Light-Driven Reconfiguration of a Xanthophyll Violaxanthin in the

Article pubs.acs.org/JPCB

Light-Driven Reconfiguration of a Xanthophyll Violaxanthin in the Photosynthetic Pigment−Protein Complex LHCII: A Resonance Raman Study Wojciech Grudzinski,† Ewa Janik,†,‡ Joanna Bednarska,†,⊥ Renata Welc,† Monika Zubik,†,§ Karol Sowinski,†,∥ Rafal Luchowski,† and Wieslaw I. Gruszecki*,† †

Department of Biophysics, Institute of Physics, Maria Curie-Skłodowska University, 20-033 Lublin, Poland Department of Cell Biology, Institute of Biology and Biochemistry, Maria Curie-Sklodowska University, ul. Akademicka 19, 20-033 Lublin, Poland § Department of Metrology and Modelling of Agrophysical Processes, Institute of Agrophysics of Polish Academy of Sciences, Doswiadczalna 4, 20-290 Lublin, Poland ∥ Chair and Department of Synthesis and Chemical Technology of Pharmaceutical Substances, Faculty of Pharmacy, Medical University, Chodzki 4a, 20-093 Lublin, Poland ‡

S Supporting Information *

ABSTRACT: Resonance Raman analysis of the photosynthetic complex LHCII, immobilized in a polyacrylamide gel, reveals that one of the protein-bound xanthophylls, assigned as violaxanthin, undergoes light-induced molecular reconfiguration. The phototransformation is selectively observed in a trimeric structure of the complex and is associated with a pronounced twisting and a trans−cis molecular configuration change of the polyene chain of the carotenoid. Among several spectral effects accompanying the reconfiguration there are ones indicating a carotenoid triplet state. Possible physiological importance of the light-induced violaxanthin reconfiguration as a mechanism associated with making the pigment available for enzymatic deepoxidation in the xanthophyll cycle is discussed.

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INTRODUCTION

the complex is presented in the Supporting Information Figure S1. Under certain conditions the Vio molecule can be replaced in its binding site by zeaxanthin (Zea). Understanding of the structure−function relationship in this complex, and in particular the structural and dynamic properties of pigment− protein interactions, is an important element of progress in photosynthesis research. Resonance Raman scattering is a powerful technique that enables the in situ analysis of molecular configuration of xanthophyll pigments bound to LHCII.3−10 This is owing to the fact that the absorption spectra of the LHCII-bound carotenoids are shifted on the wavelength scale and therefore different pigments may be probed by applying different, selected laser lines.7 Precise resonance Raman studies provided unique information on light-induced, in situ molecular configuration changes of the LHCII-bound xanthophylls. It has been shown that upon illumination with strong light a twist in the molecular configuration of Neo takes place, which has been

Life on Earth is powered by the energy of light reaching our planet from the Sun but direct utilization of electromagnetic radiation energy to drive biochemical reactions is not possible. Photosynthesis is a process that converts the energy of photons to energy accumulated in chemical bonds, which can be utilized by living organisms. The primary photosynthetic reactions take place in the functional protein complexes called reaction centers but efficient operation of photochemical reactions is possible thanks to activity of numerous pigment−protein complexes called antenna, which collect photons and transfer electronic excitations toward the reaction centers. The largest photosynthetic antenna complex of plants, LHCII (lightharvesting pigment−protein complex of photosystem II), comprises virtually half of the chlorophyll molecules on Earth and is the most abundant membrane protein in our biosphere.1,2 In natural conditions, LHCII appears as a trimer. A monomer of the complex comprises 14 molecules of chlorophylls and 4 molecules of xanthophylls: 8 chlorophyll a (Chl a), 6 chlorophyll b (Chl b), 2 lutein (Lut), 1 neoxanthin (Neo), and 1 violaxanthin (Vio).1,2 The molecular structure of © 2016 American Chemical Society

