fluorescence-excitation spectrum:

which the horizontal axis corresponds to photon energy, the vertical axis corresponds to the polarization angle, and the intensity is given by the color code. Between two successive scans, the linear polarization of the excitation beam was rotated over 3°. Integration over all polarizations yields the spectrum shown by the black line in Figure 1c, which features an asymmetric band with a width of 479 cm−1 (fwhm) and a maximum at 13476 cm−1. Clearly, over the entire spectral band, the two-dimensional representation of the data reveals a modulation of the fluorescence intensity with 180° periodicity as a function of the polarization of the incident radiation. Considering that this spectrum is generated by the interaction of hundreds of thousands of transition-dipole moments, the observed polarization dependence is most remarkable, indicating that collective excitations dominate the spectrum. This feature was

F(E , Θ) = B +

∑ Ak cos2(Θ − φk)Lk(E − Ek) k

(1)

Here B is a small constant to account for background signals, k counts the spectral contributions associated with the individual chlorosome, Θ is the polarization angle of the incident light, and φk represents the angle of the projection of the transitiondipole vector of the respective spectral component onto the sample plane with respect to some arbitrary reference axis. Finally, Ak and Lk(E − Ek) give the amplitudes and lineshapes C


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The statistics clearly show that there are two pairs of spectral components energetically close to each other, one in the lowenergy part of the spectrum (E1 = 13468 ± 26 cm−1 and E2 = 13502 ± 41 cm−1) and another one in the high-energy part of the spectrum (E3 = 13685 ± 39 cm−1 and E4 = 13747 ± 57 cm−1). These pairs of two transitions will be referred to as the low-energy doublet and the high-energy doublet, both featuring distributions for their spectral splittings. Nevertheless, for an individual chlorosome, the spectral separations within the lowenergy doublet and the high-energy doublet, ΔE12 and ΔE34, are correlated, Figure 2f and Figure S3a. Such correlations are not observable for other combinations ΔEij vs ΔEkl (for i, j ≠ k, l), Figure S3b,c. Moreover, from the fwhm distributions, it follows that high-energy doublet transitions are broader than the low-energy components. This suggests that the underlying structure gives rise to some degree of coupling between these electronic transitions, so that the states of the higher-lying doublet may relax to the lower one. The distributions for the ratios of the oscillator strength and the mutual angles between the transition-dipole moments of the various bands are shown in Figure 2d and e, and the statistical data are summarized in Table 2. It is clear that certain optical transitions that underlie the observed spectra are polarized more or less perpendicular to each other, while others are polarized nearly parallel to each other. In particular, the two spectral components in the lowand high-energy doublets are strongly perpendicularly polarized to each other, as demonstrated by the narrowness of the distributions for ΔΦ12 and ΔΦ34. The observed polarization properties are consistent with, and a strong independent indication for, an underlying tubular symmetry for the overall BChl arrangement within an aggregate.24,27,34 The strong resonant excitation-transfer interactions between the individual BChl molecules lead to the formation of Frenkel excitons, i.e., collective electronic excitations shared by many molecules. The observations reflect the robustness of excitons in self-assembled nanotubes against the localization effects of random disorder in their microscopic parameters, such as the transition energies of the individual molecules as a result of random solvent shifts.35 This robustness can be traced back to the behavior of the exciton density of states near the band bottom and results in strongly delocalized exciton states whose wave functions (and thus excitation densities) wind around the tube at least once, which in turn leads to a clear distinction between optical transitions with a polarization perpendicular and parallel, respectively, to the axis of the tube.35 This interpretation is confirmed by cryoEM imaging, which indeed reveals a tubular arrangement of the BChl molecules within the aggregates, in which a repeat distance of 1.24 ± 0.06 nm in the direction of the cylinder axis is observed from power spectra of individual chlorosome images (Figure S5 and Figure 3a). This indicates the presence of syn−anti dimers as the repeating unit in the BChl stacks, a situation similar to what was observed for WT chlorosomes.22 The widths of the various distributions in Figure 2 reflect small variations in the arrangements and (or) the microscopic disorder realizations of the light-harvesting aggregates in individual chlorosomes, as will be demonstrated below. The distributions for the mutual polarization angles between transitions in the low-energy doublet and transitions in the high-energy doublet are broader than those for ΔΦ12 and ΔΦ34, although there still seems to be some preference for parallel or perpendicular orientations. A likely explanation for

