Estimation of the bacteriochlorophyll c oligomerisation extent in Chloroflexus aurantiacus chlorosomes by very low-frequency vibrations of the pigment molecules: A new approach

Biophysical Chemistry 240 (2018) 1–8

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Estimation of the bacteriochlorophyll c oligomerisation extent in Chloroflexus aurantiacus chlorosomes by very low-frequency vibrations of the pigment molecules: A new approach ⁎

A.G. Yakovleva, , A.S. Taisovaa, V.A. Shuvalova,b, Z.G. Fetisovaa, a b

T

⁎

Lomonosov Moscow State University, Belozersky Institute of Physico-Chemical Biology, Leninskie Gory, 119991 Moscow, Russian Federation Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, Moscow 142290, Russian Federation

H I GH L IG H T S

G R A P H I C A L A B S T R A C T

low-frequency vibrations in Cfx. • Very aurantiacus chlorosomes are sensitive to oligomerisation.

of coupled oscillators predicts • Theory the very low-frequency BChl c vibrations.

The very low-frequency BChl c vibra• tions depends on growth-light-intensity.

unit building blocks are built up • The from quasi-linear chains of several BChl c pigments.

A R T I C LE I N FO

A B S T R A C T

Keywords: Photosynthesis Chloroflexus aurantiacus Chlorosome Coherent oscillations

In green photosynthetic bacteria, the chlorosomal bacteriochlorophyll molecules are organized via self-assembly and do not require proteins to provide a scaffold for efficient light harvesting. Despite numerous investigations, a consensus regarding the spatial structure of chlorosomal antennae has not yet been reached. For the first time, we demonstrated by coherent femtosecond spectroscopy at cryogenic temperature that the very low-frequency (~101 cm−1) vibrations of bacteriochlorophyll c pigments in isolated Chloroflexus aurantiacus chlorosomes are sensitive to their oligomerisation extent which depends on the light intensity during the growth of the cell cultures. We explained this sensitivity in terms of the coupling of delocalised vibration modes of BChl c molecules aggregated into chains within their antenna unit building blocks. These findings, together with previously obtained spectroscopy and microscopy data, confirmed that the unit building blocks functioning within Chloroflexus aurantiacus chlorosomal antenna are built up from the rather short (2–5 BChl c pigments) quasilinear chains. The approach presented here seems to be perspective since it directly reveals structural and dynamical properties of the oligomeric systems.

Abbreviations: ΔA, light−dark absorbance changes; BChl, bacteriochlorophyll; Cb, Chlorobaculum; Cfx, Chloroflexus; Chl, chlorophyll; C, Chlorobium; CMC, chlorosome-membrane complexes; FWHM, full width at half maximum; OD, optical density; RC, reaction centre; Rba, Rhodobacter ⁎ Corresponding authors. E-mail addresses: [email protected] (A.G. Yakovlev), [email protected] (Z.G. Fetisova). https://doi.org/10.1016/j.bpc.2018.05.004 Received 2 April 2018; Received in revised form 14 May 2018; Accepted 19 May 2018 Available online 22 May 2018 0301-4622/ © 2018 Elsevier B.V. All rights reserved.