Received: February 17, 2016 Revised: April 29, 2016 Published: May 1, 2016 4373

DOI: 10.1021/acs.jpcb.6b01641 J. Phys. Chem. B 2016, 120, 4373−4382

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The Journal of Physical Chemistry B proposed to trigger the cascade of fine conformational changes in the protein environment opening way to effective photoprotection.11 It has also been demonstrated, that another LHCII-bound xanthophyll, tentatively assigned as Vio, undergoes light-driven molecular reconfiguration.5 The results of the recent studies based on molecular dynamic simulations have confirmed exceptional motional freedom of Neo and Vio in LHCII.12 It is possible that both the light-induced molecular reconfiguration of the LHCII-bound xanthophylls reported are physiologically significant in modulation of the photosynthetic activity of the complex. Occupation of the xanthophyll-binding sites in LHCII by proper pigments13 and in a proper molecular configuration14 appears to be essential for molecular structure, organization, and physiological activity of the complex. LHCII is a principal photosynthetic antenna pigment−protein complex, harvesting energy utilized to drive photochemical reactions. On the other hand, under strong light conditions light-harvesting activity appears unnecessary and even deleterious as it may cause photo-oxidative damage of the photosynthetic apparatus. It appears that under such conditions, LHCII can switch its activity toward quenching excessive excitations.15,16 One of the elements associated with the photoprotective activity in LHCII is directly linked to the enzymatic deepoxidation of Vio leading to accumulation of Zea in the photosynthetic apparatus subjected to light stress.17 The reactions take place in the thylakoid membranes and are referred to as the xanthophyll cycle. Operation of the xanthophyll cycle requires Vio to be detached from the protein site and to be directly present in the lipid phase of the thylakoid membranes where enzymatic deepoxidation takes place.18 Molecular mechanisms associated with making Vio available for deepoxidation are not known. The Vio-binding site in LHCII is located at the interface of monomers constituting a trimer.1,2 It is therefore possible that availability of Vio to deepoxidation is higher in LHCII monomers than in trimers and that this process is related to light-driven LHCII trimer to monomer transition.19,20 It is also very likely that light-induced reconfiguration of Vio is associated with the pigment destabilization in the protein bed because the V1 xanthophyll-binding site in LHCII is optimized to host Vio in the alltrans molecular configuration.21 In the present work, we apply resonance Raman scattering spectroscopy to explore this interesting and important molecular mechanism.

Lipid-LHCII Samples with Exogenous Xanthophylls. MGDG, DGDG (chloroform/methanol 1:1 (v/v) solvent system) and exogenous Vio or Zea (dissolved in ethanol) were mixed. The mixture was dried to a thin film under a nitrogen stream and then placed under vacuum (less than 10−5 bar) for 30 min in order to remove organic solvents. Next, the LHCII in buffer (20 mM Tricine, 10 mM KCl, pH 7.6) containing 0.1% β-D-dodecylmaltoside (DM) was added and the mixture was sonicated in an ultrasonic bath for 30 min to form a lipid−protein multibilayer. Next, the detergent was removed from the samples by incubation with BioBeads SM-2 Adsorbents (Bio-Rad) for 12 h. After incubation, the samples were centrifuged at 15 000×g for 5 min in order to separate LHCII complexes imbedded into lipids multibilayers. The pellet was resuspended in a buffer (20 mM Tricine, 10 mM KCl, pH 7.6). The molar ratio of LHCII/lipids/exogenous xanthophyll in the control sample was 1:20:0, and in the sample with exogenous violaxanthin or zeaxanthin the molar ratio of LHCII/lipids/exogenous xanthophyll was 1:20:3. Spectroscopic characteristic and xanthophyll composition of the lipid-LHCII samples were presented in detail previously.21 Native Electrophoresis. The LHCII samples prepared according to the procedure described above were separated using nondenaturing gel electrophoresis. The samples, diluted in 0.1% DM, were mixed with 60% sucrose solution in a ratio of 2:1 (v/v). Thirty microliters of the mixture (Chl a + Chl b concentration of 140 mg/mL) was deposited in each well. Polyacrylamide (8% and 3%) was used as a separating and stacking gel, respectively. The samples were electrophoresed at a constant current of 4 mA per gel for approximately 1.5 h at 4 °C in 96 mM glycine, 12.5 mM Tris, and 0.05% LDS running buffer. After electrophoretic separation, the gels were subjected to Raman imaging and spectral analysis. Pigment Analysis. After separation, gel slices containing LHCII were crushed in 20 mM tricine and 10 mM KCl buffer (pH 7.6) containing 0.03% DM and vortexed for 5 min in order to extract LHCII complexes from polyacrylamide gel. Next, the mixture was centrifuged at 15 000×g for 15 min in order to remove gel fragments. The supernatant was mixed with nbutanol (3:2, v/v) to ensure complete extraction of pigments. Next, xanthophylls were analyzed chromatographically. The entire procedure of LHCII enrichment with exogenous xanthophylls and details of chromatographic analysis are described previously.21 Raman Imaging Microscopy. Raman spectroscopy was carried out using an inVia confocal Raman microscope (Renishaw, U.K.) with an argon laser (Stellar-REN, ModuLaser, U.S.A.) operating at 514 nm (from 1.75 (5%) to 35 mW (100%) laser power at the sample) and 488 nm (from 0.8 (5%) to 16 mW (100%) laser power at the sample), equipped with a 20× short distance objective (N PLAN EPI, NA 0.40, Leica). The optical image of an electrophoresis gel slice below each well was obtained with the function “image-montage” of WiRE 4.1 software (Renishaw, U.K.). On the basis of such an image, areas of 400 μm × 23 mm for Raman scanning were selected. Eight Raman images were recorded on each slot with 488 and 514 nm laser lines at 5, 10, 50, and 100% intensity. In all the experiments presented in this work, the resonance Raman spectra were recorded with a light intensity sequence from the lowest to the highest. Moreover, each spectrum was recorded from a different sector of the electrophoretic gel. At each point (3 × 230 points), the spectra were recorded at about 1 cm−1 spectral resolution (2400 lines/mm grating) in the spectral