of the spectral bands. The latter is assumed to have a Gaussian form characterized by the energetic center position Ek and a fwhm denoted by Wk. The experimental 2D polarization-resolved spectra are fitted to eq 1 using a standard, nonlinear fitting approach based on the least-squares method (see Materials and Methods). The number of spectral contributions taken into account was restricted to four, because this is the minimal number of transitions necessary to reproduce all the features of the experimental spectra reasonably well. These considerations also resulted in consistent and physically reasonable values for the fit parameters (see below). Note that we have a total of 16 independent fitting parameters associated with these four spectral bands. Figure 1c displays the decomposition of the polarization-averaged spectrum obtained from Figure 1b (black line) into four Gaussian transition bands. For future reference, these bands are labeled 1−4, in order of increasing energy. For the example in Figure 1, this analysis revealed broad absorption bands with energy positions (fwhm) of E1 = 13437 cm−1 (W1 = 226 cm−1), E2 = 13484 cm−1 (W2 = 280 cm−1), E3 = 13629 cm−1 (W3 = 487 cm−1), and E4 = 13716 cm−1 (W4 = 464 cm−1). The mutual polarization angles between the various absorption bands, determined as ΔΦij = |φi − φj| if the result is less than 90° and ΔΦij = |180° − |φi − φj|| otherwise, are given by ΔΦ12 = 79.4°, ΔΦ13 = 4.4°, ΔΦ14 = 80.7°, ΔΦ23 = 83.8°, ΔΦ24 = 1.3°, and ΔΦ34 = 85.1°. The fitted 2D polarization spectrum for the measurement presented in Figure 1b is shown in Figure 1d (see also the Supporting Information movie). Following this procedure, we analyzed 66 individual chlorosomes, out of which 58 complexes could be decomposed into four spectral contributions similar to the example given in Figure 1c (Supporting Information, see Figure S2 for three additional examples). The remaining eight chlorosomes could not be analyzed in this way, and they were not used for further analysis. Figure 2 shows the statistics for the (a) spectral peak positions, (b) fwhms, (c) energetic separations ΔEij between the spectral peaks, (d) ratio of the oscillator strengths, (e) mutual polarization angles of the various transitions that follow from the fit routine, and (f) correlation between the energetic separations ΔE12 and ΔE34. The distribution of the peak positions of the total (polarization averaged) spectra shown in gray scale in Figure 2a is characterized by a mean of E = 13609 cm−1 and a width (standard deviation) of 33 cm−1, which is about a factor of 2 smaller than the 59 cm−1 that have been found for the width of the corresponding distribution of a wildtype sample.33 This indicates a more homogeneous overall absorption band for the chlorosomes of the bchR mutant, which is also reflected in the statistics of the underlying spectral contributions, revealing narrow distributions for the energy positions and fwhms as summarized in Table 1. Table 1. Mean Values and Standard Deviations of the Distributions for the Peak Positions, fwhms, and the Oscillator Strength of the Transition Bands 1−4 Obtained for the 58 Chlorosomes Analyzed as Described in the Texta i

1

2

3

4

Ei (cm−1) Wi (cm−1) Oi (au)

13468 ± 26 233 ± 35 52 ± 13

13502 ± 41 270 ± 41 59 ± 15

13685 ± 39 495 ± 66 98 ± 22

13747 ± 57 501 ± 63 82 ± 22

a

The distributions for Ei and Wi are given in Figure 2. D


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Table 2. Mean Values and Standard Deviations Obtained from the Distributions of the Spectral Separations, Ratios of the Oscillator Strengths and Mutual Orientations of the Transition-Dipole Moments of the Various Pairs of Transition Bands 1−4a

a

ij

12

13

14

23

24

34

ΔEij (cm−1) Oi/Oj ΔΦij (deg)

34 ± 31 0.95 ± 0.35 78 ± 16

217 ± 28 0.57 ± 0.24 28 ± 27

279 ± 51 0.69 ± 0.26 62 ± 28

182 ± 45 0.63 ± 0.22 67 ± 26

245 ± 38 0.78 ± 0.32 23 ± 26

62 ± 53 1.24 ± 0.30 87 ± 3

The distributions for ΔEij, Oi/Oj, and ΔΦij are given in Figure 2.

