Fluorescence Excitation Spectra from Individual Chlorosomes of the

Letter pubs.acs.org/JPCL

Fluorescence Excitation Spectra from Individual Chlorosomes of the Green Sulfur Bacterium Chlorobaculum tepidum Marc Jendrny,† Thijs J. Aartsma,‡ and Jürgen Köhler*,† †

Experimental Physics IV and Bayreuth Institute of Macromolecular Research (BIMF), Universität Bayreuth, Universitätsstr. 30, 95447 Bayreuth, Germany ‡ Leiden Institute of Physics, Leiden University, Niels Bohrweg 2, 2333 CA Leiden, The Netherlands S Supporting Information *

ABSTRACT: We performed polarization-resolved fluorescence excitation spectroscopy on individual chlorosomes from the photosynthetic green sulfur bacterium Chlorobaculum tepidum. The experiments were conducted at room temperature and under cryogenic conditions. All spectra showed a strong intensity modulation as a function of the polarization of the incident radiation, and we determined the modulation ratio as a function of the excitation energy. Under ambient conditions this ratio shows only little variation across the absorption band, whereas the low-temperature experiments clearly revealed that the broad absorption band around 740 nm consists of several spectral contributions.

SECTION: Spectroscopy, Photochemistry, and Excited States

I

authors suggested a lamellar arrangement of the pigments.15,16 For the system studied here, that is, wild-type chlorosomes from the green sulfur bacterium Chlorobaculum tepidum, recent cryo-EM data revealed multilayer tubular domains that are embedded in less-ordered lamellar domains of lower curvature.11 An even more detailed model for the pigment arrangement has been achieved by combining cryo-EM and nuclear magnetic resonance (NMR) data.17 It is suggested that the BChl molecules of chlorosomes from Chlorobaculum tepidum form concentric multitubular aggregates that are oriented parallel to the long axis of the chlorosome. Optical spectroscopy has been widely used to study structure−function relationships of supramolecular aggregates.18−26 The large degree of structural disorder among the chlorosomes was confirmed by single-molecule studies.27−32 From absorption linear dichroism (at a single excitation wavelength of 733 nm (13 643 cm−1)) and 2D polarizationfluorescence microscopy (at three distinct excitation wavelengths, 458 nm (20619 cm−1), 633 nm (15 798 cm−1), and 750 nm (13 333 cm−1)) on single chlorosomes,31,32 it was concluded that the dominant contributions to the absorption spectrum stem from exciton states with transition-dipole moments preferentially aligned along the principal axis of the chlorosome and that the overall pigment organization within a chlorosome is consistent with the concentric multitubular synanti chlorophyll aggregate organization found in ref 17.

n photosynthesis, solar radiation is absorbed by a lightharvesting apparatus that acts as an antenna, and the excitation energy is then transferred efficiently to a reaction center. Among the many organisms that perform photosynthesis, green (non) sulfur bacteria feature a light-harvesting machinery of unparalleled efficiency that allows them to thrive in low-light environments.1−4 The main light-harvesting antennae of green (non) sulfur bacteria are chlorosomes. These are considered to be sacks with a length of 100−210 nm, a width of 40−100 nm, and a height of 10−40 nm that accommodate hundreds of thousands of bacteriochlorophyll (BChl) c, d, or e molecules depending on the bacterial species and growth conditions.2−7 In contrast with light-harvesting complexes from other types of bacteria there is no protein scaffold as structure-determining element. Rather, the chromophores are organized in stacked structures that are formed by self-assembly and stabilized by hydrogen-bonding interactions. The excitonic interactions between the aggregated BChl molecules lead to a strong absorption around 740 nm (13 514 cm−1) that is significantly red-shifted with respect to the monomer Qy absorptions at 650 nm (15 385 cm−1). However, because chlorosomes are large and compositionally very heterogeneous, structural information with atomic resolution about the supramolecular organization of the BChl molecules within the chlorosome is not available. This is therefore the subject of a long-standing debate, and in the past many different model structures (lamellas, rods, multiwalled rods, spirals, etc.) have been proposed for the BChl arrangement within the chlorosomes. From various studies,7−14 it was concluded that the interior of the chlorosomes is filled with closely packed rod-shaped structures, whereas other © 2012 American Chemical Society