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EXPERIMENTAL SECTION Materials. n-Dodecyl-β-D-maltoside (DM) was purchased from Sigma Chem. Co. (U.S.A.). All other chemicals used in the preparations were of analytical grade. Isolation of LHCII and Xanthophylls. LHCII was isolated from fresh spinach (Spinacia oleracea L.) leaves according to a slightly modified method5 of Krupa et al.22 Preparation purity was monitored using HPLC and electrophoresis methods.5 The Chl a/Chl b molar ratio in the preparations was 1.33. The chlorophyll concentration was determined in 80% acetone as described elsewhere.23 All-trans stereoisomers of Vio and Zea were extracted from spinach leaves and from berries of Lycium barbarum respectively. 9-cis Vio was isolated from blossoms of Viola tricolor. Xanthophylls were purified using HPLC and identified on the basis of retention times and UV−vis absorption spectra. 24,25 Monogalactosylodiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) were purchased from Lipid Products Company (U.K.). 4374


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The Journal of Physical Chemistry B

Figure 1. Optical image of one electrophoresis gel slice overlaid by a Raman intensity map. Optical image of one electrophoresis gel slice (in background) with visible green bands corresponding to the LHCII trimer, LHCII monomer, and free pigment fraction (indicated) superimposed with a Raman intensity map (based on the integration of the ν1 band in the spectral range 1520−1530 cm−1). Raman image was recorded in fast scanning mode (0.1 s per point). The Raman image was recorded with a 488 nm laser.

Figure 2. Resonance Raman spectra recorded with a 488 nm laser line from the trimeric and monomeric LHCII. Resonance Raman spectra recorded with a 488 nm argon laser line (100%, 16 mW power) from the samples of LHCII in a trimeric (red dashed line) and monomeric (black solid line) form in polyacrylamide electrophoresis gel. The spectra were normalized at the ν1 band maximum. Selected bands are marked.

region of 440−1800 cm−1 using an EMCCD detection camera Newton 970 from Andor, U.K. Images were acquired by use of the Renishaw WiRE 4.1 system high-resolution mapping mode (HR maps). Acquisition time for a single spectrum was 0.1 s. All spectra were preprocessed by cosmic ray removal, noise filtering, and baseline correction using WiRE 4.1 software from Renishaw, U.K.

an experimental protocol is the stability of supramolecular organization of LHCII. It means that it is very unlikely that the trimeric LHCII structures, immobilized in a polyacrylamide gel matrix, will be photoconverted to monomers during laser scanning. Light-induced LHCII trimer to monomer transition is a process that was reported to operate efficiently and can significantly change the molecular organization state of the protein both in suspension and in a lipid membrane environment.19,20 Raman spectra of LHCII were recorded during imaging of electrophoretic gels (see Figure 1). Figure 2 presents the resonance Raman spectra recorded with a 488 nm argon laser from the samples of LHCII in trimeric and monomeric forms. Four particularly distinctive spectral bands of carotenoids can be resolved in the spectra:9,26,27