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1. Introduction

type arrangement of the BChl aggregates in chlorosomes based on X-ray structure analysis [24]. Concentric helical nanotubes of pairs of alternating syn-antiligated BChl c and d stacks were proposed for the green sulphur bacterium, Cb. tepidum, mutant [25]. Besides, a concentric-roll structure of antennae was proposed in [26,27]. The variety of structural models can be a consequence of structural heterogeneity of the chlorosomes. Availability of the various types of molecules (BChl c, d, e) and large variations in the size of their aggregates strongly complicated the use of X-ray structure analysis. This inherent heterogeneity affects the use of various optical spectroscopy methods due to ensemble averaging of important spectral features. The single-particle spectroscopy of individual chlorosomes also confirmed the strong structural disorder of them. A combination of several techniques, such as mutagenesis, single-particle spectroscopy, and cryo-EM imaging is useful for study the structural arrangement of the aggregated BChl molecules inside the chlorosome [25]. Despite numerous investigations, a consensus regarding the 3D structure of the chlorosomal antennae has not yet been reached (for review, see [21] and the references therein). Previously, the growth-light-controlled variability of the aggregation extent of peripheral antenna pigments was studied in isolated Cfx. aurantiacus chlorosomes by steady-state and time resolved spectroscopy of the BChl c Qy absorption bands [16,19–21]. The experimental findings were theoretically explained in terms of unit building blocks from which the rod-like structures of BChl c aggregates are composed in Cfx. aurantiacus chlorosomes. Each rod was considered as a linear chain of the unit building blocks in accordance with the freeze-fracture electron microscopy data [16,22,23]. In its turn, each unit building block was composed of several short BChl c chains. The size of the BChl c unit building block as well as the number of BChl c pigments in quasi-linear chains within the unit building block were estimated using the standard exciton theory. The tubular model of 6 exciton-coupled BChl c chains within the unit building block and inter-chain distances of ~2 nm ensured the best fit of the experiment and approximated the in vivo BChl c low packing density [16,19–21]. The number of BChl c molecules per each chain within the unit building block was found to be not > 6–7 [21]. This model was able to explain several key spectroscopic properties of Cfx. aurantiacus chlorosomes, such as the exciton level structure of BChl c aggregates, revealed by spectral hole burning experiments, the antenna-size-dependent exciton dynamics in the BChl c chlorosomal antenna, the temperature dependence of the steady state fluorescence line shape of the BChl c band and the femtosecond pumpprobe spectra of the BChl c chlorosomal antenna. In the present work, we suggest a new approach to estimation of the BChl c oligomerisation extent in Cfx. aurantiacus chlorosomes by study of low-frequency vibrations of the aggregates. A set of intermolecular vibrational frequencies provides a deep insight into the structure of the molecular aggregate. There are at least three essential problems in this approach. First, it is clear that the true intermolecular vibrations of a large molecular assembly have very low frequencies. Two widely used techniques such as stimulated Raman and coherent pump-probe spectroscopy are not able to reveal modes with frequencies lower than several cm−1. Second, a real motion of large molecular aggregates is an extremely complicated mixture of inter- and intramolecular motions, and it is not clear how to separate them. Third, the known low-frequency BChl c modes of Cfx. aurantiacus chlorosomes are not assigned. Nevertheless, an approach presented here seems to be perspective because it directly reveals structural and dynamical properties of the nuclear subsystem. The present study compared the coherent components of the kinetics measured in the BChl c Qy bands of Cfx. aurantiacus chlorosomes, which were isolated from cells grown under different light intensities and therefore had different sizes of BChl c antennae and their unit building blocks [19–21]. Pronounced coherent low-frequency (50–250 cm−1) oscillations were previously found in Cfx. aurantiacus chlorosomes at room [28] and cryogenic [29] temperatures. The weaker low-frequency oscillations were found in chlorosomes isolated

Photosynthesis is the key natural process which underlies of life on Earth. Solar energy is converted into the energy of stable chemical compounds in this biological process. Photosynthesis is the main source of oxygen and organic compounds on Earth. Solar energy refers to an inexhaustible and ecologically pure form of energy. Photosynthesis starts from absorption of light quanta in light-harvesting chlorophyll complexes. Then the excitation energy is transferred to the photosynthetic reaction centres, where primary conversion of the light energy into the energy of separated charges occurs. These primary events trigger a long and very complicated series of biochemical reactions resulted in a synthesis of stable chemical compounds. The quantum efficiency of primary steps of natural photosynthesis is close to 100% [1] due to the rigid optimization of the photosynthetic apparatus structure according to the functional criteria [2,3]. The requirements for structural optimization become more stringent with increasing the antenna size [4]. Therefore the mechanisms of primary processes and the features of the photosynthetic apparatus that ensure its high efficiency for light energy conversion must be explored. This subject has compelling importance for both natural and artificial photosynthesis. Previously, it was shown by the model calculations, that antenna pigment oligomerization is biologically expedient strategy for light harvesting in photosynthesis [5,6]. Oligomerization of antenna pigments is possible due to intrinsic donor-acceptor properties of all chlorophylls. These key properties of chlorophylls make possible selfaggregation of the pigments [7,8]. The most amazing example of long-range ordered natural light harvesting antennae are the chlorosomes of green photosynthetic bacteria. Chlorosomes are the largest among all known photosynthetic light-harvesting structures (~104–105 pigments in the aggregated state). A chlorosome body of ellipsoidal shape (100–200 × 40–100 × 10–30 nm) is attached to the inner surface of the cytoplasmic membrane. In the green nonsulphur thermophilic bacterium, Chloroflexus (Cfx.) aurantiacus, the chlorosome is the peripheral antenna that is typically comprised of approximately 104 bacteriochlorophyll (BChl) c pigments [9]. Direct experimental proof of oligomeric organization of chlorosomal pigments in intact cells of the green bacteria Cfx. aurantiacus, Chlorobium (C.) limicola and Chlorobaculum (Cb.) pheovibrioides was obtained by the spectral hole-burning method [10–12]; and the chlorosomal aggregate has been shown to be built from quasi-linear chains of BChl c molecules. A long-range molecular order of chlorosomal BChl c of the green bacteria Cfx. aurantiacus and C. limicola was shown by picosecond fluorescence polarisation spectroscopy [13,14]. Besides BChl c/d/e, chlorosomes of all green bacteria also contain carotenoids and a small amount of BChl a [15]. This BChl a antenna connects the chlorosomal BChl c antenna with the cytoplasmic membrane, in which the core BChl a antenna and reaction centres are located [9,15]. The harvested energy is transferred from BChl c aggregates to the BChl a baseplate within 10–40 ps, depending on the size of the BChl c antenna [16]. As with most photosynthetic organisms, these bacteria are able to adapt to low light intensities by drastically increasing their peripheral antenna size [17–21, this work]. The most interesting and attractive feature of the chlorosome is the fact that BChl molecules self-aggregate into ordered structures without participation of proteins as a structural matrix. It has been widely accepted that the neighbouring molecules in the chlorosomal BChl aggregates couple with each other via coordination bonds between the C31 hydroxy group and the central Mg atom and via hydrogen bonds between the C13 carbonyl group and the C31 hydroxy group [7,8]. Structural models of the chlorosomal BChl aggregates have been extensively investigated [14,16,22–27]. Some models used a rod-like arrangement of BChl c aggregates based on freeze-fracture electron microscopy data [16,22,23]. Conversely, some models used a lamellar2