Received: November 6, 2012 Accepted: December 3, 2012 Published: December 3, 2012 3745

dx.doi.org/10.1021/jz301808h | J. Phys. Chem. Lett. 2012, 3, 3745−3750

The Journal of Physical Chemistry Letters

Letter

In total, we studied 24 individual chlorosomes at room temperature, and more examples of fluorescence excitation spectra from single chlorosomes are shown in the Supporting Information; see Figure S1. For each chlorosome, we determined the normalized modulation ratio M, defined as M = (Imax − Imin)/(Imin + Imax), as a function of the excitation energy. Here Imax (Imin) refers to the maximum (minimum) of the fluorescence intensity as a function of the polarization of the incident radiation. For clarity, the result is shown in Figure 1c only for a selection of 15 typical chlorosomes. The absolute values of M show only relative small variations between the individual chlorosomes, and for each individual chlorosome the modulation ratio is about constant above an excitation energy of 13 320 cm−1. At excitation energies below that value, M is affected by the relative increase of the background level with respect to the fluorescence signal. Therefore, for each individual chlorosome, the modulation ratio was averaged only over all excitation energies above 13 320 cm−1, which yields the distribution shown in Figure 1d. It features a mean of 0.75 and a standard deviation of 0.08. The mean value is in good agreement with the value of 0.77 that has been obtained in ref 32 at a single excitation wavelength of 750 nm (13 300 cm−1), yet the width of our distribution is larger with respect to what has been observed in ref 32, which presumably reflects different sample preparation conditions. To decrease the line widths of the spectral contributions, we performed experiments similar to that described in Figure 1 at 1.5 K. The pattern in Figure 2a displays a stack of 250 consecutively recorded spectra, where the incident polarization has been rotated by 3° between two successive scans. The summed spectrum is shown as the black line in Figure 2b and features a broad asymmetric band peaking at 13 632 cm−1 with a shoulder at 13 382 cm−1. It has an overall width of 548 cm−1 (fwhm). The blue and red lines in Figure 2b correspond to individual fluorescence excitation spectra recorded at the polarizations, indicated by the red and blue lines in Figure 2a, and reveal two spectral contributions to the summed spectrum, one peaking at 13 351 cm−1 with a width of 683 cm−1 (red) and the other one peaking at 13 666 cm−1 with a width of 462 cm−1 (blue). For four distinct spectral positions across the band, Figure 2c shows the fluorescence intensity of the emission as a function of the polarization of the excitation (dots). These spectral positions are indicated by the arrows in Figure 2b and correspond to the peak positions of the two individual polarization-resolved spectra and to two spectral positions (13 120 cm−1 and 13 840 cm−1) that are far in the wing of each line. The latter have been chosen to minimize the crosstalk because the two bands are not orthogonally polarized with respect to each other. For all spectral positions, the polarization-induced intensity variations are consistent with a cos2 dependence (solid black lines in Figure 2c). The relative phase angles with respect to the modulation at 13 120 cm−1 are 5.7°, 29.7°, and 30.6° in the order of increasing photon energy. For 8 out of 25 individual chlorosomes, the fluorescence excitation spectra could be decomposed into two distinct spectral components due to their polarization properties, as shown above. Comparison of these spectra yields a large degree of heterogeneity. The spectra differ from each other with respect to the spectral peak positions, the width of the bands, and the mutual angle between the transition-dipole moments of the two spectral components. For the remaining 17 chlorosomes, we find fluorescence excitation spectra consisting of a featureless broad band that

