Controlling Photosynthetic Excitons by Selective Pigment

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

Controlling Photosynthetic Excitons by Selective Pigment Photooxidation Kristjan Leiger, Juha Matti Linnanto, Margus Rätsep, Kõu Timpmann, Aleksandr A. Ashikhmin, Andrey A. Moskalenko, Tatiana Fufina, Azat Gabdulkhakov, and Arvi Freiberg J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b08083 • Publication Date (Web): 13 Dec 2018 Downloaded from http://pubs.acs.org on December 17, 2018

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The Journal of Physical Chemistry

Controlling Photosynthetic Excitons by Selective Pigment Photooxidation Kristjan Leigera, Juha Matti Linnantoa, Margus Rätsepa, Kõu Timpmanna, Aleksandr A. Ashikhminb, Andrey A. Moskalenkob, Tatiana Y. Fufinab, Azat G. Gabdulkhakovc, and Arvi Freiberg*a, d aInstitute

bInstitute

of Physics, University of Tartu, W. Ostwaldi 1, Tartu 50411, Estonia

of Basic Biological Problems, Russian Academy of Sciences, 142290 Pushchino, Russia

cInstitute

dInstitute

of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Russia

of Molecular and Cell Biology, University of Tartu, Riia 23, Tartu 51014, Estonia

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ABSTRACT:

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As a basis of photosynthesis, photo-induced oxidation of (bacterio)chlorophyll

molecules in the special reaction center complexes has been a subject of extensive research. In contrast, the generally harmful photooxidation of antenna chromoproteins has received much less attention. Here, we have established the permanent structural changes in the LH2 antenna bacteriochlorophyll-protein

complex

from

a

sulfur

photosynthetic

purple

bacterium

Ectothiorhodospira haloalkaliphila taking place at physiological conditions upon intense optical irradiation. To this end, a crystal structure of the LH2 complex from Ectothiorhodospira haloalkaliphila was first resolved by X-ray diffraction to 3.7 Å, verifying a great similarity with the earlier structure from Phaesporillum molischianum. Analysis of the various steady-state and picosecond time-resolved optical spectroscopy data and related model simulations then confirmed that the major spectral effects observed – bleaching and blue-shifting of the B850 exciton band, and correlated emergence of a higher-energy C700 exciton band – are associated with photooxidation of increasing numbers of B850 bacteriochlorophylls into 3-acetyl-chlorophylls with no noticeable damage to the pigment-binding protein scaffold. A prospective non-invasive method for an in situ optical control of excitons by selective photooxidation of pigment chromophores was thus revealed and demonstrated in a structurally well-defined native system.

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INTRODUCTION Photooxidation is a natural part of photosynthesis and as such has been subject to extensive research.1 Yet this mostly concerns primary photochemical events in the so called reaction center proteins. Photooxidation of light-harvesting (or antenna) chromoprotein complexes, mostly considered harmful, has received much less attention.2–9 One of the research areas where oxidation is a common burden is single-particle (-molecule, -protein, -organelle or -cell) spectroscopy.10 The high optical excitation intensity necessarily used in these studies often results in serious modifications of the sample spectra. Mechanistic aspects of these changes, which involve bleaching, shifting, broadening, etc. of spectral bands, and depending on the experimental conditions, appear either reversible or permanent,2,9,11–13 have rarely been thoroughly explained. Filling in this gap of knowledge using complementary experimental and theoretical methods was the primary aim of the present work. While finding a suitable model system is critical for achieving this goal, we choose to investigate a LH2 peripheral antenna complex from a phototrophic sulfur purple bacterium Ectothiorhodospira (Ect.) haloalkaliphila. This complex is distinguished from other possible candidates by unique photo-sensitivity of its optical spectra in the presence of oxygen.3,9 However, since the LH2 complex from Ect. haloalkaliphila had not been structurally defined, one of our first tasks was to determine spatial structure of this complex to a sufficient resolution. As will be shown below, the structure of the LH2 complex from Ect. haloalkaliphila determined by X-ray diffraction analysis displays considerable similarity with that from Phaesporillum (Phs.) molischianum.14 Therefore, the near-IR optical spectrum of the LH2 complex of Ect. haloalkaliphila can be considered to arise from singlet excitons in two cyclic arrangements of bacteriochlorophyll a (BChl) pigment chromophores designated as B850 and B800. These designations refer to the positions of the lowest-energy singlet exciton absorption maxima, being 3



related to the Qy singlet electronic transitions of the BChl pigments (see refs 15–17 for comprehensive reviews). Following a generally accepted model, photooxidation of the BChl molecules requires promoting of the dissolved oxygen molecules into the singlet excited state by energy transfer from triplet states of BChls. The thus photosensitized singlet state of molecular oxygen is long lived and chemically reactive, triggering a variety of potentially destructive processes.18,19 The photosynthetic antenna complexes are believed to be safeguarded from the singlet oxygen formation by the carotenoid molecules.4 Located close to the BChl molecules,14 they are very efficiently promoted to the triplet state by triplet-triplet energy transfer from the BChl pigments. As the triplet states of carotenoids lie energetically below the singlet excitation level of oxygen, the latter can no longer receive the energy. At very high excitation intensity, however, some of the BChl triplet excitations may still “leak over” to close-by oxygen molecules, rendering the carotenoid protection insufficient. Oxidation of LH2 complexes has usually been marked by bleaching and blue-shifting of the B850 absorption band, accompanied by accumulation of oxidation products around 700 nm.2,3,5,6,9,11,12 The latter spectral signature was in refs 3,5,9 related to aggregated 7,8didehydrobacteriochlorophyll a. This green product of BChl dehydrogenation, better known as 3acetylchlorophyll a (abbreviated as AcChl19), shows a Qy absorption band that in acetone peaks at 677 nm.18 Specifically, for the LH2 of Ect. haloalkaliphila, it was concluded that oxidation takes place without significant disruption of the protein scaffold.3,9 This fact encouraged us to have a closer look at the correlated changes in the spectra of pigment chromophores, in a hope of clarifying the hitherto unknown mechanisms of photooxidation taking place in these complexes.

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By extensive experimental studies and related theoretical analyses that importantly involved numerical simulations of disordered exciton spectra, we succeeded to show that the major spectral effects observed in LH2 complexes under the intense light exposure can be distinctly associated with photooxidation of various numbers of BChl molecules in their B850 domain.

