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We have prepared lycopene aggregates with low scattering in an acetone-water suspension. The aggregates exhibit highly distorted absorption, extending from the UV up to 568 nm, as a result of strong excitonic interactions. We have investigated the structural organization of these aggregates by resonance Raman and TEM, revealing that the lycopene aggregates are not homogeneous, containing at least four different aggregate species. Transient absorption measurements upon excitation at 355, 515, and 570 nm, to sub-select these different species, reveal significant differences in dynamics between each of the aggregate types. The strong excitonic interactions produce extremely distorted transient electronic signatures, which do not allow an unequivocal identification of the excited states at times shorter than 60 ps. However, these experiments demonstrate that all the lycopene aggregated species form long-living triplets via singlet fission., Competing Interests: Competing interests: The authors have no competing (include financial AND non-financial) interests as defined by Nature Research, or other interests that might be perceived to influence the results and/or discussion reported in this paper., (© 2025. The Author(s).)

In high light, the antenna system in oxygenic photosynthetic organisms switches to a photoprotective mode, dissipating excess energy in a process called non-photochemical quenching (NPQ). Diatoms exhibit very efficient NPQ, accompanied by a xanthophyll cycle in which diadinoxanthin is de-epoxidized into diatoxanthin. Diatoms accumulate pigments from this cycle in high light, and exhibit faster and more pronounced NPQ. The mechanisms underlying NPQ in diatoms remain unclear, but it can be mimicked by aggregation of their isolated light-harvesting complexes, FCP (fucoxanthin chlorophyll-a/c protein). We assess this model system by resonance Raman measurements of two peripheral FCPs, trimeric FCPa and nonameric FCPb, isolated from high- and low-light-adapted cells (LL,HL). Quenching is associated with a reorganisation of these proteins, affecting the conformation of their bound carotenoids, and in a manner which is highly dependent on the protein considered. FCPa from LL diatoms exhibits significant changes in diadinoxanthin structure, together with a smaller conformational change of at least one fucoxanthin. For these LL-FCPa, quenching is associated with consecutive events, displaying distinct spectral signatures, and its amplitude correlates with the planarity of the diadinoxanthin structure. HL-FCPa aggregation is associated with a change in planarity of a 515-nm-absorbing fucoxanthin, and, to a lesser extent, of diadinoxanthin. Finally, in FCPb, a blue-absorbing fucoxanthin is primarily affected. FCPs thus possess a plastic structure, undergoing several conformational changes upon aggregation, dependent upon their precise composition and structure. NPQ in diatoms may therefore arise from a combination of structural changes, dependent on the environment the cells are adapted to., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

We provide direct evidence of singlet fission occurring with water-soluble compounds. We show that perylene-3,4,9,10-tetracarboxylate forms dynamic dimers in aqueous solution, with lifetimes long enough to allow intermolecular processes such as singlet fission. As these are transient dimers rather than stable aggregates, they retain a significant degree of disorder. We performed a comprehensive analysis of such dynamic assemblies using time-resolved absorption and fluorescence spectroscopy, nuclear magnetic resonance spectroscopy, and theoretical modelling, allowing us to observe the characteristic signatures of singlet fission and develop a model to characterize the different species observed. Our findings reveal that structure fluctuations within perylene-3,4,9,10-tetracarboxylate associations are key in favoring either singlet fission or charge separation. The efficiency of triplet formation is higher than 100%, and the disordered system leads to triplets living in the nanosecond time range., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Carotenoids are conjugated linear molecules built from the repetition of terpene units, which display a large structural diversity in nature. They may, in particular, contain several types of side or end groups, which tune their functional properties, such as absorption position and photochemistry. We report here a detailed experimental study of the absorption and vibrational properties of allene-containing carotenoids, together with an extensive modeling of these experimental data. Our calculations can satisfactorily explain the electronic properties of vaucheriaxanthin, where the allene group introduces the equivalent of one C═C double bond into the conjugated C═C chain. The position of the electronic absorption of fucoxanthin and butanoyloxyfucoxanthin requires long-range corrections to be found correctly on the red side of that of vaucheriaxanthin; however, these corrections tend to overestimate the effect of the conjugated and nonconjugated C═O groups in these molecules. We show that the resonance Raman spectra of these carotenoids are largely perturbed by the presence of the allene group, with the two major Raman contributions split into two components. These perturbations are satisfactorily explained by modeling, through a gain in the Raman intensity of the C═C antisymmetric stretching mode, induced by the presence of the allene group in the carotenoid C═C chain.