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2.2. Spectroscopy

from the green sulphur bacterium, C. tepidum [29,30]. The nature of this phenomenon is still under debate; however, the closeness of most of the oscillation frequencies to the frequencies revealed by resonance Raman spectroscopy [31] argues in favour of their vibrational origin. Being a complicated mixture of inter- and intramolecular motions, some of these low-frequency vibrations may reflect the structural peculiarities of the chlorosomal aggregates. Indeed, some low-frequency vibrational modes of in vitro BChl a oligomers were found to be sensitive to oligomerisation [32]. In the present work, the very low-frequency parts of the Fourier spectra of the coherent oscillations of Cfx. aurantiacus chlorosomes were also shown to be sensitive to oligomerisation. Together with model calculations, this finding provides a novel approach to study the structure of the BChl c aggregates in Cfx. aurantiacus chlorosomes.

Ground state absorption spectra of chlorosomes were recorded at room temperature with a Hitachi-557 spectrophotometer (Japan). Time-resolved absorption difference (ΔA, light − dark) spectra were measured with a fs laser spectrometer including a mode-locked Ti:sapphire generator (Spectra Physics, USA), a multipass Ti:sapphire amplifier (Avesta, Russia) and an optical multichannel analyser (Oriel, France). Amplified pulses were focused into an H2O–D2O mixture to produce a smooth white-light continuum. A small part of the continuum was used for probing, and the spectrally-filtered main part was used for pumping. The pump wavelength was 750 nm, and the optical density (OD) of the samples was 0.5–1.0. The pump intensity was attenuated to avoid multiphoton effects. The pulse duration was 30 fs. The magic angle of 54.7° was used between the planes of polarisation of the pump and probe beams. At each pump-probe time delay, the ΔA spectrum was collected and averaged over several thousand measurements to provide a spectral measurement accuracy of approximately 10−4 OD. The ΔA kinetics were plotted at fixed wavelengths on the base of the measured ΔA spectra. Group velocity dispersion was compensated to < 35 fs. All measurements were carried out at 90 K. All samples were mixed with glycerol (65% v/v) to provide good transparency.

2. Materials and methods 2.1. Sample preparation 2.1.1. Growth All experiments were performed on the filamentous nonsulfur thermophilic green bacterium Chloroflexus aurantiacus (Cfx. aurantiacus) strain Ok-70-fl (collection of Leiden University, The Netherlands). Cells of Cfx. aurantiacus were cultivated anaerobically in batch cultures at 55 °C in 1-liter screw cap bottles at different light intensities. Different light intensities with photon flux densities of 5 μE m−2 s−1 (low-light intensity), 10 μE m−2 s−1 (middle-light intensity) and 50 μE m−2 s−1 (high-light intensity) were used. Different light intensities were achieved by varying the number of lamps and the distance from the light-source. We used the following medium for the growth of bacteria [17]: yeast extract (Difco) 1.0 g, vitaminfree casamino acids (Difco) 2.0 g, glycyl–glycine 0.8 g, 100 ml mineral basis (Medium D) [33,Supplement], distilled water ad 1000 ml, pH 8.2.