MATERIALS AND METHODS Sample preparation for optical measurements. The LH2 antenna complexes of Ect. haloalkaliphila, purified into detergent (2.5% of β-DDM) micelles, were prepared as described in ref 20 and stored at –78 °C in a deep freezer. Before use the concentrated samples were diluted with 50 mM Tris buffer (pH 8.0) to obtain the required optical density. No attempt was made to manipulate oxygen content in the samples, except in control measurements. In latter cases, an oxygen scavenger including 1% w/v glucose, 100 units/ml of glucose oxidase, and 1000 units/ml of glucose catalase (all from Sigma) was added to the sample solution to minimize the concentration of dissolved oxygen. The sample was deposited into a cuvette formed from a holed piece of a double-sided sticker tape on the microscope base glass and closed by a coverslip. Deliberate photooxidation of the samples was achieved at ambient temperature of 295 K by illumination into the carotenoid absorption range of the LH2 complex. The filtered (415 - 573 nm) light of a microscope halogen incandescent lamp was focused to a 5 mm diameter spot enclosing the whole sample region within the cuvette. Alternatively, the sample was illuminated by 532 nm light from Spectra Physics Millennia Prime solid state laser. In both cases, power density of ~0.2 W/cm2 (as recorded at 500 nm in case of halogen lamp illumination) was used. Nevertheless, the exposures employed in different experiments, thus the specific oxidation state achieved for different samples, were not directly comparable. 5



Crystal growth. High-quality crystals of LH2 complexes from Ect. haloalkaliphila for X-ray diffraction analysis were grown using hanging drops vapor diffusion method as described previously,21 with minor modifications. Droppings containing 10-12 mg/ml of the protein, 3.5% 1,2,3-heptanetriol, 2% dioxane, 0.05% LDAO and 1 M potassium phosphate, pH 7.8, were equilibrated against a reservoir solution of 1.65 M potassium phosphate, pH 7.8. The crystals of variable sizes (see Figure S1) were grown within 3–9 months in the dark at 16 ºC. X-ray diffraction analysis. Diffraction data of LH2 complexes from Ect. haloalkaliphila were collected at 100 K to 3.6 Å resolution on the ID23-1 beamline at the European Synchrotron Radiation Facility (ESRF), Grenoble, France22 equipped with a Pilatus 6M detector. Data collection was controlled by the MxCuBE system23 and the strategy was calculated by BEST.24 Data were processed and scaled with the XDS package.25 The data collection statistics are summarized in Table S1. It was found that the LH2 crystals studied belonged to the space group P432 with unit cell parameters a = b = c =177.24 Å and with four transmembrane chains in an asymmetric unit. Spectroscopy. Conventional absorption and circular dichroism (CD) spectra were simultaneously measured using a Chirascan Plus spectrophotometer (Applied Photophysics). Quartz cuvettes (Hellma Analytics) with 5 mm path length were used in a thermo-stabilized cell holder. The fluorescence spectra were recorded by a home-made microspectroscopy system12 composed of an Olympus IX-71 inverted microscope, Andor Shamrock 303i spectrometer, and Andor iDus 420 spectroscopic camera. Fluorescence excitation was provided either by a He-Ne random polarized laser (Melles-Griot 25-LYR-173-230) at 594 nm or by a diode laser at 407 nm. Average excitation intensity was ≤ 0.5 mW/cm2, i.e., significantly lower than used for intentional photooxidation of the samples, see above. The spectra were appropriately corrected for spectral sensitivity of the set-up.

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Most measurements were performed at ambient temperature of 295 K. For the sake of improved spectral resolution, some measurements were also executed at 5±0.5 K. In the latter case, the samples in plastic cuvettes (Brand) of 10 mm path length or gelatin capsules (Torpac) of 4 mm diameter were placed in an Utreks helium bath cryostat equipped with a temperature controller. The samples for cryogenic measurements included glycerol (in 2:1 volume ratio) to secure a transparent glassy state. Modeling of the spectra and data evaluation. Given the proven in this work similarity of crystal structures of LH2 complexes from Ect. haloalkaliphila and Phs. molischianum, the spectral simulations were based on a crystal structure of the LH2 complex from Phs. molischianum,14 as the one resolved to higher resolution. Different photooxidation complexes were generated by replacing one by one the BChl molecules in the B850 domain of LH2 with AcChl molecules. All possible oxidization configurations were accounted for, in total 32 772 different configurations. The Qy transition dipole moments equal to 6.13 and 5.04 Debye for, respectively, BChl and AcChl molecules were applied. These values correspond to experimental extinction coefficients of these pigments in organic solvents.18 Exciton structure of the absorption spectra of LH2 complexes were simulated by means of standard molecular exciton theory,26 taking into account all the 24 pigment sites and disorder broadening of the spectra. Starting site energies and exciton parameters were taken from an earlier publication.27 These parameters were subsequently scaled by comparison with experimental spectra, as discussed below. The disorder model accounted for static Gaussian fluctuations of Qy transition energies of pigment sites as well as of pigment spatial positions and orientations. The three Cartesian pigment coordinate positions were varied within 1.5 Å (x and y-directions) and 3.0 Å (z-direction), and that of the three transition dipole orientations within 10 degrees. Every single oxidized configuration 7



was an average over 1000 random disorder samples. The final simulated spectrum is a superposition of all the averaged single oxidized configurations with Gaussian homogeneous line widths (henceforth defined as the full width at half maximum, fwhm) assigned to the transitions. See Discussion for more computational details.

RESULTS Crystal structure of the LH2 complex from Ect. haloalkaliphila resolved to 3.7 Å. The structure of the LH2 complex from Ect. haloalkaliphila shown in Figure 1 was solved by molecular replacement with Phaser28 using the structure of A, B, D and E chains from the crystal structure of the LH2 complex from Phs. molischianum14 (PDB entry 1LGH). Water molecules were removed from the model. The initial model was subjected to crystallographic refinement with REFMAC5.29 Manual rebuilding of the model was carried out in Coot.30 At last cycles of refinement the resolution was cut to 3.7 Å. The coordinates and structure factors are deposited in the Protein Data Bank, PDB ID 6Q53. As seen, the LH2 complex from Ect. haloalkaliphila shows a fair amount of similarity with that from Phs. molischianum14. It forms a cylinder of C8 symmetry, where a tetrametric unit cell is composed of 4 α-helical transmembrane polypeptide chains, non-covalently associated with 6 BChl and 2 carotenoid (lycopene) pigment chromophores. In the complete assembly the BChls organize into two rings: 8 BChls of the B800 ring in the hydrophilic part and 16 BChls of the B850 ring in the hydrophobic part of the complex. Eight carotenoid molecules reinforce the LH2 structure by interconnecting the B850 and B800 BChl rings. To uncover possible differences between the positions and orientations of the BChl pigments in LH2 complexes from Ect. haloalkaliphila and Phs. molischianum, an overlay of the B800 and

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B850 pigment arrangements is shown in Figure S2. As seen, the pigment architectures in these two complexes are almost identical.