Figure 3. (a) Averaged power spectrum from 16 individual cryo-EM images of bchR mutant chlorosomes (Figure S5). The red arrow indicates the presence of a layerline at 1/1.24 nm−1. (b) Distribution of the angles β12 and β34 that characterize the orientation of the molecular transition dipoles with respect to the symmetry axis of a cylindrical aggregate. (c) Basic structural element used for the BChl arrangement within a molecular aggregate within a chlorosome as obtained from NMR and molecular modeling.22 The grid features a unit cell with lattice parameters a and b that are inclined by an angle γ with respect to each other. Each unit cell contains two molecules whose transition dipoles make an angle η with the a-axis and are tilted by small alternating angles ± α out of the lattice plane (green and red arrows, respectively) as a result of the syn-anti stacking; see also top view at the bottom. This alternation is so small that optically it may be neglected, so that the structure effectively may be considered as having just one molecule per (smaller) unit cell. (d−f) Model structures for BChl aggregates obtained by wrapping the grid under an angle of δ with respect to the a-axis onto a cylinder. The grid as well as the structures for parts e and f have been adapted from ref 22.

from the statistics of the oscillator strengths presented in Figure 2d using the aforementioned relationship. The dipole angles have mean values (standard deviation) of β12 = 56.1 (4.7)° and β34 = 52.2 (3.4)°, suggesting that tubes responsible for the low and high energy doublet transitions indeed have slightly different structural arrangements. Structural models for tubular aggregates can be obtained by wrapping a two-dimensional lattice of molecules on a cylindrical surface.36 In order to associate the spectroscopic and cryo-EM imaging data with a microscopic model, we used as a starting point the type of lattice deduced by Ganapathy et al. from NMR and molecular modeling for the chlorosomes from the WT and the bchQRU mutant.22 Thus, we consider an oblique two-dimensional lattice, as depicted in Figure 3c. This lattice is rolled on a cylindrical surface along the chiral vector that makes an angle δ with the a-axis. In order to obtain a seamless tube, the chiral vector should connect two lattice points; its length equals the tube’s circumference (Supporting Information, Figure S6). We used the above model to simulate the polarizationresolved fluorescence−excitation spectra. For the particular example shown in Figure 4, we used two cylindrical structures with radii r1 = 14.7 nm and r2 = 9.7 nm to account for the lowand high-energy doublets of the spectrum, respectively. In order to consider the (possibly) different electrostatic local environments, we used slightly different (1.5%) site energies for the BChl molecules in the two tubular structures.

this is that the low- and high-energy doublets originate from different cylindrical arrangements that either are not aligned perfectly parallel to each other or have different disorder characteristics. In principle, structural models that contain two molecules per unit cell also can give rise to four optical peaks in total, two of which are parallel and two (2-fold degenerate ones) that are perpendicular to the cylinder axis. In that case, it is expected that the distributions of the mutual angles between the various transitions are all very similar, which is in contrast to the data displayed in Figure 2e. Hence, on the basis of the statistics presented, transitions 1−4 likely do not all belong to the same cylinder. The low-energy doublet, consisting of transitions 1 and 2, arises from one type of tubular structure, while transitions 3 and 4, which form the high-energy doublet, are associated with a slightly different tubular arrangement. Note that, in a molecular aggregate with tubular symmetry, a direct relationship exists between the ratio of the oscillator strengths of the exciton transitions polarized parallel and perpendicular to the axis of the tube and the angle of the molecular transition dipoles β with respect to this axis, O∥/O⊥ = 2 cos2(β)/sin2(β).36 Here the additional factor of 2 accounts for the fact that the polarization-resolved measurements are on average sensitive to only half of the perpendicular oscillator strength, because it is known that the chlorosomes are oriented with their long axis parallel to the surface of the substrate.21 Figure 3b shows the distribution for the molecular dipole angle associated with the low- and high-energy doublet, denoted by β12 and β34, respectively. These distributions were obtained E