Resonance Raman spectroscopy is one of the most powerful techniques in analytical science due to its molecular selectivity, high sensitivity, and the fact that, in contrast to IR absorption spectroscopy, the presence of water does not hamper or mask the results. Originating in physics and chemistry, the use of Raman spectroscopy has spread and now includes a variety of applications in different disciplines, including biology. In this chapter, we introduce the basic principles of Raman and resonance Raman scattering, and show resonance Raman can be applied to study carotenoid molecules, in complex biological or chemical matrices. We describe the type of information that can be extracted from resonance Raman spectra, illustrating the power of this method by a series of example applications., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The siphonaxanthin-siphonein-chlorophyll-a/b-binding protein (SCP), a trimeric light-harvesting complex isolated from photosystem II of the siphonous green alga Codium fragile, binds the carotenoid siphonaxanthin (Sx) and/or its ester siphonein in place of lutein, in addition to chlorophylls a/b and neoxanthin. SCP exhibits a higher content of chlorophyll b (Chl-b) than its counterpart in green plants, light-harvesting complex II (LHCII), increasing the relative absorption of blue-green light for photosynthesis. Using low temperature absorption and resonance Raman spectroscopies, we reveal the presence of two non-equivalent Sx molecules in SCP, and assign their absorption peaks at 501 and 535 nm. The red-absorbing Sx population exhibits a significant distortion that is reminiscent of lutein 2 in trimeric LHCII. Unexpected enhancement of the Raman modes of Chls-b in SCP allows an unequivocal description of seven to nine non-equivalent Chls-b, and six distinct Chl-a populations in this protein., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Carotenoids are lipophilic plastidial isoprenoids highly valued as nutrients and natural pigments. A correct balance of chlorophylls and carotenoids is required for photosynthesis and therefore highly regulated, making carotenoid enrichment of green tissues challenging. Here we show that leaf carotenoid levels can be boosted through engineering their biosynthesis outside the chloroplast. Transient expression experiments in Nicotiana benthamiana leaves indicated that high extraplastidial production of carotenoids requires an enhanced supply of their isoprenoid precursors in the cytosol, which was achieved using a deregulated form of the main rate-determining enzyme of the mevalonic acid (MVA) pathway. Constructs encoding bacterial enzymes were used to convert these MVA-derived precursors into carotenoid biosynthetic intermediates that do not normally accumulate in leaves, such as phytoene and lycopene. Cytosolic versions of these enzymes produced extraplastidial carotenoids at levels similar to those of total endogenous (i.e. chloroplast) carotenoids. Strategies to enhance the development of endomembrane structures and lipid bodies as potential extraplastidial carotenoid storage systems were not successful to further increase carotenoid contents. Phytoene was found to be more bioaccessible when accumulated outside plastids, whereas lycopene formed cytosolic crystalloids very similar to those found in the chromoplasts of ripe tomatoes. This extraplastidial production of phytoene and lycopene led to an increased antioxidant capacity of leaves. Finally, we demonstrate that our system can be adapted for the biofortification of leafy vegetables such as lettuce., (© 2020 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

We have investigated the photophysics of aggregated lutein/violaxanthin in daffodil chromoplasts. We reveal the presence of three carotenoid aggregate species, the main one composed of a mixture of lutein/violaxanthin absorbing at 481 nm, and two secondary populations of aggregated carotenoids absorbing circa 500 and 402 nm. The major population exhibits an efficient singlet fission process, generating μs-lived triplet states on an ultrafast timescale. The structural organization of aggregated lutein/violaxanthin in daffodil chromoplasts produces well-defined electronic levels that permit the energetic pathways to be disentangled unequivocally, allowing us to propose a consistent mechanism for singlet fission in carotenoid aggregates. Transient absorption measurements on this system reveal for the first time an entangled triplet signature for carotenoid aggregates, and its evolution into dissociated triplet states. A clear picture of the carotenoid singlet fission pathway is obtained, which is usually blurred due to the intrinsic disorder of carotenoid aggregates.