3. Results and discussion 3.1. Spectra, kinetics and oscillations Ground state absorption (room temperature) and transient ΔA (90 K, 0.3 ps time delay) spectra of Cfx. aurantiacus chlorosomes isolated from cultures grown at low-, middle- and high-light intensities are shown in Fig. 1. The Qy absorption bands at ~740 nm belong to chlorosomal BChl c. The negative ΔA bands at ~745 nm (Fig. 1B) were mostly formed by BChl c absorption band bleaching and red-shifted stimulated emission [28]. The ps ΔA kinetics at 755 nm for Cfx. aurantiacus chlorosomes isolated from cultures grown at low-, middle- and high-light intensities are shown in Fig. 2A. Pronounced complicated oscillations were seen within approximately 2 ps after excitation in all three samples. To extract oscillatory components, the kinetics were fitted by exponents and an instrumental function. The minimal number of exponents necessary for a good approximation was found to be three. The major component had time constants of 11 (49%, high-light samples), 18 (52%, middlelight samples) and 32 (55%, low-light samples) ps. The time constants of the two minor components were 0.1 (19%, all samples) and 1.3 (32%, low-light samples; 29%, middle-light samples; 26%, high-light samples) ps. The time constants of these components agreed with those revealed in previous room-temperature experiments [28]. It was generally accepted that the major component reflected the energy transfer from BChl c aggregates to BChl a baseplate and that the faster minor components reflected the migration of excitation energy inside the chlorosomal BChl c antenna [28]. It should be noted that the fit utilised in this model was not unique (i.e. the time constants and relative contributions of the three components could be slightly varied without aggravating the fit). The pure oscillations were obtained by subtracting the fit from the original kinetics (Fig. 2B). The overall oscillatory features were similar in all three samples; for example, the strong initial oscillations were quickly damped within 0.5 ps; however, the weaker lower-frequency oscillations were seen within 2–3 ps. The phase of the oscillations varied with the wavelength in a complicated manner (data not shown). The 120–150 cm−1 oscillations had approximately reversed phases in the short- (< 745 nm) and long-wavelength (> 745 nm) areas of the ΔA spectra. The phases of the 9 and 41 cm−1 oscillations also varied with wavelength; however, their signs remained unchanged. The Fourier transformation of the oscillations revealed several bands within 5–400 cm−1 (Fig. 2C). The strongest bands were at

2.1.2. Isolation of chlorosome-membrane complexes Chlorosome-membrane complexes (CMC) were prepared from fresh Cfx. aurantiacus cells cultures with different BChl c contents by the method of Ma et al. in slight modification [34]. Fresh cells were harvested by centrifugation at 8,500 × g for 20 min, washed with 10 mM Tris-HCl buffer, pH 8.0 and suspended in TA buffer (10 mM Tris-HCl buffer pH 8.0 containing 10 mM sodium ascorbate). The suspension was disrupted by two passes through a French pressure cell at 20,000 psi in the presence of 2 mM phenylmethylsulphonyl fluoride. Unbroken cell and large debris were removed by centrifugation at 20,000 × g for 30 min at 4 °C. The supernatant was centrifugated for 90 min at 180,000 × g (45,000 rpm, Ti 50) at 4 °C, and the resulting pellet was resuspended in a small volume of TA buffer. It contained the cytoplasmic membranes with attached chlorosomes and will be referred to as the chlorosome-membrane complexes (CMC). 2.1.3. Chlorosome isolation Chlorosomes were isolated from Cfx. aurantiacus CMC suspension in TA buffer in a twofold successive continuous sucrose gradient (55–20% (wt/wt) and 45–15% (wt/wt) as described earlier in modification for CMC [35]. Continuous sucrose density gradients were prepared in 50 mM Tris-HCl-buffer, pH 8,0, and 2 M NaSCN (Tris-thiocyanate buffer) in the presence of 10 mM sodium ascorbate in centrifuge tubes each containing a total of 38 ml. 1,5–2,0 ml of the CMC suspension with optical density 150–170 optical units at 740 nm was carefully added on top of each sucrose gradient. After centrifugation for 20 h at 135,000 × g (28,000 rpm, SW-28) at 4 °C the chlorosomes banding between 28 and 30% sucrose were collected (based on the amplitude of absorption at 740 nm). We used only freshly isolated samples of chlorosomes in all spectroscopic experiments. Before measurements, the chlorosomes were incubated for 30 min with 18 mM sodium dithionite at 4 °C in order to assure strongly reduced conditions. 3

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Fig. 1. Ground state absorption (A, room temperature) and transient ΔA (B, 90 K, 0.3 ps time delay) spectra of high-, middle- and low-light Cfx. aurantiacus chlorosomes. The spectra are normalised and vertically shifted for clarity. Zero absorption and ΔA are shown by dash lines.