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cryo-EM measurements reported by us for the bchR mutant is to use 0 < δ < 90°, which gives rise to the tube depicted schematically in Figure 3d. For the WT and the bchQRU mutant, rolling angles of δ = 90° and δ = 0° were concluded, respectively (Figure 3e and f and Supporting Information, Figure S6), leading to spatial periodicities given by the lattice constants a for the WT and b·sin γ for the triple mutant. These numbers showed good agreement with the stacking distances of 1.22 ± 0.03 and 0.83 ± 0.03 nm observed in cryo-EM for these two species.22 It should be emphasized that the above conclusions are based on quantities obtained from the fluorescence excitation experiments on individual chlorosomes. Hence, the distributions for the values of these quantities obtained from the singleparticle fluorescence experiments, see Figure 2, correspond to the distribution of a macroscopic ensemble of chlorosomes, for which each individual entry represents an average over the small ensemble of aggregates that constitutes the particular chlorosome under study (intrachlorosome variation). Their widths reflect the fact that, even for the mutant species, heterogeneity is still an important factor. Sources of heterogeneity may be variation in the structural model parameters as well as random disorder in the exciton Hamiltonian resulting from local fluctuations. This issue is addressed more quantitatively in the Supporting Information (Figure S8, Table S3). For aggregates of tubular symmetry, the energetic separation between mutually orthogonal polarized spectral bands, i.e., ΔE12 and ΔE34, is reciprocally related to the radius of the structure;34 see also Figure S9. Simulations reveal that the variations in ΔE12 and ΔE34 observed in Figure 2 reflect variations in the average radii of the cylindrical BChl aggregates between individual chlorosomes extending from 3.7 to 16.6 nm (Supporting Information, Table S4 and Figure S9). Because the observation of a distribution for these energetic separations translates directly into a distribution of curvatures for the structural elements, this finding manifests a high degree of structural heterogeneity between individual chlorosomes. In Figure 5, we show schematic sketches of two concentric cylinders that are consistent with the ratio of the energetic splittings ΔE12/ΔE34 that correspond to the entries in the lower left (left) and upper right (right) corner of the inset of Figure 5. Accordingly, the average curvature of the structural elements within a particular chlorosome displays strong variations from one chlorosome to the other (interchlorosome variation). Given the widths of the distributions for ΔE12 and ΔE34 on the

Figure 4. Simulation of the polarization-resolved fluorescence− excitation spectrum from two cylindrical aggregates. Each cylinder has a length of 30 nm. The molecules are arranged according to the proposed structure for the bchR mutant; see Figure 3d. Further details about the simulations are given in Table 3 and the Supporting Information. Left: Two-dimensional polarization-resolved representation. Right: Gaussian profiles of the underlying transitions from cylinder 1 (blue, red) and cylinder 2 (green, cyan).

More details about the model parameters are provided in Table 3, together with the available information for the WT and the bchQRU mutant, and in the Supporting Information (Figure S7, Table S2). The simulated spectra show a reasonable agreement with the measured ones in Figure 1b and c; in particular, the energy spacing (both magnitude and direction) between the transitions in the low- and high-energy doublets from the analysis of the experimental data (ΔE12 = 34 cm−1; ΔE34 = 62 cm−1, see Figure 1c and Table 2) is reproduced rather well by the simulations (ΔE12 = 39 cm−1; ΔE34 = 59 cm−1, see Figure 4a). For both arrangements, the spatial periodicity (stacking distance of equivalent BChl molecules) along the a-axis agrees within experimental accuracies with the results from cryo-EM. It should be pointed out that the modeling is performed for single aggregates, whereas the spectra have been taken for single chlorosomes that represent an assembly of aggregates. In performing the simulation of Figure 4, fluctuations in the structure and model parameters for a single aggregate, such as random transition energy disorder for molecules within an aggregate, have not been taken into account (see below). Furthermore, the value used for the lattice constant a was allowed to differ, by at most a few percent, from that reported by Ganapathy et al.22 for the WT and bchQRU mutant. Given the different side groups of the BChl molecules, such a small variation is not unreasonable. The important difference with the work of ref 22 lies in the rolling angle δ. The only way to explain both the fluorescence excitation experiments and the

Table 3. Summary of the Values of the Model Parameters for the WT and the bchQRU Mutant from ref 22 as Well as the Values Used for Simulating the Spectra from the bchR Mutanta

a The two entries for the values of a, δ, and d for the bchR mutant refer to cylinder 1 and cylinder 2, respectively. The red boxes emphasize the similarities of distinct parameters between the wild type and the bchR mutant and the bchR mutant and the bchQRU mutant, respectively. *From single-molecule spectroscopy.32 **From single-molecule spectroscopy, this work. ***From cryo-EM imaging, this work.