When plants are exposed to high-light conditions, the potentially harmful excess energy is dissipated as heat, a process called non-photochemical quenching. Efficient energy dissipation can also be induced in the major light-harvesting complex of photosystem II (LHCII) in vitro, by altering the structure and interactions of several bound cofactors. In both cases, the extent of quenching has been correlated with conformational changes (twisting) affecting two bound carotenoids, neoxanthin, and one of the two luteins (in site L1). This lutein is directly involved in the quenching process, whereas neoxanthin senses the overall change in state without playing a direct role in energy dissipation. Here we describe the isolation of an intermediate state of LHCII, using the detergent n-dodecyl-α-D-maltoside, which exhibits the twisting of neoxanthin (along with changes in chlorophyll-protein interactions), in the absence of the L1 change or corresponding quenching. We demonstrate that neoxanthin is actually a reporter of the LHCII environment-probably reflecting a large-scale conformational change in the protein-whereas the appearance of excitation energy quenching is concomitant with the configuration change of the L1 carotenoid only, reflecting changes on a smaller scale. This unquenched LHCII intermediate, described here for the first time, provides for a deeper understanding of the molecular mechanism of quenching., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The EAG ( ether-à-go-go ) family of voltage-gated K + channels are important regulators of neuronal and cardiac action potential firing (excitability) and have major roles in human diseases such as epilepsy, schizophrenia, cancer, and sudden cardiac death. A defining feature of EAG (Kv10-12) channels is a highly conserved domain on the N terminus, known as the eag domain, consisting of a Per-ARNT-Sim (PAS) domain capped by a short sequence containing an amphipathic helix (Cap domain). The PAS and Cap domains are both vital for the normal function of EAG channels. Using heme-affinity pulldown assays and proteomics of lysates from primary cortical neurons, we identified that an EAG channel, hERG3 (Kv11.3), binds to heme. In whole-cell electrophysiology experiments, we identified that heme inhibits hERG3 channel activity. In addition, we expressed the Cap and PAS domain of hERG3 in Escherichia coli and, using spectroscopy and kinetics, identified the PAS domain as the location for heme binding. The results identify heme as a regulator of hERG3 channel activity. These observations are discussed in the context of the emerging role for heme as a regulator of ion channel activity in cells., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Burton et al.)

Calculating the spectroscopic properties of complex conjugated organic molecules in their relaxed state is far from simple. An additional complexity arises for flexible molecules in solution, where the rotational energy barriers are low enough so that nonminimum conformations may become dynamically populated. These metastable conformations quickly relax during the minimization procedures preliminary to density functional theory calculations, and so accounting for their contribution to the experimentally observed properties is problematic. We describe a strategy for stabilizing these nonminimum conformations in silico , allowing their properties to be calculated. Diadinoxanthin and alloxanthin present atypical vibrational properties in solution, indicating the presence of several conformations. Performing energy calculations in vacuo and polarizable continuum model calculations in different solvents, we found three different conformations with values for the δ dihedral angle of the end ring ca. 0, 180, and 90° with respect to the plane of the conjugated chain. The latter conformation, a nonglobal minimum, is not stable during the minimization necessary for modeling its spectroscopic properties. To circumvent this classical problem, we used a Car-Parinello MD supermolecular approach, in which diadinoxanthin was solvated by water molecules so that metastable conformations were stabilized by hydrogen-bonding interactions. We progressively removed the number of solvating waters to find the minimum required for this stabilization. This strategy represents the first modeling of a carotenoid in a distorted conformation and provides an accurate interpretation of the experimental data.