Figure 1. Crystal structure of the LH2 complex from Ect. haloalkaliphila refined to 3.7 Å resolution at 100 K. (A) Side view of four transmembrane chains in an asymmetric unit cell of the crystal: α-peptides - yellow and magenta, β-peptides – cyan and blue, BChls – green, lycopene –

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orange. (B) Top view from cytoplasmic side of the membrane of the full LH2 complex, obtained by rotation around the fourth order axis. Figures were prepared using PyMOL.31

Spectral response of LH2 complexes to intense optical irradiation. Figure 2A shows the spectral changes occurring in the near-IR absorption range of the LH2 complex from Ect. haloalkaliphila upon a broadband illumination into the carotenoid absorption region. As already indicated in Introduction, this absorbance is mainly due to the Qy exciton transitions of BChls in the B800 and B850 domains (see Figure 3A below for a wide-scale absorption spectrum of the sample). The principal effects observed are the bleaching and blue-shifting of the B850 absorption band, and the simultaneous formation of a new band around 700 nm. This latter band will be subsequently designated as C700. These specific effects take place on the background of diminishing sample absorbance, setting in within a few minutes after the beginning of exposure (see Supporting Information, Figure S2). This is reminiscent of illumination effects in solubilized BChls where the major photo-products had low absorption in the visible and near-IR spectral range.19 Differently from B850, the decrease of the B800 band intensity takes place with no change of its spectral position. Removal of oxygen from the sample constrains the bleaching process considerably, see inset of Figure 2C. This result ensures that the effects observed in LH2 complexes were of a photooxidative nature.

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Figure 2. (A) Absorption spectra of LH2 antenna complexes from Ect. haloalkaliphila in Qy range as a function of increasing exposure to oxidizing light between 415 and 573 nm. The light intensity applied, as measured at 500 nm, was 0.2 W/cm2. (B) Same as in part (A) but for absorption spectra normalized with respect to the peak value of the B800 band. (C) Same as in part (A) but for fluorescence spectra. Excitation at 594 nm into the Qx absorption band was employed. Inset of (C) presents the absorption spectra of LH2 complexes with oxygen scavenger added, oxidized similarly 11



over a time span of 1 hour (normalized to B800 band). The spectra were measured at ambient temperature; similar color code is used in all panels; vertical arrows indicate directions of major changes of the spectra; the inclined arrow points out that an impurity representing the LH1 absorption band has become visible in strongly bleached samples.

To follow the correlated photo-induced changes of the B850 and C700 bands more clearly, and to isolate them from the overall bleaching, the absorption spectra of Figure 2A were normalized with respect to the B800 band peak value (Figure 2B). The bleaching of the B850 absorption band and correlated growth of the C700 band with increasing light exposure is now very transparent, as is the blue shift of the B850 band. The latter shift, noticed already in ref 7, was originally explained by dimeric BChl antenna model in which the dimer excitons were by photooxidation progressively converted into a monomeric state. In this work, by virtue of an improved knowledge about the LH2 structure, we develop a more complete model, whereby 16 BChls in the B850 compartment are gradually replaced by AcChl oxidation products. As will be explained in detail in the Discussion, basic changes of the exciton absorption spectra observed in Figures 2A and 2B reflect the modifications of electronic interactions between all the 24 pigment chromophores of the B800 and B850 domains of LH2. The largely unaffected shape of the B800 band is worth special attention. It importantly confirms that photooxidation essentially only concerns the pigments in the B850 compartment, leaving the B800 domain almost intact. The enhanced stability of the B800 chromophores may be explained by their very short excited state lifetime (0.7 ps32) compared with the B850 chromophores (~1.01.5 ns, depending on temperature and the fluorescence recording wavelength33,34). The B800 exciton lifetime in untreated (i.e., with no preliminary exposure to oxidizing light) LH2 complexes

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is primarily shortened due to the very efficient excitation energy transfer to the overlapping B850 exciton states. Emission of the sample (Figure 2C) is consistent with its absorption. Initially, only the fluorescence from the thermalized B850 exciton states is observed that peaks at 865.5 nm. A weak blue-side step visible around 800 nm is due to a small population of the B800 exciton states. This contribution quickly vanishes with lowering the temperature33 (see also Figure 4A). The fluorescence becomes rapidly quenched with increasing exposure to oxidizing light. At the same time, emission bands around 700 and 800 nm start accumulating. These bands are apparently related to the C700 and B800 absorption bands, respectively. The C700 emission looks relatively very weak. As can be seen in Figure 3B, this is because the 594 nm excitation wavelength used is good for exciting the BChl chromophores and not the AcChl oxidation products. A comparison of Figures 2A and 2C clearly shows that the quenching of the B850 fluorescence occurs significantly faster than bleaching of the corresponding absorbance. This suggests that along irradiation some kind of excitation traps have been created in the B850 compartment. Earlier, a BChl+● mono-cation radical that absorbs at around 900 nm was proposed as a potential fluorescence quencher in bacterial antennas.8 The presence of this one-electron oxidation product in our samples cannot be excluded. However, its amount should be rather small. Otherwise we would have seen a red shift of the absorption/fluorescence spectra, which was not observed. Temporal red-shifting of the B850 fluorescence in single LH2 complexes from purple non-sulfur bacteria at ambient temperature has, however, been regularly detected.2,11,12 Obviously, in the present samples and under current experimental conditions, the mono-cations become rapidly converted into AcChls,5 significantly diminishing the concentration of the BChl+● intermediates (see analysis of the low-temperature spectra below).

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Figure 3. (A) Absorption spectra of LH2 complexes upon illumination with increasing doses of 532 nm laser light of 0.2 W/cm2 intensity. (B) Differential absorption spectrum (35 min minus 0 min exposure time) of LH2 complexes. The initial absorption spectrum was subtracted from the spectrum upon saturating illumination. The spectra were recorded at ambient temperature; indicated are differential bands related to various (Qy, Qx, and Soret) molecular transitions of BChl (red) or AcChl (blue) origin.

The modifications that occur upon irradiation in a broad spectral range of 260-960 nm are demonstrated in Figure 3. The main absorption bands in untreated and oxidized LH2 complexes shown in Figure 3A are known to be related to either BChl (peaks at 846, 796, 591, and 372 nm) or carotenoid (500 nm) pigment excitons. Dominant loss of the lowest-energy B850-Qy exciton