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chlorosomes is extracted, for example, differences in chlorosome structure during the course of growth and biogenesis of these structures. On the other hand, within an individual chlorosome, the correlation between ΔE12 and ΔE34 indicates a structural hierarchy in the pigment organization, which is consistent with a multilayer arrangement of the BChl aggregates within chlorosomes. For instance, the aggregates of radii r1 and r2 used in the above modeling may well be concentric tubes (as depicted in Figure 5); this, in turn, would be consistent with a coupling between both transition doublets as concluded above from their line widths. We finally stress that the above variations in system parameters do not include variations within an individual aggregate, such as the occurrence of random shifts in molecular transition energies or intermolecular interactions. Such variations are also likely to occur and will lead to some degree of localization of the exciton states.38 Evidently, the current level of modeling does not allow us to extract estimates for the exciton delocalization size in the aggregates making up a chlorosome. It is conceivable that such estimates can be obtained by making more detailed global fits of all measured distributions presented in Figure 2 based on particular models of intra-aggregate disorder. While very interesting, this requires an extensive study, which goes beyond the scope of the present work. Nevertheless, the clear polarization properties reported above do demonstrate that the typical delocalization sizes must be large enough to span the circumference of typical cylinders. In summary, combining information from single-particle spectroscopy and cryo-EM with previous data from NMR and molecular modeling, we have been able to elucidate the dominant structure as well as structural variation of the lightharvesting aggregates in chlorosomes of the bchR mutant of Cba. tepidum. In order to establish the importance of combining the optical and EM experiments to pinpoint the precise structure, we focus on two important pieces of information that are derived from these two types of experiments. The optical data yield the angle β, which is directly linked to the rolling angle δ via β = |(90° − δ + η) mod 180°|. The EM data yield the stacking distance along the axis of the tube, which is given by d = a·sin δ. As is clear, the close agreement between the stacking distance for the bchR mutant reported here and the WT (Table 3, lower red box) suggests that both chlorosome arrangements are compatible with the same structural model, whereas the values found by us for the molecular dipole angles

Figure 5. Concentric cylinders with a ratio of their radii that is consistent with the correlation of the energetic separations of the lowand high-energy doublet as indicated by the color code in the inset.

one hand and the reasonable degree of correlation between their values in an individual chlorosome on the other hand, the structures shown in Figure 5 provide information about the extremes of the variations of the structural elements between the individual chlorosomes. Hence, it is conceivable that for some chlorosomes the dominating spectral contributions stem from structural elements with rather similar curvatures, Figure 6a, whereas for other chlorosomes the dominating spectral contributions stem from structural elements with a higher degree of variation, Figure 6b. However, in a macroscopic ensemble of chlorosomes, this will be manifested as a contribution to the inhomogeneous line broadening. As shown by the Vacha group,20 for sufficiently large curvatures, it suffices to consider only a quarter-cylinder as the primary structural element to reproduce similar spectral signatures as obtained for a closed cylinder. Hence, we may finally speculate that curved structural elements other than closed cylinders, i.e., partial cylinders, lamellae, or spirals, Figure 6c, could contribute to the observed spectral properties. This variation may reflect different growth conditions for the different bacteria from which the macroscopic ensemble of

Figure 6. Speculation about the variation of the overall supramolecular arrangement of the BChl molecules between individual chlorosomes. (a) Cylinders with an overall similar curvature. (b) Cylinders with strongly varying curvatures. (c) Cylinders and curved lamellae with strongly varying curvatures. The gray structure indicates the phospholipid envelope. G