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RESULTS Raman spectra of two molecular organization forms of LHCII, monomers and trimers, were recorded directly after the electrophoretic separation in a polyacrylamide gel without transfer to a detergent solution, which may influence the protein organization status. The additional consequence of such 4375


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Figure 3. Resonance Raman spectra recorded with a 514 nm laser line from the trimeric and monomeric LHCII. Resonance Raman spectra recorded with a 514 nm argon laser line (100%, 35 mW power) from the samples of LHCII in a trimeric (red dashed line) and monomeric (black solid line) form in polyacrylamide electrophoresis gel. The spectra were normalized at ν1 band maximum. Selected bands are marked.

Table 1. Xanthophyll Concentration in the Samplesa pigment

LHCII suspension 0.1% DM

LHCII monomer

LHCII trimer

LHCII monomer + Zea

LHCII trimer + Zea

LHCII trimer + Vio

Vio Neo Lut Zea

0.5 ± 0.06 0.90 ± 0.10 2 0

0.15 ± 0.02 0.94 ± 0.09 2 0

0.16 ± 0.03 0.97 ± 0.05 2 0

0.12 ± 0.02 0.95 ± 0.08 2 0.04 ± 0.04

0.17 ± 0.04 0.95 ± 0.15 2 0.11 ± 0.10

0.23 0.83 2 0

a

Xanthophyll stoichiometry per monomer (2 Lut) determined for LHCII suspended in 0.1% DM, monomeric and trimeric, with and without exogenous Zea or Vio separated with native electrophoresis. The data represents an average from three different samples ±SD. In the case of LHCII trimers enriched with exogenous violaxanthin, the results of analysis of the sample subjected to Raman analysis is presented.

1. The ν1 band, centered at 1527 cm−1, assigned to the CC stretching vibrations in the conjugated double bond system; 2. The ν2 band, between 1230 and 1100 cm−1, assigned to the CC stretching coupled either to the CH in-plane bending or to the CCH3 stretching modes; 3. The ν3 band, centered at 1005 cm−1, representing the CH3 in-plane rocking vibrations; 4. The ν4 band, between 945 and 977 cm−1, corresponding to the out-of-plane wagging modes of the CH groups. As can be seen, the spectra presented in Figure 2 are very similar to each other. This means that chemical structure and molecular configuration of the protein-bound xanthophylls, which are in resonance at this particular wavelength, are not sensitive to organization of the complex into trimers. On the other hand, distinctive differences can be observed in the resonance Raman spectra recorded from monomeric and trimeric LHCII with application of a 514 nm argon laser (Figure 3). On the basis of the exact position of the in situ absorption spectra of LHCII-bound xanthophylls, on a wavelength scale one can conclude that Neo and one of the luteins (Lut1) are particularly in resonance at 488 nm while

another lutein (Lut2) and Vio are in resonance at 514 nm.28 According to the Raman excitation profiles,29−32 the effect of resonance Raman enhancement is particularly high at wavelengths corresponding to the 0−0 vibrational transition in a carotenoid absorption spectrum and several times weaker when excited at other wavelengths absorbed by a carotenoid. For example, in the case of the ν1 mode of β-carotene in solution the resonance effect in the 0−0 vibrational band is higher by a factor of 2.8, as compared to the 0−1 band.29,30 In the case of the ν2 mode, such a factor is even higher (∼6).29,30 A strong resonance enhancement at wavelengths corresponding to the 0−0 transition also has been observed for spheroidene in a microcrystalline form.31,32 In such a case, the resonance effect in the 0−0 vibrational band was higher than in the 0−1 band by a factor of ca. 2.31,32 Owing to the selective resonance enhancement observed at 0−0 excitation wavelengths, application of different laser lines, absorbed by carotenoids in the red edge of their absorption spectra in a system comprising several carotenoid constituents, enables selective analysis of different pigments characterized by absorption spectra shifted on a wavelength scale.7 Nevertheless, it has to be taken into 4376