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intensity is accompanied by proportional decrease of the successive higher-energy (B800-Qy, B800-850-Qx, B800-850-Soret) exciton intensities. Oxidation both bleaches and amplifies certain parts of the absorption spectrum, as visualized by differential absorption (oxidized (35 min) minus initial (0 min)) spectrum of Figure 3B. In this figure, negative sign designates a bleaching and positive sign a gain in the signal intensity. The gain is most notable between 400 and 450 nm and around the C700 band. The present photooxidation results seem to be well consistent with previous chemical oxidation data.4,6,9,18 With additional literature support,35–37 we are now able to assign all the major spectral structures in the differential absorption spectrum of Figure 3B to various electronic states of the BChl and AcChl pigment chromophore assemblies. In accordance with refs 3 and 9, no absorbance or fluorescence corresponding to solubilized BChl pigments, spectrally located around 770780 nm, was observed. This is good evidence that the photooxidation in our sample does not compromise the protein scaffold holding the pigments together in their respective binding pockets. A similar conclusion with respect to chemical oxidation of LH2 complexes from Ect. haloalkaliphila was made in ref 9. Absorption and fluorescence spectra recorded at 5 K. Absorption and fluorescence spectra for untreated (Figure 4A) and oxidized (Figure 4B) samples were also measured at 5 K. The spectral changes (overall narrowing and red shift of the B850 band) that follow the decrease of temperature are rather typical for all bacterial antenna complexes, see e.g. ref 17, except for the width of the inhomogeneous distribution function (IDF), which appears significantly broader in LH2 from Ect. haloalkaliphila (254±38 cm-1) than in that from Rhodobacter (Rba.) sphaeroides, for example (~150 cm-1).38–40 This speaks for a rather large heterogeneity of the present samples.

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Figure 4. (A) Comparison of the absorption (blue curves) and fluorescence (red curves) spectra of untreated LH2 complexes recorded at ambient temperature (dashed lines) and at 5 K (solid lines). Shown in green in the red edge of the low-temperature absorption spectrum is a Gaussianapproximated IDF. The fluorescence spectra were normalized by the peak intensity. The inset shows a deconvolution of the Qx absorption band (turquoise line) into two Gaussian thin-line components peaking at 591 and 601 nm due to B850 and B800 BChl assemblies, respectively. The bold black line designates sum of the fitting Gaussian components. (B) Absorption (blue curve) and fluorescence (red curve) spectra of oxidized LH2 complexes recorded at 5 K. The photooxidation was performed with a 532 nm laser light of 0.2 W/cm2 intensity during 84 min. The spectra were normalized with respect to B800 peak intensities. Shown for reference with dashed line is the fluorescence spectrum of Chl a,36 normalized to C700 intensity and shifted by 26.5 nm toward longer wavelengths to compensate for its exciton shift. Fluorescence was excited at 407 nm

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in both panels. Grey bars at the bottom show approximate fluorescence recording ranges used in the measurements of fluorescence excitation spectra in the corresponding panels of Figure 5 below.

A small but distinct step is visible in the red side slope of the low-temperature absorption spectrum around 900-910 nm of untreated samples. The corresponding fluorescence spectrum peaks at ~915 nm (Figure 4B). This must be the same structure that emerged in the spectrum of strongly bleached samples at ambient temperature (Figure 2A). Because of its presence both in untreated and oxidized samples, it is most probably related to trace LH1 complexes rather than to BChl+● mono-cation radical intermediates. The drastically changed low-temperature spectra upon photooxidation are shown in Figure 4B. The three absorption bands that at ambient temperatures peak at 598, 696, and 793 nm reveal at low temperatures noticeable internal spectral structure, visible both in absorption and fluorescence spectra. The C700 fluorescence in Figure 4B is much amplified compared with the ambienttemperature spectrum of Figure 2C. This is due to different excitation wavelength used (407 nm instead of 594 nm), which, as we learned in Figure 3B, appears more favorable in terms of excitation of the C700 fluorescence. Excitation into the Qx absorption band is usually considered non-selective with respect to the B800 and B850 BChl assemblies. Figure 4A shows that this is hardly the case at low temperatures, where the band appears clearly split into two components. Red-shifting of this band upon selective photo-bleaching of the B850-BChls determines that the blue-side component peaking at ~591 nm is due to B850, while the red-side component at ~601 nm, due to B800 BChls, see the inset of Figure 4A. In an attempt to confirm the origin of the fluorescence bands observed in oxidized samples (Figure 4B), excitation spectra of these bands were measured, as shown in Figure 5. The excitation 17



spectra of untreated samples related to B800 and B850 emissions quite expectedly involve just the Soret (around 372 nm) and Qx (591 nm) bands of BChls (Figures 5C and 5D). Additional contribution by carotenoids (at 505 nm) supports existing energy transfer between the carotenoid and BChl pigment compartments of LH2. In the oxidized sample, in contrast, the dominant emission of C700 is most strongly excited around 440 nm, in the Soret band of AcChls,18 and only weakly around the Soret band of BChl. The Soret contributions of BChl and AcChls are roughly equal when both the C700 and B800/B850 emissions are simultaneously recorded (Figure 5C).

Figure 5. Fluorescence excitation spectra measured at ambient temperature of untreated (blue curves) and oxidized (red curves, photooxidation with a 532 nm laser light of 0.2 W/cm2 intensity during 84 min) LH2 complexes measured at ambient temperature. The fluorescence was recorded at selected emission regions shown, see also Figure 3B.

According to Figures 4A, 5A and 5B, the C700 emission from untreated samples is very weak, though not exactly zero. This indicates that even untreated samples include a small proportion of

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oxidized LH2 complexes. Due to high sensitivity of these samples toward photooxidation, the preoxidation might take place either during preparation of the samples or during the measurements, despite the relatively dim excitation light used. CD spectra of LH2 complexes in visible and near-IR range. CD spectroscopy is a powerful method for exploring excitonically coupled molecular assemblies.41 Therefore, to elucidate the nature of the C700 band, CD spectra in the actual spectral range were measured as a function of light exposure. We note that light-induced changes observed by CD generally correlate well with those seen in absorption and fluorescence spectra. The initial CD spectrum in Figure 6 shows an exciton structure typical for LH2 complexes of Phs. molischianum. The red part of this structure is firmly known27,41 to be associated with the Qy transitions of BChls in cyclic B800 and B850 arrangements. Under the increasing light exposure, the shape of the B850 spectrum gets readily weakened as well as blue-shifted, whereas the changes observed in B800 range during the first 35 min are relatively minor. Similar spectral transformations during photooxidation were observed in chromatophores from the carotenoid-less mutant strain R26 of Rba. sphaeroides.7

Figure 6. Dependence of the CD spectra of LH2 complexes on the illumination time at ambient temperature. The inset shows zoomed-in section of the spectrum around 700 nm, normalized by 19



the intensity of the C700 absorption band obtained after 35 min bleaching. The main frame spectra were normalized by initial/untreated B850 band intensity. The sample as well as color code is the same as used in Figure 3A.

The loss of CD signal related to B850 pigment chromophores parallels with a rise of weaker CD feature around 700 nm, see inset of Figure 6. A similar band arising upon chemical oxidation was in ref 9 associated with C700 excitons. We stay with the same explanation, despite the fact that chemical and photooxidation are rather different processes. One might notice that the B700 signal measured after 35 min is larger than the one measured after 84 min. This appears consistent with overall bleaching of the sample spectrum, including the C700 band.