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The Journal of Physical Chemistry B of the bchR mutant suggest a close resemblance between the two mutant structures (Table 3, upper red box). However, having both experimental techniques at hand, we find for the dominant structural element a tubular arrangement of the BChl molecules that is neither compatible with the model for the WT nor the model for the bchQRU mutant as proposed in ref 22. Interestingly, our simulations also reveal that the influence of the long-range order of the BChl arrangement such as variations of the curvature of the cylindrical surface or a spread in the rolling angle are not decisive for the details in the ensemble absorption spectra of the chlorosomes. A scatter in these parameters manifests itself mainly in the widths of the absorption bands. Given that we observe distributions for these parameters, we propose that a macroscopic ensemble of chlosoromes is best represented by a mixture of organelles in which each individual chlorosome accommodates differently curved suprastructures; see Figures 5 and 6. This might also explain some of the debates in the literature concerning different supramolecular organisations of the BChl molecules.14−21 Finally, because the current models of the WT and bchQRU chlorosomes have not yet been compared to spectroscopic data from single chlorosomes, it might be worthwhile to consider deviations of the rolling angle δ from the reported values of 90 and 0°, respectively, also for the chlorosomes from these strains.

light was provided by a tunable titanium:sapphire laser (Coherent, 899-01) (Coherent) that was pumped by a frequency doubled continuous-wave neodynium-yttrium-vanadat (Nd:YVO4) laser (Coherent Verdi V10). The laser wavelength was scanned, with a rate of 2.8 nm/s (≈47 cm−1/ s) by rotating a birefringent filter via a motorized micrometer screw (Melles Griot Nanomover). In order to suppress nonamplified fluorescene from the laser crystal, the output from the laser passed a band-pass filter (BP 736/128; AHF Analysetechnik). The excitation intensity was adjusted to about 3 W/cm2. Between two successive scans, the polarization of the excitation light was rotated in steps of 3° by a waveplate (Thorlabs Inc.) operated by a stepper motor (OWIS GmbH). Focusing of the laser beam and collection of the emission from the sample was accomplished by an objective (Mikrothek, NA = 0.85 or Edmund Optics, NA = 0.85) that was mounted in the cryostat. After passing band-pass or long-pass filters (BP 850/ 80, LP 780) (AHF Analysetechnik, AG, Tübingen, Germany) to block residual laser light, the emission of a well isolated chlorosome was focused onto a single-photon counting avalanche photodiode (SPCM-AQR-15, PerkinElmer, Waltham, MA). For the localization of single chlorosomes, a wide-field fluorescence image was recorded from the sample prior to the excitation spectroscopy experiments. Therefore, a lens that defocused the excitation light to a spot size of about 2 μm in diameter was introduced into the excitation path. The widefield fluorescence image was registered with a charge-coupled device camera (Andor LUCAEM R 604; Andor Technology Ltd.). Model Simulations. The model for the microscopic structure of a cylinder is obtained by wrapping in a helical way the 2D lattice structure obtained previously from NMR and molecular modeling22 onto a cylindrical geometry. The electronically excited states (excitons) are found using a Frenkel exciton model that accounts for the molecular excitation energy and resonance transfer interactions between the molecules within the point dipole approximation. For multiwalled cylinders, the dipole interactions between molecules situated on different walls are neglected. Numerical diagonalization of the Frenkel Hamiltonian yields the exciton eigenstates and exciton energies, which in turn may be used to calculate the fluorescence excitation spectrum using standard techniques. More details are provided in the Supporting Information. Values for the molecular and structural model parameters are taken from previous publications22 and in some cases (indicated in the main text) were fine-tuned to improve the fit to experiment; the remaining unknown parameters (rolling angle and several optical line widths) were determined from fitting the experimental excitation spectra. Cryo-EM Imaging and Analysis. Aliquots of purified chlorosomes (3 μL) were applied to 3.5/1 Qantifoil grids and were plunge-frozen in liquid ethane at 83 K with a Vitrobot vitrification system (FEI, Eindhoven, The Netherlands). Cryoelectron microscopy was performed with a Tecnai G2 Polara electron microscope (FEI, Eindhoven, The Netherlands) equipped with a Gatan energy filter and a specimen temperature of 80 K. Images were recorded at 115,000× magnification in the zero-loss imaging mode, using a slit-width of 20 eV, with a slow-scan CCD camera at 1 μm underfocus, to have optimal phase contrast transfer at 300 kV for details with a periodicity of about 2 nm. Power spectra were calculated from