Here we report about both room-temperature and lowtemperature polarization-resolved fluorescence excitation spectroscopy on individual chlorosomes from wild-type Chlorobaculum tepidum. Experimental details are given in the Supporting Information. Whereas the room-temperature spectra reproduce previously obtained results,31,32 the low-temperature approach allows to decompose the absorption band for some chlorosomes into two spectral components by virtue of their polarization properties. This reveals that the relative weight with which these transitions contribute to the absorption varies as a function of the spectral position within the band. In both temperature regimes, we find for all individual chlorosomes a clear modulation of the fluorescence intensity as a function of the polarization of the incident radiation. An example of a room-temperature polarization-resolved fluorescence excitation spectrum is shown in Figure 1 in a 2D

Figure 1. (a) Stack of 70 fluorescence excitation spectra from an individual chlorosome as a function of the polarization of the excitation light in a 2D representation. Between two successive scans, the polarization was rotated by 12° and the fluorescence intensity is indicated by the color code. (b) Average of 70 individual spectra (black line) and two individual laser scans for distinct polarization angles: 84° (red) and 144° (blue). The corresponding individual spectra are indicated by the horizontal lines in part a of the Figure. For better visibility, the line within each spectrum corresponds to a gliding average over 11 data points of the respective spectrum. (c) Modulation ratio M from individual chlorosomes as a function of the excitation energy. For clarity, four examples have been highlighted by different colors, and the display has been restricted to 15 chlorosomes. The room-temperature fluorescence excitation spectrum from an ensemble of chlorosomes is shown by the bold black line at the top for better comparison. The vertical dotted line corresponds to a photon energy of 13 320 cm−1. (d) Distribution of the modulation ratio from 24 individual chlorosomes, each one averaged over all excitation energies above 13 320 cm−1.

representation. The horizontal axis corresponds to photon energy, the vertical axis corresponds to the polarization of the incident radiation, and the intensity of the spectrum is given by the color code. Between two successive scans, the polarization of the excitation light has been rotated by 12°. The summed spectrum is shown as the black line in Figure 1b and features a broad asymmetric band peaking at 13 691 cm−1 with an overall width of 695 cm−1 (fwhm). The blue and red lines in Figure 1b correspond to individual fluorescence excitation spectra recorded at the polarizations indicated by the red and blue lines in Figure 1a. Unfortunately, these cannot be distinguished and do not provide new information. 3746



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Figure 2. (a,d) Stack of 250 fluorescence excitation spectra from an individual chlorosome as a function of the polarization of the excitation light in a 2D representation. Between two successive scans the polarization was rotated by 3° and the fluorescence intensity is indicated by the color code. (b,e) Average of 250 individual spectra (black line) and two individual laser scans for distinct polarization angles: (b) 138° (blue), 186° (red); (e) 87° (blue), and 156° (red). The corresponding individual spectra are indicated by the horizontal lines in parts a and d of the Figure. For better visibility, the line within each spectrum corresponds to a gliding average over 11 data points of the respective spectrum. (c,f) Fluorescence intensity (dots) as a function of the polarization of the excitation at the spectral positions given in the Figure and marked by the arrows in parts b and e together with cos2-type functions (solid black lines) fitted to the data.