Fig. 2. ΔA kinetics (A), their oscillatory components (B) and Fourier spectra of the oscillatory components (C) of high-, middle- and low-light Cfx. aurantiacus chlorosomes measured at 755 nm at 90 K. The samples were pumped by 30-fs pulses at 750 nm. The curves are vertically shifted for clarity. Zero lines in (C) are shown by dash.

approximately 9, 41 and 135 cm−1 for the low-, middle- and high-light samples. The Fourier spectra of all samples were similar in the area above 40 cm−1, although the spectral positions and amplitudes of several peaks clearly varied. The total number of spectral peaks in this area did not change with growth-light-intensity. Conversely, the Fourier spectra of all three samples were noticeably different in the spectral area below 40 cm−1 (Fig. 3A). For example, the high-light spectrum contained one small peak at 26 cm−1 in addition to the strong peaks at 9 and 41 cm−1. The middle-light spectrum contained two additional peaks between the 9 and 41 cm−1 peaks, namely, at 18 and 28 cm−1. The low-light spectrum contained a set of small distinct strongly-overlapping peaks at 5, 9, 15, 24, 31, 38 and 41 cm−1 superimposed on the broad background. Rapid damping of the kinetics within 2–3 ps prevented better resolution of the lowest-frequency Fourier components. The Fourier spectra of the ΔA kinetics measured at 747 and 740 nm were qualitatively similar to those measured at 755 nm, although the spectral positions and amplitudes of the peaks varied insignificantly with wavelength (Fig. 3B and C). The same difference between the Fourier spectra of low- and high-light samples is seen at these wavelengths.

The frequency of this oscillation is equal to the vibrational frequency. The oscillation vanishes in the centre of the stimulated emission band. The oscillation phase has the opposite sign in the short- and long-wavelength parts of this band. Also, a vibrational wave packet can be created in the ground state by impulse Raman scattering. In this case, the oscillation vanishes in the centre of the absorption band. On the other hand, nonvibrational oscillations can be produced by beating between different excitonic states contributing to the total BChl c Qy band. Such electronic beats were recently found at 620 cm−1 in Cb. limnaeum chlorosomes at cryogenic temperature using 2D spectroscopy [37]. It is likely that vibrational and electronic oscillations are mixed that greatly complicates their analysis. The strong argument in favour of the vibrational nature of the lowfrequency coherent oscillations in chlorosomes is a closeness of their frequencies to those revealed by Raman spectroscopy. The resonance Raman scattering in Cfx. aurantiacus chlorosomes revealed several vibrational modes below 400 cm−1, namely, at 63, 94, 120, 148, 162, 183, 214, 272, 291, 315, 360 and 384 cm−1, with the strongest modes at 94, 120 and 148 cm−1 [31]. Most of the Fourier modes (Fig. 3) had frequencies close to the vibrational Raman frequencies, except for the modes with frequencies below 40 cm−1 which were not presented in the Raman spectrum. It is interesting that modes with similar frequencies were found in reaction centres of green and purple photosynthetic bacteria [38,39] and in artificial chlorophyll a oligomers [32]. Such a similarity may be a consequence of the similar tetrapyrrole structure of all (bacterio)chlorophylls. It should be emphasised that no coherent oscillations were found in the absorption band of monomeric

3.2. Low-frequency vibrations The nature of low-frequency oscillations in the ΔA spectra of chlorosomes is not completely understood; however, most of the oscillation modes are thought to be vibrational. Each of the vibrational oscillations can be rationalized in terms of the vibrational wave packet. The coherent excitation of vibrational manifold creates the wave packet oscillating along the potential energy surface of the excited state [36]. 4

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slightly. In other words, the tetrapyrrole ring and phytol tail move like rigid molecules in different directions to each other in the 8.6-cm−1 mode. For this reason, the 8.6-cm−1 mode can be considered as an analogue of the intermolecular vibrational mode [40]. The characteristic frequency of this intermolecular-like motion is not dependent on the structure of side chains and is thus similar in other types of BChls [40]. The other calculated lowest-frequency modes of BChl a were at 3, 20, 23, 27, 35, 40, 44 and 47 cm−1 [40]. All of these modes were strongly delocalised over 10–40 atoms. The 35-cm−1 strong mode was the most intensive in the region below 50 cm−1 and represents deformations of the macrocycle plane along with librations of the acetyl group. Another calculated strong-frequency mode at 146 cm−1 was only delocalised on the atoms of the tetrapyrrole macrocycle and corresponded with the mutual displacement of the central Mg atoms and pyrrole rings [40]. The phytol tail and tetrapyrrole ring can be treated as rigid molecules only for the four lowest-frequency normal modes calculated at 3, 9, 20 and 23 cm−1 [40]. In the higher-frequency modes, the motions of these two parts of the BChl molecule are strongly mixed. An atomistic ab initio calculation of spectral density for the model chlorosomes consisting of multilayered rolls revealed strong vibrational modes above 1000 cm−1 and much weaker modes below 1000 cm−1 [27]. The low-frequency modes calculated in [27] were ascribed to intermolecular vibrations in a BChl layer. All calculated modes appearing in the spectral density were strongly coupled with the optical transition [27]. The lowest-frequency BChl modes may be sensitive to the composition and spatial configuration of BChl aggregates due to their strong delocalisation. As far as the lowest-frequency modes of single BChl molecules have some intermolecular features [40], coupling between the same modes of neighbouring molecules may be assumed to occur in the chlorosomal BChl c aggregates. This coupling may originate from H bonds and Van der Waals interactions between closely-spaced BChl c molecules within an aggregate. In principle, the direct inclusion of some atoms from neighbouring BChl c molecules into collective motions cannot be discounted; however, it seems to be a more unlikely possibility.