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Figure 4. Resonance Raman spectra recorded with a 514 nm laser line from the LHCII with exogenous violaxanthin and zeaxanthin. Resonance Raman spectra recorded with a 514 nm argon laser line (100%, 35 mW power) from the samples of trimeric LHCII with added exogenous zeaxanthin (black solid line) and violaxanthin (red dashed line) in polyacrylamide electrophoresis gel. The spectra were normalized at ν1 band maximum. Selected bands are marked.

isomerization. According to our analysis of the results, this is the case with Vio present in LHCII at substoichiometric conditions. It is very likely that Vio is particularly sensitive to association of monomeric LHCII units into the trimeric structure owing to the fact that this xanthophyll is localized just at the monomer−monomer interface.1,2 In sharp contrast to Lut and Neo, the isolation procedure can considerably affect stoichiometry of the xanthophyll cycle pigments in LHCII.21,33 Interestingly, the supplementation of the LHCII samples with exogenous Vio and Zea (possible because the majority of the V1 xanthophyll sites of LHCII in the preparations are not occupied, see Table 1) leads to appearance of the spectral forms of trimeric LHCII very closely corresponding to control experiment trimeric and monomeric forms, respectively (compare Figures 3 and 4). Such an effect supports interpretation that Vio is a xanthophyll with a molecular configuration particularly sensitive to association of LHCII monomers into trimers. Resonance Raman spectra recorded with the 514 nm laser line represent two LHCII-bound xanthophylls, one of two Lut and Vio. The position of the band representing the CC stretching on a wavenumber scale depends on both the conjugation length and chromophore environment. The conjugation length in the case of Lut (N = 10) is higher than in the case of Vio (N = 9) and therefore one may expect a slight shift of the CC band corresponding to Lut toward lower frequencies. Owing to the fact that the concentration of Vio is not higher than 0.2 of Lut that is in resonance at 514 nm, the CC stretching band is basically constituted by Lut and broadened due to the contribution of Vio. On the other hand, the intensity of Raman scattering

consideration that carotenoids characterized by slightly shifted absorption spectra give rise to combined resonance Raman spectra, representing the superposition of spectral components. The positions of the most red-shifted (0−0) vibrational maxima for the LHCII-bound xanthophylls are as follows: 488 nm for Neo, 489 nm for Lut1, 495 nm for Lut2, and 492 nm for Vio.28 Indeed, an argon laser tuned to 488 nm has been successfully applied to study selectively the xanthophyll Neo in the LHCII environment.11 The chromatographic analysis of the xanthophyll composition in LHCII in the bands of electrophoretic analysis representing trimeric and monomeric organization of the complex from the same preparations are very similar to each other (the data are presented in Table 1). Owing to this fact, the differences observed in the resonance Raman spectra of LHCII monomers and trimers should be assigned to the molecular organization of the complex rather than to their pigment composition. Spectral differences associated with LHCII trimerization are observed while probing with a 514 nm laser (Figure 3) but not with a 488 nm laser (Figure 2; the spectral differences can also be observed in the Figures S2 and S3). This effect points to one of the lutein molecules (Lut2) and/or Vio as the pigment(s) being in resonance and sensitive to LHCII trimerization. In the authors opinion, contribution of Lut2 will be higher from the contribution of Vio at 514 nm due to the differences in concentration of both pigments (see Table 1). This conclusion applies to principal Raman bands, for example, ν1. On the other hand, the bands that represent vibrations that are not associated with pronounced polarizability changes, such as ν4, can become Raman active upon molecular reconfiguration, including twisting and/or trans-to-cis 4377


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Figure 5. Resonance Raman spectra recorded with a range of intensities of the 514 nm laser line from the LHCII with violaxanthin. Resonance Raman spectra recorded with a 514 nm argon laser line from the samples of trimeric LHCII form with added exogenous violaxanthin in polyacrylamide electrophoresis gel. The spectra were recorded for a range of laser intensities (5, 10, 50, and 100%, as indicated). The spectra were normalized at the ν1 band maximum. Selected bands are marked and magnified. The spectra have been recorded from different sectors of the sample with the laser power set from the lowest to the highest values.