DISCUSSION Here an assessment of the experimental observations will be given based on model simulations of exciton absorption spectra in the course of photooxidation. As already indicated, we assume selective and progressive oxidation of the B850 BChls to AcChls upon exposure to intense light. Since only minor modifications of the B800 spectra were observed (at least at limited exposures), the replacement of BChls in the B800 domain was not considered. The successive exchange of BChls to AcChls should lead to a gradual blue-shift of the B850 exciton band. This is because of the significantly higher Qy transition energy in AcChl compared with that in BChl. In different normal solvents the Qy absorption maximum of AcChl is found around 677-681 nm,18 while that of BChl, at 770-775 nm.36 As schematically shown in Figure 7, the mixed B800-850/B800-C700 exciton states are for the most part expected to be located in between the B800-850 and B800-C700 states in, respectively, the LH2 complexes with fully reduced BChls or fully oxidized AcChls in the B850 binding pockets of native protein.

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Figure 7. Calculated energy level schemes for idealized (no disorder) native LH2 complex (left, B800-850) and a complex where all the BChls in the B850 compartment have been exchanged to AcChl (right, B800-C700). Shown in center is an intermediate situation (designated as B800850/B800-C700) for a randomly selected spatial conformation with half (8) of the B850 BChls exchanged. Highlighted by red are the exciton states mainly localized on B800-BChl sites. Arrows indicate major channels of fluorescence decay observed at cryogenic temperatures.

In order to produce Figure 7, the exciton Hamiltonian matrix elements (diagonal site energies and non-diagonal coupling energies) were scaled by comparison with the experimental absorption (Figures 2 and 3) and polarized fluorescence excitation spectra (Figure S4). This yielded 14860, 12635 and 12530 cm-1 for the Qy site energies of C700-AcChl, B850-BChl, and B800-BChl pigment chromophores, respectively. The nearest neighbor intra-dimer/inter-dimer exciton coupling energies were then estimated as follows: 385/302 cm-1 in B850 and 250/200 cm-1 in C700.

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The couplings between B800 pigments are relatively very weak (~15 cm-1). This causes clumping of all the 8 exciton states highlighted by red in Figure 7 around the B800-BChl site energy. Similarly, the couplings between B800 and B850/C700 pigment sites are rather weak (35-36 cm1).

This explains why there is little influence on the energetics of the B800 exciton states upon the

replacement of BChls with AcChls. The exciton spectral simulations for successive numbers of oxidized pigment sites are shown in Figure 8. Each specific situation (with 0 to 16 oxidized sites) presented is a statistical mix of a large number of different possible spatial configurations. Spectral disorder was additionally accounted for by static Gaussian fluctuations of Qy transition energies of pigment sites as well as of pigment spatial positions and orientations, as specified in the Materials and Methods. The widths of the disorder distributions used were 250 cm-1 and 100 cm-1, respectively, in the case of B850/C700 and B800 sites. To obtain realistic shapes of the spectra, the stick exciton spectra were “dressed” with Gaussian homogenous band shapes of similar 320 cm-1 fwhm. The spectra in Figure 8 reproduce the main features of the experimental spectra in Figures 2A and 3A very well. In both the initial spectrum (black, corresponding to zero oxidized BChl) and the final spectrum (magenta, all B850 BChls oxidized to AcChl), only two bands are visible: B850 and B800, and B800 and C700, respectively. Upon illumination the spectra gradually evolve between these limiting forms. Exciton spectra of pigment aggregates are generally asymmetric. In Figure 8, this is very obvious in case of the C700 band, which tails toward higher energy up to 630 nm. A similar high-energy tail in case of B850 excitons is eclipsed by overlap with the B800 band (but see Figure 9A). The latter appears rather symmetric because of the weak coupling between the B800-BChls, as explained above.

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Figure 8. Simulated exciton absorption spectra of LH2 complexes as a function of increasing number (from 0 to 16) of the B850-BChl pigments replaced by AcChl. Drawn with bold black and magenta lines are, respectively, the initial spectrum with zero oxidized sites and final spectrum with all 16 sites oxidized. See text for further explanations.

Moreover, notable in Figure 8 is the opposite shift of the B850 and C700 bands. The major blue shift of B850, also observed experimentally, can be readily explained by average shortening of the BChl antenna oligomers and the related coherence length of B850 excitons. Concomitantly, the red shift of the C700 band is caused by average growth of the AcChl oligomers, and increase of the corresponding exciton coherence length. This shift, revealing itself primarily at initial oxidation phases (1-3 sites), was in fact not experimentally detected. Figure 9 demonstrates the rather excellent quantitative agreement achieved between experimental and simulated spectra. A step around 750 nm in the calculated spectrum of Figure 9A designates the high-energy edge of the B850 exciton band mentioned in relation with Figure 8 above. According to Figure 9B, in the maximally oxidized sample statistically about 15 BChls have been replaced by AcChl molecules.

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Figure 9. Comparison of the experimental (black lines) and simulated (colored lines: red for fully oxidized B850 ring, blue for 14 oxidized B850-BChls) absorption spectra of untreated (A) and maximally photo-oxidized (B) samples at ambient temperature. Please notice the reciprocal (linear in energy) wavelength scale, see text for further explanations.

The discrepancy observed in Figure 9A around 700-790 nm and in Figure 9B around 720780 nm can be readily explained by neglect of vibrational sidebands in model simulations. As was shown in refs 42–44, the prominence of the vibrational sidebands in cyclic antenna complexes depends on exciton coupling strength. For strongly coupled B850 excitons they are almost

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completely suppressed, while much less so in case of weakly coupled B800 excitons. Extended vibronic structure related to B800 excitons is clearly observable in the absorption spectra measured at cryogenic temperatures, see Figures 4A and S5. The red-side deviation between calculated and experimental spectra can be mostly assigned to impurity LH1 absorption, as already noted while describing Figure 2. The significantly broader width of the C700 band compared with that of B850 in Figure 9 is unexpected, provided the smaller estimated exciton coupling energy, as reported above. We assign this effect to greater inhomogeneous broadening of the C700 band. This appears reasonable, given the potential rearrangements of the methyl and ethyl groups at positions C-7 and C-8, respectively, of the chlorin skeleton ring B upon photooxidation of BChl.9,19 Slightly different orientation of the AcChl molecules in their protein binding pockets, changing pigment-protein interactions, may also contribute into this broadening. Emission properties of the samples (Figures 2C, 4 and S7) generally match the expectations (Figure 7) based on qualitative analysis of absorption, fluorescence excitation and CD spectra (Figures 2-6). In untreated samples, the lowest-energy excited states belong to B850 excitons and the fluorescence observed is principally originating from the relaxed, Boltzmann-populated B850 exciton states (Figure 4A). In the opposite limit of fully oxidized samples, the B800 excitons should occupy all the lowest-energy positions. Although the raising contribution of the B800 fluorescence was indeed observed (Figures 2C, 4B and S7), there always existed relatively strong background emission from complexes in different oxidation states. This heterogeneity, inevitable for real sample ensembles, significantly obstructs assessment of the excitation energy relaxation pathways and rates between the exciton states that belong to different pigment pools (C700, B800 and B850) in the LH2 complexes of various oxidation status. Resolution of this dilemma requires more challenging measurements on single complex level. 25