Fig. 3. Fourier spectra of the oscillatory components of the ΔA kinetics of high-, middle- and low-light Cfx. aurantiacus chlorosomes measured at 755 (A), 747 (B) or 740 (C) nm, 90 K in a frequency range of 0–60 cm−1. The spectra are vertically shifted for clarity. Zero lines are shown by dash.

BChl a of Cfx. aurantiacus chlorosomal baseplate at ~800 nm at cryogenic (this study) or room [28] temperature. Notice that the resonance Raman spectroscopy studies the ground state modes while the coherent pump-probe spectroscopy reveals the modes in both the excited and ground states. In contrast to high-frequency (> 1000 cm−1) vibrational modes, low-frequency modes in chlorosomes and other light-harvesting complexes or reaction centres have not been identified. It is currently widely accepted that these modes are strongly delocalised and may represent both intra- and intermolecular nuclear motions of (bacterio) chlorophylls with possible participation from the nearest molecular environment. Characteristic intermolecular vibrational frequencies of such supramolecular complexes are very low. Low-frequency modes with intrachromophore character were observed and identified in chlorophylls and porphyrins in vitro. These delocalised modes included motions of central metal atoms, neighbouring nitrogen atoms, ethyl groups and ring distortions. These modes were likely influenced by intermolecular interactions including the interaction of the pigment molecule with its environment. Normal mode analysis of individual BChl a molecules was used to calculate tens of low-frequency vibrational modes in the 3–400 cm−1 region [40]. Among them, the 8.6 cm−1 mode was the most delocalised, with a delocalisation index of approximately 55 (i.e. this mode was approximately delocalised over the entire molecule). This mode represents the libration of the tetrapyrrole macrocycle plane of the BChl a molecule relative to the axis passing through the atoms of C5MgC10 as well as the libration of the phytol tail. During libration, the spatial structures of the tetrapyrrole macrocycle and phytol tail change

3.3. Estimation of BChl c oligomerisation extent Focus should now be placed on the lowest-frequency part of the Fourier spectra of oscillations (Fig. 3). At least two possibilities can be considered. First, the difference between the low- and high-light Fourier spectra may be a consequence of the change in the spatial configuration of BChl c oligomer chains. It should be noted that both the coherent pump-probe and Raman spectroscopy methods reveal only those vibrational modes that are coupled with a specific optical transition. The coupling strength and, consequently, the amplitude of each Fourier or Raman mode may depend on the chlorosome structure. Thus an enhancement of some of the lowest-frequency modes in the low-light Cfx. aurantiacus chlorosomes (Fig. 3) may be explained by the geometry change of BChl c oligomer chains. It seems unlikely that the change in the growth-light intensity could be responsible for the noticeable change in the BChl c intramolecular structure. Further analysis of the possible dependence of the 3D chlorosomal structure on growth-light intensity is strongly restricted by the fact that all low-frequency chlorosomal modes are not assigned. Second, the experimentally observed oscillation at 9 cm−1 can be assumed to represent the theoretical vibrational 8.6 cm−1 BChl mode [40], and self-assembly of the BChl c molecules can be assumed to produce coupling of the 9 cm−1 vibrational mode of the neighbouring BChl c molecules. In this context, a simple case of linear chain of N coupled identical oscillators can be considered as a preliminary model. According to the theory of coupled oscillators (classical or quantum), frequency eigenvalues of this chain can be written as [41]: 5