the out-of-plane wagging vibrations of the CH groups are not associated with pronounced polarizability changes.5,7,9 On the other hand, such vibrations are associated with significant changes in the electric dipole moment, which gives rise to a relatively intensive band in the IR absorption spectra.26 Interestingly, the fact that xanthophylls bound to LHCII adopt a twisted molecular configuration results in an increased intensity of the ν4 band in resonance Raman spectra.5,11 A comparison of the resonance Raman spectra of Vio in LHCII and in solution is presented in Figure S4. In light of these arguments, the more intensive and more structured ν4 band in the Raman spectrum of a trimeric LHCII, as compared to the spectrum of a monomer, can be interpreted in terms of a certain molecular configuration change (most probably a twisting) of the Vio molecule upon formation of the trimeric structure. The same mechanism may be responsible for the increase in intensity of the band at 1131 cm−1, because this band is particularly intense in the case of carotenoids in a cis molecular configuration and practically not observed in the case of an extended molecular configuration of the polyene chain.30,34 Interestingly, the shoulder in the ν1 band spectral region at 1555 cm−1 is particularly intensive in the case of trimeric LHCII probed with the 514 nm excitation (Figure 3). This spectral feature is typical for shorter polyenes and for long polyenes in a cis molecular configuration.27,30,34 An example of such an effect is presented in the Supporting Information Figure S5 in which the resonance Raman spectra are compared for Vio in the molecular configurations all-trans and 9-cis. Additional effect is associated with appearance of new spectral

bands not only depend on pigment concentration but also on polarizability changes associated with vibrations of analyzed groups. Moreover, it can be significantly influenced by the molecular configuration of the entire chromophore of a carotenoid. Such an effect can be particularly observed in the ν2 band in the case of a trans−cis reconfiguration30 or in the ν4 band in the case of a polyene chain twisting.7 In the authors opinion, exogenous Vio and Zea, present in relatively small concentrations (see Table 1), do not considerably affect the position of the principal CC stretching band but clearly influence the shape of the Raman bands sensitive to the molecular configuration of protein-bound xanthophylls. Moreover, the xanthophyll spectra recorded with the 514 nm excitation display a very clear band at 1254 cm−1. Interestingly, the Raman bands in this spectral region have been reported and referred to as a fingerprint of a carotenoid in a central cis molecular configuration, for example, at 1245 cm−1 in the case of the 15-cis β-carotene and at 1268 cm−1 in the case of the 11cis β-carotene.30,34 The other differences in the resonance Raman spectra recorded with a 514 nm laser line, associated with LHCII trimerization are the following (see Figure 3): 1. More intensive and structured ν4 band; 2. Rise in intensity of the component of the ν2 band at 1131 cm−1, associated with a decrease in intensity of the principal component of this band (at 1155 cm−1); 3. Appearance of additional spectral components at the lower and the higher wavenumber edges of the ν1 band. In general, the ν4 band is not particularly intensive in the Raman scattering spectra of carotenoids owing to the fact that 4378


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The Journal of Physical Chemistry B components in the ν1 band region. Such an effect is relatively small, as compared to the ν1 band intensity, but highly reproducible. The spectral component at 1496 cm−1 (Figures 3 and 4) can be interpreted as a manifestation of a carotenoid excitation to the triplet state.4 Interestingly, the resonance Raman spectra recorded from trimeric LHCII with a 514 nm laser line, assigned to a large extent to Lut and Vio, are strongly dependent on the intensity of the probing light beam (Figure 5 and Figure S6). Such a distinct light-dose-dependency, particularly pronounced in the 1254 cm−1 spectral region, has not been observed in the case of the LHCII trimers probed with a 488 nm laser line (see Figure S7). On the other hand, certain small spectral changes can be observed in the 1131 cm−1 region, both with the application of the 488 nm- and 514 nm-emitting lasers (Figures S7 and S8). The changes observed by scanning with 488 nm, both in monomeric and trimeric LHCII can be assigned to lightinduced reconfiguration of Neo.