the absolute values found for M can vary between 0.1 (chlorosome no. 6 at 13 951 cm−1) and 0.96 (chlorosome no. 22 at 13 706 cm−1), but also individually the chlorosomes feature a heterogeneous variation of M across the absorption band. For some examples (nos. 2, 17, 21) the variations of M are less than 20%, whereas for other chlorosomes (nos. 1, 15, 19, 23) M varies up to 65% as a function of the excitation energy. This variation for the individual complexes is substantially larger than what has been observed at room temperature; see the Supporting Information Figure S3. The modulation of the fluorescence excitation spectra is often expressed as an apparent angle γ defined via γ = arctan((Imin/Imax)1/2), which is a measure for the distribution of the orientations of the transition-dipole moments that contribute to the absorption band. For example, γ = 0° corresponds to a situation where all transition-dipole moments are oriented parallel with respect to each other, and γ = 45° corresponds to an isotropic orientation. For the individual chlorosomes, the apparent angles are displayed in Figure 3d,e as a function of the excitation energy. For some individual chlorosomes γ is nearly constant, whereas for other chlorosomes γ varies substantially across the absorption band. In general, we observe pronounced differences between individual chlorosomes, and the values found for γ are distributed over the range from ∼15° to 40° and show some accumulation around 30°. A natural starting point for the analysis of the electronic excitations of supramolecular aggregates like chlorosomes considers a Frenkel-exciton Hamiltonian.12,33−37 The resulting eigenstates correspond to Frenkel excitons, that is, delocalized electronic excitations that are coherently shared by a large number of molecules. For assemblies with regular geometries, model calculations have shown that only a few exciton states with distinct polarization properties are optically accessible and that their transition strength depends crucially on the morphology of the aggregate.34 The situation changes drastically for any deviation from the perfectly symmetric case caused, for instance, by dispersions in the transition

shows no significant change in shape if the polarization of the excitation is varied. An example is shown in Figure 2d−f. The setup of this part of the Figure is similar to the setup of Figure 2a−c. Summing up the individual scans results in the spectrum shown as the black line in Figure 2e. It peaks at 13681 cm−1 and has a width of 603 cm−1 (fwhm). The individual spectra feature maxima at 13 676 cm−1 (red) and 13 693 cm−1 (blue) in close coincidence with the spectral position of the maximum of the total spectrum. The widths of the bands are 570 cm−1 (red) and 667 cm−1 (blue), respectively. The fluorescence intensity of the emission as a function of the polarization of the excitation (dots) is shown in Figure 2f for three distinct spectral positions that correspond to the spectral peak position of the total spectrum and to the two spectral positions chosen previously on either side of the maximum. For all three spectral positions, the polarization-induced intensity variations are consistent with a cos2 dependence (solid black lines), and a phase shift between the individual spectra is not observable. More examples of lowtemperature fluorescence excitation spectra from single chlorosomes are provided in the Supporting Information; see Figure S2. As before, we determined for each chlorosome the normalized modulation ratio M as a function of the excitation energy. The result is shown in Figure 3. The cases in Figure 3a refer to the chlorosomes that show two clearly resolved spectral contributions as a function of the polarization, cf. Figure 2a−c, whereas the cases in Figure 3b refer to the chlorosomes with excitation spectra that could not be decomposed into distinct components due to their polarization properties, cf. Figure 2d− e. Our data reveal that at low temperatures the modulation ratio M can vary substantially across the absorption band that is shown by the bold black line at the top of Figure 3a,b for better comparison, yet these variations are not correlated with our distinction of the chlorosomes according to their polarization (un-) resolved spectral contributions. For each individual chlorosome studied, the bar in Figure 3c refers to the span of the variation of M across the spectral range from 13 100 cm−1 to 14 000 cm−1. For different chlorosomes, 3747