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variable in size with growth-light-intensity from 3–4 exciton-coupled BChl c molecules in high-light chlorosomes to 6–7 BChl c molecules in low-light chlorosomes. Thus the number of BChl c molecules per chain in the unit building blocks of low-light chlorosomes is approximately two-fold higher than in high-light chlorosomes; however, the number of BChl c molecules does not exceed several molecules per chain. Thus, as the light intensity decreases, the size of the BChl c antenna increases thus compensating light deficit by enhancing the light-absorbing capability of the antenna. An inevitable drop in the efficiency of the antenna functioning with increasing its size, in turn, is compensated by an increase in the size of the unit BChl c aggregate thus ensuring the high efficiency of energy transfer within the antenna regardless of its size [16,19–21,42]. This subject is highly important for both natural and artificial photosynthesis. 3.5. Comparison with dimeric and monomeric BChl vibrations Native BChl dimers (P) and monomers (B) are presented in reaction centres (RCs) of bacterial photosynthesis. In contrast to chlorosomes, the RC pigments are fixed in the protein matrix. In spite of evident structural diversity between the RCs and chlorosomes, it seems useful to compare the lowest oscillatory frequencies of them in order to test a validity of our conclusions. In the RCs of purple bacterium Rhodobacter (Rba.) sphaeroides as well as Cfx. aurantiacus, the BChl a dimer and monomer play a key role in charge separation as primary electron donor and primary electron acceptor, respectively. Pronounced oscillations were observed in the P* stimulated emission band and BA− absorption band of these RCs in the coherent pump-probe experiments [e.g. 36,39]. In the Rba. sphaeroides RCs, the four lowest frequency modes of the oscillations were at 8–9, 28–33, 43–45 and 66–69 cm−1 in the P* and BA− oscillations. Among these modes, the strongest one was at 8–9 cm−1 in the P* oscillations and at 28–33 cm−1 in the BA− oscillations. Unfortunately, it is impossible to extract the pure monomeric oscillations, because the BA− oscillations mainly repeat the P* ones. It should be noticed that no Fourier modes were observed between 8–9 and 28–33 cm−1. Recently, molecular dynamics simulations showed that the nearest protein environment of the P and B pigments is directly involved into the low-frequency collective nuclear motion [43]. This finding complicates the direct comparison between chlorosomal and RC Fourier spectra of the oscillations. Nevertheless, it is evident that the 9 cm−1 mode is very conservative with respect to the structural changes since it was observed in both the chlorosomal (Fig. 3) and RC [39] oscillations. If to assume that the 26 and 41 cm−1 modes of high-light Cfx. aurantiacus chlorosomes (Fig. 3) are analogous to the 28–33 and 43–45 cm−1 modes of RCs, then one can conclude that high-light chlorosomal aggregates mainly composed from BChl dimers. The theoretical model (formula (1)) led to the same conclusion. Usually, the resonance Raman spectroscopy provides much better accuracy in determination of vibrational frequencies. The lowest-frequency modes of P and B are at 36 and 85 cm−1, respectively, in the Raman spectra measured at 95 K [38]. In Cfx. aurantiacus chlorosomes, the lowest-frequency Raman mode is at 63 cm−1 [31]. On the other hand, an absence of the 9 cm−1 mode in the Raman spectra of chlorosomes and RCs may be explained by assignment of this mode to the corresponding excited states. Pure BChl or Chl monomers can be obtained in vitro by dissolving the samples in MeOH. Recent study of the Q-bands of Chl a monomers by fs 2D electronic spectroscopy showed the absence of strong vibronic and vibrational bands at frequencies below 260 cm−1 [44]. The resonance Raman spectroscopy of the Chl monomers is extremely complicated in the frequency area below 300 cm−1 due to the strong fluorescence [32]. The lowest-frequency Raman modes of Chl a in vitro monomers and oligomers were reported to be at 115 and 50 cm−1, respectively [32]. No coherent oscillations were found in the fs ΔA spectra of monomeric BChl a of Cfx. aurantiacus chlorosomal baseplate at cryogenic (this study) or room [28] temperature. Thus, information

Fig. 4. Theoretical vibrational spectra of the linear chain of coupled identical oscillators calculated by formula (1) for different number of oscillators (N). The eigenfrequency of each oscillator (ω0) = 9 cm−1, and the coupling frequency (ω1) = 37 cm−1. All modes were assumed to have the same amplitude and Gaussian line shape (FWHM = 5 cm−1). The spectra are vertically shifted for clarity.

ωn 2 = ω0 2 + ω12 sin2 (πn/2N ), n = 0, 1, 2, …, N − 1

(1)