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MATERIALS AND METHODS Chlorosome Preparations. Chlorosome isolation was basically performed as previously described.37 Cultures of Chlorobaculum tepidum mutant bchR were harvested after 7 days. Cells were centrifuged (7500 × g, 20 min) and were resuspended in isolation buffer (10 mM Tris−HCl pH 7.5, 2.0 M NaSCN, 5.0 mM EDTA, 1.0 mM PMSF, 2.0 mM DTT). Lysozyme (3 mg mL−1) was added to the cell suspension which was then incubated at room temperature for 30 min. Afterward, the cells were mechanically disrupted using a chilled French press at 138 MPa and for at least three cycles. Large cell debris and unbroken cells were removed by centrifugation (10,000 × g for 20 min). The chlorosomes and membrane vesicles in the supernatant were concentrated by ultracentrifugation at 220,000 × g for 2 h. Chlorosomes were separated from membranes on a continuous sucrose density gradient with 7− 47% linear gradients prepared in isolation buffer by ultracentrifugation at 220,000 × g for 18 h at 4 °C. Subsequently, the chlorosomes were washed twice with 4 volumes of phosphate buffer saline (10 mM potassium phosphate pH 7.2, 150 mM NaCl) and pelleted by ultracentrifugation at 220,000 × g for 1.5 h. The isolated chlorosomes were resuspended in 1−2 mL of phosphate buffer containing 1.0 mM PMSF and 2.0 mM DTT and stored at 4 °C until further required.37 Preparation of Single-Particle Sample. The stock solution (OD733 = 48 cm−1) was stored in the dark at −20 °C in a buffer (5 mM dipotassium phosphate (K2HPO4), 5 mM monopotassium phosphate (KH2PO4), 150 nM sodium chloride (NaCl), pH 7.2 at room temperature). For singleparticle experiments, this solution was diluted in the same buffer to 10−9 M, and about 10 μL of the sample solution was adsorbed onto a SiO2 glass substrate under a nitrogen atmosphere for 30 min and mounted in a helium cryostat. Single-Particle Spectroscopy. For polarization-resolved fluorescence−excitation spectroscopy, a previously described home-built widefield/confocal microscope was used. Excitation H


Article

The Journal of Physical Chemistry B the Fourier transforms of boxed areas of 256 × 256 pixels of the individual chlorosome images.