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with spectral position. Likewise, the variation of γ as a function of the excitation energy reflects the change of the orientation of the transition-dipole moments that contribute to the spectrum (caused by a change of the spectral composition of the band). According to previous research,38 the number of rods per chlorosome varies between 10 and 30 and is determined by the rod dimensions and the volume of the chlorosome. When long rods predominate in a chlorosome or when the chlorosome volume is small, the orientation of the rods is largely determined by their orientation along the long axis of the chlorosome. For this situation, we expect a larger modulation ratio because the rods are preferentially organized in a parallel fashion. When there are short rods in a chlorosome, or when the chlorosome volume is large, the constraints of the boundary of the chlorosome influence the orientation of the rods to a lesser extent manifested by a smaller modulation ratio. We infer that at cryogenic temperatures an individual chlorosome will be trapped in a particular conformation, imposing a distinct energy landscape for the electronically excited states. Hence, the lowtemperature variations observed for the modulation ratio reflect the degree of structural and/or energetic disorder in a chlorosome, especially concerning the size and orientation of pigment rods that are present in its interior. At elevated temperatures, more conformational states become thermally accessible, and the structural fluctuations become faster. Because the experimental time resolution for acquiring a sufficient number of photons is finite, the outcome of a roomtemperature experiment represents an (temporal) average over many more conformational states as the cryogenic experiment does. As a consequence, this leads to the observation of a more homogeneous distribution for the orientation of the absorbing transition-dipole moments across the chlorosome. This is in line with the observation that the values obtained for M at room temperature are larger and more homogeneous with respect to those found at low temperature. An independent piece of information that supports this interpretation is provided by averaging the apparent angles found at low temperatures over both excitation wavelengths and chlorosomes. This corresponds to averaging over all conformations and yields 31 ± 4° (mean ± sdev), which reproduces the roomtemperature value found in ref 31 for a single excitation wavelength within the absorption band. Exploiting single-molecule spectroscopic techniques allowed us to record fluorescence excitation spectra from individual chlorosomes from Chlorobaculum tepidum and to uncover variations of the excitonic composition of the spectra between the individual chlorosomes. A more extensive analysis of these data requires a detailed investigation of the spectroscopic properties of single BChl c aggregates with well-defined orientation and size. Such information could then possibly be used to assess the chlorosome data more quantitatively in terms of a composition of aggregate sizes.

Figure 3. Modulation ratio M as a function of the excitation energy (a) from individual chlorosomes that feature polarization-resolved spectral contributions (chlorosomes 1−8) and (b) from individual chlorosomes that do not feature polarization resolved spectral contributions (chlorosomes 9−25). The bold black line at the top corresponds to the ensemble fluorescence excitation spectrum recorded at 1.5 K that is overlaid for comparison. (c) Variation of M for all individual chlorosomes across the spectral range from 13 100 cm−1 to 14 000 cm−1. (d,e) Apparent angles γ as a function of the excitation energy from individual chlorosomes (d) that feature polarization-resolved spectral contributions (e) and from individual chlorosomes that do not feature polarization-resolved spectral contributions. For clarity, only four examples from each group have been highlighted by different colors. In general, identical colors refer to the same individual chlorosome throughout the Figure.

energies of the monomers (diagonal disorder), by variations in the strength of the interactions between the monomers (offdiagonal disorder), or by geometric distortions. This yields a redistribution of the oscillator strength, lifts degeneracies of the exciton states, and leads to a localization of the excitation on a segment of the supramolecular structure.12,33 As a consequence of this, higher, initially forbidden exciton states become optically accessible, which further increases the complexity of the absorption spectrum and makes it even more difficult to obtain information about the excitonic level structure. Deviations from M = 0 provide evidence of a nonrandom ordering of the transition-dipole moments of the monomers within the chlorosome, whereas deviations from M = 1 tell us that the underlying spectrum consists of overlapping, spectrally unresolved contributions from two or more transitions that feature nonparallel transition-dipole moments. The actual value of M is determined by the relative weight with which each exciton state contributes to the total spectrum. From the variation of M as a function of the wavelength that has been observed at low temperature, we can conclude that the relative contributions of the exciton states to the total spectrum change

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ASSOCIATED CONTENT

S Supporting Information *

We provide more examples of single chlorosome spectra, describe the optical setup, the fluorescence excitation experiments, and the sample preparation. This material is available free of charge via the Internet at http://pubs.acs.org. 3748



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AUTHOR INFORMATION

Corresponding Author

*Tel: +49 921 55 4000. Fax: +49 921 55 4002. E-mail: juergen. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

We thank Dre de Wit for the preparation of the samples. Financial support from the German Science Foundation (DFG/GRK 1640) and the State of Bavaria within the initiative “Solar Technologies go Hybrid” is gratefully acknowledged.

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Fluorescence Excitation Spectra from Individual Chlorosomes of the

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