Where ω0 is an eigenfrequency of each oscillator, and ω1 is a coupling frequency. In the classical model of physical pendulums coupled by springs, ω02 = g/l, and ω12 = 4 k/m, where g is the acceleration of gravity, l is the length of the pendulum, k is the coefficient of elasticity and m is the mass of the pendulum. Mathematical details of the theory are available in Supplement. According to formula (1), all frequencies fall within ω0 and (ω02 + ω12)0.5; moreover, most are near both boundaries of this spectral area. For example, the frequency spectrum of the chain of N identical oscillators is shown in Fig. 4 for different N. For small N, an increase in the chain length (i.e. an increase in the N value) simply produced a proportional increase in the total number of frequency peaks. For larger N, the spectrum consisted of two major peaks formed by the overlapped modes, whose frequencies were close to both boundaries, and a few smaller peaks located between the two. For modes with Gaussian line shape and a full width at half maximum (FWHM) of 5 cm−1, overlapping of them became noticeable when N > 5 if ω0 = 9 cm−1 and ω1 = 37 cm−1 (Fig. 4). The BChl c aggregates seem to be small in all samples, because only a few modes were observed near the initial 9-cm−1 mode (Fig. 3). If the aggregates are quasi-linear chains [13,14,21], then the number of molecules per chain can be easily estimated as follows. According to model calculations (Fig. 4), the appearance of new modes in addition to the initial 9-cm−1 mode reflects an increase in the number N of the aggregated BChl c molecules. For small N, the addition of one BChl c molecule to the aggregate simply results in the appearance of one new mode. For high-light chlorosomes, the number N seems to be 2, as only one additional mode was observed at 26 cm−1 (Fig. 3). For middle-light chlorosomes, two additional modes at 18 and 28 cm−1 correspond to N = 3. Finally, four additional modes at 15, 24, 31 and 38 cm−1 correspond to N = 5 in the low-light samples. 3.4. Correlation with previous findings in Cfx. aurantiacus chlorosomes The estimated sizes of the BChl c aggregates correlates with the previous data [16,19–21,42]. Using different spectroscopy methods, it was shown that each unit building block in the Cfx. aurantiacus BChl c antenna consists of several short quasi-linear chains. These chains are 6

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about possible monomeric BChl vibrations at frequencies ~101 cm−1 is not available now.

AGY and ZGF wrote the manuscript. All authors discussed the results. Appendix A. Supplementary data

3.6. Comparison with Cb. tepidum chlorosomal vibrations Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bpc.2018.05.004.

The chlorosome of the green sulphur bacterium, Cb. tepidum, contains ~105 BChl c/d molecules in the aggregated state that is ~10 times greater than in the Cfx. aurantiacus chlorosome. This bacterium is perfectly adapted to very low light intensities. The size of Cb. tepidum chlorosomes is somewhat larger than that of Cfx. aurantiacus ones. The rods of Cb. tepidum chlorosome have ~10 nm diameter versus ~5 nm diameter of the Cfx. aurantiacus rods. Some substituents on the BChl c chlorine rings are different in the chlorosomes of both families. These structure differences are likely resulted in the different spectra of resonance Raman scattering as well as Fourier spectra of coherent oscillations. The very low-frequency Raman modes of Cb. tepidum chlorosomes are at 57, 89, 124, 138 and 173 cm−1 (63, 94, 120, 148, 183 and 214 cm−1 in Cfx. aurantiacus chlorosomes) [31]. The Fourier transformation of the coherent oscillations in the ΔA BChl spectra of Cb. tepidum chlorosomes revealed the modes at 5, 17, 52, 76, 124, 150, 171 and 190 cm−1 with three dominant modes at 17, 76 and 124 cm−1 [30]. Most of the Raman and Fourier Cb. tepidum modes are likely downshifted with respect to their Cfx. aurantiacus analogies. The broad 5 and 17 cm−1 Cb. tepidum Fourier modes strongly overlapped and covered a frequency range from 1–2 to 31 cm−1. Probably, several closely spaced modes are hidden in this range. To our knowledge, no information on the sensitivity of the Cb. tepidum vibrational modes to the oligomerisation of chlorosomal antenna pigments is available now.

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4. Conclusive remarks The new approach presented in this paper concerns an investigation of one of the basic structural features of the peripheral light harvesting antennae, namely, the growth-light-controlled variability of the aggregation extent of BChl c antenna pigments in chlorosomes of the green nonsulfur bacterium Cfx. aurantiacus [21]. For the first time, the very low-frequency (~10 cm−1) vibrations of BChl c in Cfx. aurantiacus chlorosomes were shown to be sensitive to their oligomerisation extent which is antenna-size-dependent. In its turn, the size of the chlorosomal antenna is known to be governed by the light intensity during the growth of the cell cultures. The low-light Fourier spectrum of the oscillations measured in the BChl c Qy band was found to contain several closely-disposed peaks at 10–40 cm−1 which were absent in the high-light spectrum. This finding was theoretically explained by the coupling of delocalised vibration modes of BChl c molecules aggregated into chains of different lengths within their antenna unit building blocks. The obtained data were found to be applicable for estimation of the number of BChl c molecules per chain. Comparative study of the very low-frequency BChl c vibrations in highand low-light Cfx. aurantiacus chlorosomes led us to the conclusion that the extent of BChl c oligomerisation was varied from ~2 to ~5 being governed by the light intensity during the cells growth. This conclusion is in line with previously obtained spectroscopy and microscopy data. To study a higher-order structure of chlorosomes, it is necessary to reveal the pigment vibrations with frequencies much lower than 10 cm−1. Acknowledgements This work was supported in part by the Russian Foundation for Basic Research (grants 15-04-02136а, 14-04-00295a). Author contributions ZGF and VAS led the project. AGY designed and performed the experiment and theoretical calculations. AST prepared the samples. 7

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