SUMMARY AND CONCLUSIONS Permanent changes following an optical irradiation at natural aerobic conditions have been observed in the exciton spectra of LH2 antenna membrane complexes isolated from the phototrophic purple sulfur bacterium Ect. haloalkaliphila. Numerical modelling of the spectroscopic evidence maintained by the crystal structure of the LH2 complex resolved to 3.7 Å revealed that the observed changes were associated with the gradual photooxidation of numbers of B850-BChls into AcChl. Such localized photooxidation processes of pigment chromophores did not cause any noticeable damage to the surrounding protein scaffold. An amazing robustness of the B800 excitons compared with the B850 excitons with respect to oxidation was explained by the three orders of magnitude shorter lifetimes of the former excitons. A possibility of noninvasive in situ optical control of photosynthetic excitons was thus demonstrated in a structurally well-characterized native model system. It remains to be seen whether the observed chromatic accommodation to intense light might be a part of natural strategy for photosynthetic light harvesting.45,46 Finally, a comment on single complex spectroscopy is in order. The literature about LH2 provides examples of both red- and blue-side spectral jumps of B850 exciton spectra. Depending on conditions, the transition energy changes observed have been either largely reversible or largely irreversible. According to the current work, a blue shift arises when the B850 BChls are replaced by their green oxidation product, AcChl. This process is according to our experience generally irreversible. A likely reason for the opposite red shift is oxidation of BChls along the radical cation (BChl+●) pathway. This latter process may be at least partially reversible.

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ASSOCIATED CONTENT

Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org: LH2 crystals for X-ray diffraction analysis and the data collection and refinement statistics. Comparison of the BChl pigment arrangements in LH2 complexes from Ect. haloalkaliphila and Phs. molischianum. Kinetic features of the photooxidation process. Estimation of the Qy exciton bandwidth. Disentangling excitonic and vibronic contributions into the absorption spectrum of the LH2 complex. Contrasting fluorescence decay kinetics in untreated and oxidized samples. (PDF) AUTHOR INFORMATION

Corresponding Author * Corresponding author: [email protected].

ACKNOWLEDGMENT

This work was supported by the Estonian Research Council (grant IUT02-28). AAA and AAM acknowledge the Russian Foundation for Basic Research (grant numbers 17-04-00929-a and 1804-00684-a) for partial financial support. The structural part of the study was supported by the program of the Presidium of the Russian Academy of Sciences “Molecular and Cell Biology and Postgenomic Technologies”. L. Reisberg, F. Valgepea, M. A. Bol’shakov, and Z. K. Makhneva participated in different early phases of this study.

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REFERENCES

(1)

Blankenship, R. E. Molecular Mechanisms of Photosynthesis, 1st ed.; Wiley-Blackwell,

2002. (2)

Bopp, M. A.; Jia, Y.; Li, L.; Cogdell, R. J.; Hochstrasser, R. M. Fluorescence and

Photobleaching Dynamics of Single Light-Harvesting Complexes. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 10630–10635. (3)

Makhneva, Z. K.; Bol’shakov, M. A.; Ashikhmin, A. A.; Erokhin, Y. E.; Moskalenko, A.

A. Influence of Blue Light on the Structure Stability of Antenna Complexes from Allochromatium minutissimum with Different Content of Carotenoids. Biochem. Moscow Suppl. Ser. A 2009, 3, 123–127. (4)

Limantara, L.; Fujii, R.; Zhang, J.-P.; Kakuno, T.; Hara, H.; Kawamori, A.; Yagura, T.;

Cogdell, R. J.; Koyama, Y. Generation of Triplet and Cation-Radical Bacteriochlorophyll a in Carotenoidless LH1 and LH2 Antenna Complexes from Rhodobacter sphaeroides. Biochemistry 1998, 37, 17469–17486. (5)

Kropacheva, T. N.; Hoff, A. J. Electrochemical Oxidation of Bacteriochlorophyll a in

Reaction Centers and Antenna Complexes of Photosynthetic Bacteria. J. Phys. Chem. B 2001, 105, 5536–5545. (6)

Bril, C. Studies on Bacterial Chromatophores II. Energy Transfer and Photooxidative

Bleaching of Bacteriochlorophyll in Relation to Structure in Normal and Carotenoid-Depleted Chromatium. Biochim. Biophys. Acta 1963, 66, 50–60.

28


Page 28 of 45

Page 29 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(7)

Rafferty, C. N.; Bolt, J.; Sauer, K.; Clayton, R. K. Photooxidation of Antenna

Bacteriochlorophyll in Chromatophores from Carotenoidless Mutant Rhodopseudomonas sphaeroides and the Attendant Loss of Dimeric Exciton Interaction. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 4429–4432. (8)

Law, C. J.; Cogdell, R. J. The Effect of Chemical Oxidation on the Fluorescence of the LH1

(B880) Complex from the Purple Bacterium Rhodobium marinum. FEBS Lett. 1998, 432, 27–30. (9)

Makhneva, Z. K.; Ashikhmin, A. A.; Bolshakov, M. A.; Moskalenko, A. A. 3-Acetyl-

Chlorophyll Formation in Light-Harvesting Complexes of Purple Bacteria by Chemical Oxidation. Biochem. Moscow 2016, 81, 176–186. (10) Moerner, W. E.; Orrit, M. Illuminating Single Molecules in Condensed Matter. Science 1999, 283, 1670–1676. (11) Rutkauskas, D.; Olsen, J.; Gall, A.; Cogdell, R. J.; Neil Hunter, C.; van Grondelle, R. Comparative Study of Spectral Flexibilities of Bacterial Light-Harvesting Complexes: Structural Implications. Biophys. J. 2006, 90, 2463–2474. (12) Leiger, K.; Reisberg, L.; Freiberg, A. Fluorescence Micro-Spectroscopy Study of Individual Photosynthetic Membrane Vesicles and Light-Harvesting Complexes. J. Phys. Chem. B 2013, 117, 9315–9326. (13) Kunz, R.; Timpmann, K.; Southall, J.; Cogdell, R. J.; Freiberg, A.; Köhler, J. Exciton Self Trapping in Photosynthetic Pigment–Protein Complexes Studied by Single-Molecule Spectroscopy. J. Phys. Chem. B 2012, 116, 11017–11023.