exogenous Vio, scanned with higher intensity laser light, display an additional component in the higher wavenumber region of the ν1 spectral region (at 1555 cm−1). The light-induced molecular reconfiguration of the LHCII-trimer-bound Vio does not involve further changes in the ν4 spectral region (Figure 5). Interestingly, the light-intensity-related spectral effects involve the appearance of a band in the low frequency edge of the ν1 band (at 1496 cm−1), which can be attributed to the xanthophyll triplet state.4 Such clearly pronounced changes are neither observed in monomeric LHCII scanned with a 514 nm laser line nor with application of the 488 nm laser line for scanning of both LHCII monomers and trimers (Figures S7 and S8). Relatively small spectral changes, observed with the application of a 488 nm laser line, can be assigned to Neo. The light-intensity-dependent spectral effects observed with a 514 nm probing light mean that either excited triplet forms are observed on Lut2, but preferentially in the trimeric form of LHCII, or that the triplet excitations are located on Vio in the monomer−monomer interface of a trimeric structure. If so, it could be speculated that light-induced molecular configuration changes of the LHCII−trimer-bound Vio are mediated via pigment excitation to the triplet state. Such an excitation could be a direct consequence of the triplet−triplet excitation energy transfer from a chlorophyll to a xanthophyll, a photophysical reaction recognized to be a basic one in the photoprotective activity of carotenoids.35 Very recently, we have reported that the trimeric organization of the major photosynthetic pigment−protein antenna complex LHCII provides unique conditions for efficient excitation energy transfer, which is in contrast to the protein monomers.20 It has been demonstrated that upon illumination with strong light LHCII trimers disassemble to monomeric units.19,20 This light-induced reaction has been proposed to be mediated via the so-called thermo-optic mechanism.19 Under the same conditions, we have observed the partial trans−cis photoisomerization of the Vio pool in the LHCII preparations.36 It is highly probable that the thermo-optic effect drives the LHCII trimer to monomer transition and that the light-induced reconfiguration of Vio trans to cis liberates Vio from the protein bed. It is also very likely that both mechanisms mentioned above act synergistically to drive the phototransition of the LHCII trimers to monomers and that the light-induced molecular configuration changes reported in the present work provide a mechanistic determinant that weakens the intermolecular interactions responsible for assembly of LHCII monomers to a trimeric structure. According to the results of the monomolecular layer studies, the lateral-pressure-induced transformation of a trimeric organization of LHCII to monomers takes place at 33 mN/m.37 Such surface pressure corresponds well to the level of lateral pressures of natural biomembranes (30−35 mN/ m),38 which means that under natural conditions trimeric organization of LHCII may be easily affected by different, variable conditions, such as steric hindrance of the proteinbound Vio or interaction dynamics of N-terminal loops. Interestingly, recent molecular dynamics studies revealed exceptionally high molecular structure freedom in the case of the LHCII-bound Vio, as compared to other xanthophylls.12 The supporting materials movie published along with this work (Video 2)12 shows that molecular flexibility of the LHCIIbound Vio involves not only a molecular twisting but also a trans−cis molecular reconfiguration. Importantly, the lightinduced trans−cis reconfiguration of Vio bound to LHCII has been also recorded experimentally.36 One of the possible

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DISCUSSION The resonance Raman analyses of LHCII pigment−protein complexes immobilized in a polyacrylamide gel show pronounced spectral effects in response to increase in probing light intensity. The most pronounced light-intensity-dependent spectral effects can be observed in the case of the trimeric forms of LHCII, probed with a 514 nm laser line. Analysis of the light absorption spectra of the photosynthetic pigments bound to LHCII implies that preferentially two xanthophylls, Vio and one of Lut, are in resonance at this particular wavelength. The fact that concentration of Vio in the LHCII preparation analyzed was considerably lower than that of Lut (see Table 1) seems to suggest that the latter xanthophyll contributes preferentially to the resonance Raman spectra recorded both in the case of the trimeric and monomeric forms (see Figures 2 and 3). On the other hand, detailed analysis, supported by the results obtained for the samples substituted with exogenous Vio and Zea (Figure 4), leads to the conclusion that the differences observed between the spectra of trimeric and monomeric LHCII, recorded with a 514 nm laser line have to be attributed to Vio localized within the trimeric structure of the protein at the interface of individual monomers. The molecular configuration changes associated with binding of Vio to the protein environment of the trimeric LHCII can be assigned to a polyene twisting giving rise to the more structured and more intense ν4 band, corresponding to the out-of-plane wagging modes of the C−H groups and increased intensity of the band at 1131 cm−1, combined with the decreased intensity of the principal maximum of the ν2 band. Interestingly, the samples of trimeric LHCII, probed at 514 nm, showed pronounced spectral effects in response to increase in intensity of probing light. In particular, the band at 1254 cm−1 has been found to gain intensity along with the increase in laser power. As mentioned above, such a band appears in the “fingerprint” spectral region of central cis isomers of carotenoids.30,34 Such changes are accompanied by the increase in intensity of the band at 1131 cm−1, combined with the decreased intensity of the principal maximum of the ν2 band. Such a spectral effect can also be interpreted in terms of a certain trans to cis molecular reconfiguration, based on literature30,34 and on the basis of the results of resonance Raman analysis of the pigment solutions in an organic solvent (see Figure S5). Such an interpretation has additional support from the fact that the samples of trimeric LHCII and LHCII supplemented with 4379