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Prokaryotes; Shively, J. M., Ed.; Microbiology Monographs; Springer: Berlin, Heidelberg, 2006; pp 79−114. (10) Holzwarth, A. R.; Griebenow, K.; Schaffner, K. Chlorosomes, Photosynthetic Antennae with Novel Self-Organized Pigment Structures. J. Photochem. Photobiol., A 1992, 65 (1−2), 61−71. (11) Balaban, S.; Tamiaki, H.; Holzwarth, A. R. Chlorins Programmed for Self-Assembly. In Supermolecular Dye Chemistry; Würthner, F., Ed.; Topics in Current Chemistry 258; Springer Verlag: Berlin, Heidelberg, 2005; pp 1−38. (12) Sengupta, S.; Ebeling, D.; Patwardhan, S.; Zhang, X.; von Berlepsch, H.; Böttcher, C.; Stepanenko, V.; Uemura, S.; Hentschel, C.; Fuchs, H.; et al. Biosupramolecular Nanowires from Chlorophyll Dyes with Exceptional Charge-Transport Properties. Angew. Chem., Int. Ed. 2012, 51 (26), 6378−6382. (13) Eisele, D. M.; Arias, D. H.; Fu, X.; Bloemsma, E. A.; Steiner, C. P.; Jensen, R. A.; Rebentrost, P.; Eisele, H.; Tokmakoff, A.; Lloyd, S.; et al. Robust Excitons Inhabit Soft Supramolecular Nanotubes. Proc. Natl. Acad. Sci. U. S. A. 2014, 111 (33), E3367−75. (14) van Dorssen, R. J.; Gerola, P. D.; Olson, J. M.; Amesz, J. Optical and Structural Properties of Chlorosomes of the Photosynthetic Green Sulfur Bacterium Chlorobium limicola. Biochim. Biophys. Acta, Bioenerg. 1986, 848 (1), 77−82. (15) Holzwarth, A.; Schaffner, K. On the Structure of Bacteriochlorophyll Molecular Aggregates in the Chlorosomes of Green Bacteria. A Molecular Modelling Study. Photosynth. Res. 1994, 41 (1), 225−233. (16) Tamiaki, H. Supramolecular Structure in Extramembraneous Antennae of Green Photosynthetic Bacteria. Coord. Chem. Rev. 1996, 148 (0), 183−197. (17) van Rossum, B.-J.; Steensgaard, D. B.; Mulder, F. M.; Boender, G. J.; Schaffner, K.; Holzwarth, A. R.; de Groot, H. J. M. A Refined Model of the Chlorosomal Antennae of the Green Bacterium Chlorobium tepidum from Proton Chemical Shift Constraints Obtained with High-Field 2-D and 3-D MAS NMR Dipolar Correlation Spectroscopy. Biochemistry 2001, 40 (6), 1587−1595. (18) Pšenčík, J.; Ikonen, T. P.; Laurinmaki, P.; Merckel, M. C.; Butcher, S. J.; Serimaa, R. E.; Tuma, R. Lamellar Organization of Pigments in Chlorosomes, the Light Harvesting Complexes of Green Photosynthetic Bacteria. Biophys. J. 2004, 87 (2), 1165−1172. (19) Linnanto, J. M.; Korppi-Tommola, J. E. I. Exciton Description of Chlorosome to Baseplate Excitation Energy Transfer in Filamentous Anoxygenic Phototrophs and Green Sulfur Bacteria. J. Phys. Chem. B 2013, 117 (38), 11144−11161. (20) Furumaki, S.; Vácha, F.; Habuchi, S.; Tsukatani, Y.; Bryant, D. A.; Vacha, M. Absorption Linear Dichroism Measured Directly on a Single Light-Harvesting System: the Role of Disorder in Chlorosomes of Green Photosynthetic Bacteria. J. Am. Chem. Soc. 2011, 133 (17), 6703−6710. (21) Tian, Y.; Camacho, R.; Thomsson, D.; Reus, M.; Holzwarth, A. R.; Scheblykin, I. G. Organization of Bacteriochlorophylls in Individual Chlorosomes from Chlorobaculum tepidum Studied by 2-Dimensional Polarization Fluorescence Microscopy. J. Am. Chem. Soc. 2011, 133 (43), 17192−17199. (22) Ganapathy, S.; Oostergetel, G. T.; Wawrzyniak, P. K.; Reus, M.; Gomez Maqueo Chew, A.; Aline; Buda, F.; Boekema, E. J.; Bryant, D. A.; Holzwarth, A. R.; de Groot, H. J. M. Alternating Syn-Anti Bacteriochlorophylls Form Concentric Helical Nanotubes in Chlorosomes. Proc. Natl. Acad. Sci. U. S. A. 2009, 106 (21), 8525−8530. (23) Gomez Maqueo Chew, A.; Frigaard, N.-U.; Bryant, D. A. Bacteriochlorophyllide c C-82 and C-121 Methyltransferases are Essential for Adaptation to Low Light in Chlorobaculum tepidum. J. Bacteriol. 2007, 189 (17), 6176−6184. (24) Eisele, D. M.; Cone, C. W.; Bloemsma, E. A.; Vlaming, S. M.; van der Kwaak, C. G. F.; Silbey, R. J.; Bawendi, M. G.; Knoester, J.; Rabe, J. P.; Vanden Bout, D. A. Utilizing Redox-Chemistry to Elucidate the Nature of Exciton Transitions in Supramolecular Dye Nanotubes. Nat. Chem. 2012, 4 (8), 655−662.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b03718. Ensemble spectra, additional excitation spectra, correlation of energy splittings, cryo-electron microscopy, simulations/model calculations, and variations of the model structure (PDF) A movie that illustrates the decomposition of the polarization-resolved fluorescence excitation spectrum of an individual chlorosome into four spectral components (AVI)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +31-50-363-4369. *E-mail: [email protected]. Phone: +49-92155-4000. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS L.M.G., M.J., and J.Kö. thankfully acknowledge financial support by the Deutsche Forschungsgemeinschaft (GRK1640, Ko 1359/27-1) and the State of Bavaria within the initiative “Solar Technologies go Hybrid”. This work was also funded by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the U.S. Department of Energy through Grant DE-FG02-94ER20137 to D.A.B.

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REFERENCES

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Structure of Light-Harvesting Aggregates in Individual Chlorosomes

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