29



(14) 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. (15) Sundström, V.; Pullerits, T.; van Grondelle, R. Photosynthetic Light-Harvesting: Reconciling Dynamics and Structure of Purple Bacterial LH2 Reveals Function of Photosynthetic Unit. J. Phys. Chem. B 1999, 103, 2327–2346. (16) Cogdell, R. J.; Gall, A.; Köhler, J. The Architecture and Function of the Light-Harvesting Apparatus of Purple Bacteria: From Single Molecules to in Vivo Membranes. Q. Rev. Biophys. 2006, 39, 227–324. (17) Freiberg, A.; Rätsep, M.; Timpmann, K. A Comparative Spectroscopic and Kinetic Study of Photoexcitations in Detergent-Isolated and Membrane-Embedded LH2 Light-Harvesting Complexes. Biochim. Biophys. Acta, Bioenerg. 2012, 1817, 1471–1482. (18) Smith, J. R. L.; Calvin, M. Studies on the Chemical and Photochemical Oxidation of Bacteriochlorophyll. J. Am. Chem. Soc. 1966, 88, 4500–4506. (19) Limantara, L.; Koehler, P.; Wilhelm, B.; Porra, R. J.; Scheer, H. Photostability of Bacteriochlorophyll a and Derivatives: Potential Sensitizers for Photodynamic Tumor Therapy. Photochem. Photobiol. 2006, 82, 770–780. (20) Ashikhmin, A.; Makhneva, Z.; Moskalenko, A. The LH2 Complexes Are Assembled in the Cells of Purple Sulfur Bacterium Ectothiorhodospira haloalkaliphila with Inhibition of Carotenoid Biosynthesis. Photosynth. Res. 2014, 119, 291–303.

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Page 30 of 45

Page 31 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(21) Ermler, U.; Fritzsch, G.; Buchanan, S. K.; Michel, H. Structure of the Photosynthetic Reaction Centre from Rhodobacter sphaeroides at 2.65 Å Resolution: Cofactors and ProteinCofactor Interactions. Structure 1994, 2, 925–936. (22) Nurizzo, D.; Mairs, T.; Guijarro, M.; Rey, V.; Meyer, J.; Fajardo, P.; Chavanne, J.; Biasci, J.-C.; McSweeney, S.; Mitchell, E. The ID23-1 Structural Biology Beamline at the ESRF. J Synchrotron Rad. 2006, 13, 227–238. (23) Gabadinho, J.; Beteva, A.; Guijarro, M.; Rey-Bakaikoa, V.; Spruce, D.; Bowler, M. W.; Brockhauser, S.; Flot, D.; Gordon, E. J.; Hall, D. R.; et al. MxCuBE: A Synchrotron Beamline Control Environment Customized for Macromolecular Crystallography Experiments. J Synchrotron Rad. 2010, 17, 700–707. (24) Bourenkov, G. P.; Popov, A. N. Optimization of Data Collection Taking Radiation Damage into Account. Acta Cryst. 2010, D66, 409–419. (25) Kabsch, W. XDS. Acta Cryst. 2010, D66, 125–132. (26) Davydov, A. S. Theory of Molecular Excitons; Plenum Press: New York, 1971. (27) Ihalainen, J. A.; Linnanto, J.; Myllyperkiö, P.; van Stokkum, I. H. M.; Ücker, B.; Scheer, H.; Korppi-Tommola, J. E. I. Energy Transfer in LH2 of Rhodospirillum molischianum, Studied by Subpicosecond Spectroscopy and Configuration Interaction Exciton Calculations. J. Phys. Chem. B 2001, 105, 9849–9856. (28) McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M. D.; Storoni, L. C.; Read, R. J. Phaser Crystallographic Software. J. Appl. Cryst. 2007, 40, 658–674.

31



Page 32 of 45

(29) Murshudov, G. N.; Skubák, P.; Lebedev, A. A.; Pannu, N. S.; Steiner, R. A.; Nicholls, R. A.; Winn, M. D.; Long, F.; Vagin, A. A. REFMAC5 for the Refinement of Macromolecular Crystal Structures. Acta Cryst. 2011, D67, 355–367. (30) Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Features and Development of Coot. Acta Cryst 2010, D66 (4), 486–501. (31) DeLano,

W.L.

The

PyMOL

Molecular

Graphics

System.

Available

online:

http://www.pymol.org/ (32) Trautman, J. K.; Shreve, A. P.; Violette, C. A.; Frank, H. A.; Owens, T. G.; Albrecht, A. C. Femtosecond Dynamics of Energy Transfer in B800-850 Light-Harvesting Complexes of Rhodobacter sphaeroides. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 215–219. (33) 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. (34) Timpmann, K.; Katiliene, Z.; Woodbury, N. W.; Freiberg, A. Exciton Self Trapping in OneDimensional Photosynthetic Antennas. J. Phys. Chem. B 2001, 105, 12223–12225. (35) Fajer, J.; Borg, D. C.; Forman, A.; Felton, R. H.; Dolphin, D.; Vegh, L. The Cation Radicals of Free Base and Zinc Bacteriochlorin, Bacteriochlorophyll, and Bacteriopheophytin. Proc. Natl. Acad. Sci. U.S.A. 1974, 71, 994–998. (36) Rätsep, M.; Linnanto, J.; Freiberg, A. Mirror Symmetry and Vibrational Structure in Optical Spectra of Chlorophyll a. J. Chem. Phys. 2009, 130, 194501.

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Page 33 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(37) Reimers, J. R.; Cai, Z.-L.; Kobayashi, R.; Rätsep, M.; Freiberg, A.; Krausz, E. Assignment of the Q-Bands of the Chlorophylls: Coherence Loss via Qx − Qy Mixing. Sci. Reports 2013, 3, 2761. (38) Freiberg, A.; Rätsep, M.; Timpmann, K.; Trinkunas, G. Excitonic Polarons in Quasi-OneDimensional LH1 and LH2 Bacteriochlorophyll a Antenna Aggregates from Photosynthetic Bacteria: A Wavelength-Dependent Selective Spectroscopy Study. Chem. Phys. 2009, 357, 102– 112. (39) Purchase, R.; Völker, S. Spectral Hole Burning: Examples from Photosynthesis. Photosynth. Res. 2009, 101, 245–266. (40) Rätsep, M.; Freiberg, A. Resonant Emission from the B870 Exciton State and Electron– Phonon Coupling in the LH2 Antenna Chromoprotein. Chem. Phys. Lett. 2003, 377, 371–376. (41) Georgakopoulou, S.; Frese, R. N.; Johnson, E.; Koolhaas, C.; Cogdell, R. J.; van Grondelle, R.; van der Zwan, G. Absorption and CD Spectroscopy and Modeling of Various LH2 Complexes from Purple Bacteria. Biophys. J. 2002, 82, 2184–2197. (42) Rätsep, M.; Pajusalu, M.; Linnanto, J. M.; Freiberg, A. Subtle Spectral Effects Accompanying the Assembly of Bacteriochlorophylls into Cyclic Light Harvesting Complexes Revealed by High-Resolution Fluorescence Spectroscopy. J. Chem. Phys. 2014, 141, 155102. (43) Freiberg, A.; Timpmann, K.; Ruus, R.; Woodbury, Disordered Exciton Analysis of Linear and Nonlinear Absorption Spectra of Antenna Bacteriochlorophyll Aggregates: LH2-Only Mutant Chromatophores of Rhodobacter sphaeroides at 8 K Under Spectrally Selective Excitation. N. W. J. Phys. Chem. B 1999, 103, 10032–10041.