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Figure 6. Model of one of the possible light-induced molecular reconfiguration of Vio in LHCII. Panel A shows the crystal structure PDBID: 2BHW; panel B shows the after light-induced molecular configuration change of Vio. Protein presented in gray, Vio yellow, and PG in magenta. Model created using YASARA software. The presented model of light-induced reconfiguration of Vio represents the all-trans to 13-cis molecular configuration change, based on the fact that this light-induced process has been reported in LHCII.36 However, other reconfigurations are also possible.12

molecular reconfiguration of Vio bound at the monomer− monomer interface of the trimeric structure of LHCII is presented in Figure 6. The LHCII trimers are enclosed in the present study in a polyacrylamide gel, which effectively prevents light-induced monomerization of the protein. On the other hand, the light-induced LHCII trimer-to-monomer transformation can be observed in a protein suspension with the application of the resonance Raman technique by following the intensity and spectral shape of the ν4 band.5 The advantage of the experimental system applied in the present work is the possibility to study light-induced molecular configuration changes of xanthophylls that are specific for the LHCII trimers but may not be observed as soon as the light-driven reaction is completed and induces a trimer-to-monomer transition. The physiological importance of the light-induced molecular reconfiguration of the protein-bound Vio, reported in the present study, would be directly associated with photoprotection via regulation of a LHCII trimer-monomer equilibrium, synergistically with other molecular mechanisms that very likely control the trimeric organization of LHCII. Another important aspect of the light-driven molecular reconfiguration of Vio embedded in a trimeric LHCII is the process of making the pigment available for enzymatic deepoxidation in the xanthophyll cycle.39 In order to take part in the cycle of enzymatic reactions, Vio has to be transferred from the protein environment into the lipid phase of the thylakoid membranes.18 It is possible that the lightdriven molecular reconfiguration of Vio is associated with the process of making Vio available for deepoxidation in the thylakoid membranes by liberation of the pigment from the protein environment. Vio in a molecular configuration cis is expected to return relatively quickly back to the all-trans form in the lipid phase of the thylakoid membrane owing to the lower energy deduced from a higher stability of this molecular configuration.36

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LHCII with 488 and 514 nm laser lines, comparison of resonance Raman spectra recorded with a 514 nm laser line from violaxanthin solution and from trimeric LHCII substituted with exogenous violaxanthin, resonance Raman spectra of violaxanthin in the all-trans and 9-cis configurations, resonance Raman spectra recorded with a range of intensities of a 514 nm laser line from the trimeric LHCII, resonance Raman spectra recorded with a range of intensities of a 488 nm laser line from the trimeric LHCII, and resonance Raman spectra recorded with a range of intensities of a 514 nm laser line from the monomeric LHCII. (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +(48 81) 537 62 52. Fax: +(48 81) 537 61 91. Present Address ⊥

(J.B.) Department of Medicine, Imperial College London, Du Cane Road, London W12 0NN, United Kingdom.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research has been performed within the framework of the project “Molecular Spectroscopy for BioMedical Studies” financed by the Foundation for Polish Science within the TEAM program (TEAM/2011-7/2). The research was carried out with the equipment purchased thanks to the financial support of the European Regional Development Fund in the framework of the Development of Eastern Poland Operational Programme.

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ABBREVIATIONS LHCII, Light-harvesting pigment−protein complex of Photosystem II; Lut, lutein; Vio, violaxanthin; Zea, zeaxanthin; Neo, neoxanthin; Chl a, chlorophyll a; Chl b, chlorophyll b; MGDG, monogalactosylodiacylglycerol; DGDG, digalactosyldiacylglycerol; PG, phosphatidylglycerol

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b01641. Model of the structure of LHCII, comparison of the resonance Raman spectra recorded from monomeric LHCII with 488 and 514 nm laser lines, comparison of the resonance Raman spectra recorded from trimeric

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