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(44) 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 Complex2 Chromoproteins for Gaining Deeper Insight into Bacterial Photosynthesis. Phys. Rev. E 2015, 92, 052709. (45) Croce, R.; van Amerongen, H. Natural Strategies for Photosynthetic Light Harvesting. Nature Chem. Biol. 2014, 10, 492–501. (46) Reimers, J. R.; Biczysko, M.; Bruce, D.; Coker, D. F.; Frankcombe, T. J.; Hashimoto, H.; Hauer, J.; Jankowiak, R.; Kramer, T.; Linnanto, J.; et al. Challenges Facing an Understanding of the Nature of Low-Energy Excited States in Photosynthesis. Biochim. Biophys. Acta, Bioenerg. 2016, 1857, 1627–1640.

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TOC Graphic

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Figure 1. Crystal structure of the LH2 complex from Ect. haloalkaliphila refined to 3.7 Å resolution at 100 K. (A) Side view of four transmembrane chains in an asymmetric unit cell of the crystal: α-peptides - yellow and magenta, β-peptides – cyan and blue, BChls – green, lycopene – orange. (B) Top view from cytoplasmic side of the membrane of the full LH2 complex, obtained by rotation around the fourth order axis. Figures were prepared using PyMOL.31 84x168mm (300 x 300 DPI)


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Figure 2. (A) Absorption spectra of LH2 antenna complexes from Ect. haloalkaliphila in Qy range as a function of increasing exposure to oxidizing light between 415 and 573 nm. The light intensity applied, as measured at 500 nm, was 0.2 W/cm2. (B) Same as in part (A) but for absorption spectra normalized with respect to the peak value of the B800 band. (C) Same as in part (A) but for fluorescence spectra. Excitation at 594 nm into the Qx absorption band was employed. Inset of (C) presents the absorption spectra of LH2 complexes with oxygen scavenger added, oxidized similarly over a time span of 1 hour (normalized to B800 band). The spectra were measured at ambient temperature; similar color code is used in all panels; vertical arrows indicate directions of major changes of the spectra; the inclined arrow points out that an impurity LH1 absorption band has become visible in strongly bleached samples. 84x178mm (300 x 300 DPI)



Figure 3. (A) Absorption spectra of LH2 complexes upon illumination with increasing doses of 532 nm laser light of 0.2 W/cm2 intensity. (B) Differential absorption spectrum (35 min minus 0 min exposure time) of LH2 complexes. The initial absorption spectrum was subtracted from the spectrum upon saturating illumination. The spectra were recorded at ambient temperature; indicated are differential bands related to various (Qy, Qx, and Soret) molecular transitions of BChl (red) or AcChl (blue) origin. 84x121mm (300 x 300 DPI)


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Figure 4. (A) Comparison of the absorption (blue curves) and fluorescence (red curves) spectra of untreated LH2 complexes recorded at ambient temperature (dashed lines) and at 5 K (solid lines). Shown in green in the red edge of the low-temperature absorption spectrum is a Gaussian-approximated IDF. The fluorescence spectra were normalized by the peak intensity. The inset shows a deconvolution of the Qx absorption band (turquoise line) into two Gaussian thin-line components peaking at 591 and 601 nm due to B850 and B800 BChl assemblies, respectively. The bold black line designates sum of the fitting Gaussian components. (B) Absorption (blue curve) and fluorescence (red curve) spectra of oxidized LH2 complexes recorded at 5 K. The photooxidation was performed with a 532 nm laser light of 0.2 W/cm2 intensity during 84 min. The spectra were normalized with respect to B800 peak intensities. Shown for reference with dashed line is the fluorescence spectrum of Chl a,36 normalized to C700 intensity and shifted by 26.5 nm toward longer wavelengths to compensate for its exciton shift. Fluorescence was excited at 407 nm in both panels. Grey bars at the bottom show approximate fluorescence recording ranges used in the measurements of fluorescence excitation spectra in the corresponding panels of Figure 5 below.



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Figure 5. Fluorescence excitation spectra measured at ambient temperature of untreated (blue curves) and oxidized (red curves, photooxidation with a 532 nm laser light of 0.2 W/cm2 intensity during 84 min) LH2 complexes measured at ambient temperature. The fluorescence was recorded at selected emission regions shown, see also Figure 3B. 84x92mm (300 x 300 DPI)



Figure 6. Dependence of the CD spectra of LH2 complexes on the illumination time at ambient temperature. The inset shows zoomed-in section of the spectrum around 700 nm, normalized by the intensity of the C700 absorption band obtained after 35 min bleaching. The main frame spectra were normalized by initial/untreated B850 band intensity. The sample as well as color code is the same as used in Figure 3A. 84x61mm (300 x 300 DPI)


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Figure 7. Calculated energy level schemes for idealized (no disorder) native LH2 complex (left, B800-850) and a complex where all the BChls in the B850 compartment have been exchanged to AcChl (right, B800C700). Shown in center is an intermediate situation (designated as B800-850/B800-C700) for a randomly selected spatial conformation with half (8) of the B850 BChls exchanged. Highlighted by red are the exciton states mainly localized on B800-BChl sites. Arrows indicate major channels of fluorescence decay observed at cryogenic temperatures. 84x108mm (300 x 300 DPI)



Figure 8. Simulated exciton absorption spectra of LH2 complexes as a function of increasing number (from 0 to 16) of the B850-BChl pigments replaced by AcChl. Drawn with bold black and magenta lines are, respectively, the initial spectrum with zero oxidized sites and final spectrum with all 16 sites oxidized. See text for further explanations. 84x63mm (300 x 300 DPI)


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Figure 9. Comparison of the experimental (black lines) and simulated (colored lines: red for fully oxidized B850 ring, blue for 14 oxidized B850-BChls) absorption spectra of untreated (A) and maximally photooxidized (B) samples at ambient temperature. Please notice the reciprocal (linear in energy) wavelength scale, see text for further explanations. 84x117mm (300 x 300 DPI)



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Controlling Photosynthetic Excitons by Selective Pigment

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