We describe a molecular mechanism tuning the functional properties of chlorophyll a (Chl-a) molecules in photosynthetic antenna proteins. Light-harvesting complexes from photosystem II in higher plants - specifically LHCII purified with α- or β-dodecyl-maltoside, along with CP29 - were probed by low-temperature absorption and resonance Raman spectroscopies. We show that hydrogen bonding to the conjugated keto carbonyl group of protein-bound Chl-a tunes the energy of its Soret and Q y absorption transitions, inducing red-shifts that are proportional to the strength of the hydrogen bond involved. Chls-a with non-H-bonded keto C13 1 groups exhibit the blue-most absorption bands, while both transitions are progressively red-shifted with increasing hydrogen-bonding strength - by up 382 & 605 cm -1 in the Q y and Soret band, respectively. These hydrogen bonds thus tune the site energy of Chl-a in light-harvesting proteins, determining (at least in part) the cascade of energy transfer events in these complexes., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Carotenoid-containing oil droplets in the avian retina act as cut-off filters to enhance colour discrimination. We report a confocal resonance Raman investigation of the oil droplets of the domestic chicken, Gallus gallus domesticus. We show that all carotenoids present are in a constrained conformation, implying a locus in specific lipid binding sites. In addition, we provide proof of a recent conclusion that all carotenoid-containing droplets contain a mixture of all carotenoids present, rather than only a subset of them-a conclusion that diverges from the previously-held view. Our results have implications for the mechanism(s) giving rise to these carotenoid mixtures in the differently-coloured droplets., Competing Interests: The authors have declared that no competing interests exist.

The soil chromophyte alga Xanthonema (X.) debile contains only non-carbonyl carotenoids and Chl-a. X. debile has an antenna system denoted Xanthophyte light-harvesting complex (XLH) that contains the carotenoids diadinoxanthin, heteroxanthin, and vaucheriaxanthin. The XLH pigment stoichiometry was calculated by chromatographic techniques and the pigment-binding structure studied by resonance Raman spectroscopy. The pigment ratio obtained by HPLC was found to be close to 8:1:2:1 Chl-a:heteroxanthin:diadinoxanthin:vaucheriaxanthin. The resonance Raman spectra suggest the presence of 8-10 Chl-a, all of which are 5-coordinated to the central Mg, with 1-3 Chl-a possessing a macrocycle distorted from the relaxed conformation. The three populations of carotenoids are in the all-trans configuration. Vaucheriaxanthin absorbs around 500-530 nm, diadinoxanthin at 494 nm and heteroxanthin at 487 nm at 4.5 K. The effective conjugation length of heteroxanthin and diadinoxanthin has been determined as 9.4 in both cases; the environment polarizability of the heteroxanthin and diadinoxanthin binding pockets is 0.270 and 0.305, respectively.

Cyanobacteria possess a family of one-helix high-light-inducible proteins (HLIPs) that are widely viewed as ancestors of the light-harvesting antenna of plants and algae. HLIPs are essential for viability under various stress conditions, although their exact role is not fully understood. The unicellular cyanobacterium Synechocystis sp. PCC 6803 contains four HLIPs named HliA-D, and HliD has recently been isolated in a small protein complex and shown to bind chlorophyll and β-carotene. However, no HLIP has been isolated and characterized in a pure form up to now. We have developed a protocol to purify large quantities of His-tagged HliC from an engineered Synechocystis strain. Purified His-HliC is a pigmented homo-oligomer and is associated with chlorophyll and β-carotene with a 2:1 ratio. This differs from the 3:1 ratio reported for HliD. Comparison of these two HLIPs by resonance Raman spectroscopy revealed a similar conformation for their bound β-carotenes, but clear differences in their chlorophylls. We present and discuss a structural model of HliC, in which a dimeric protein binds four chlorophyll molecules and two β-carotenes.

Transient absorption studies conducted on in vitro lycopene aggregates, as well as on lycopene crystalloids inside tomato chromoplasts, reveal the appearance of a long-lived excited state, which we unambiguously identified as lycopene triplet. These triplet states must be generated by singlet exciton fission, which occurs from the lycopene 2Ag state. This is the first time the singlet fission process has ever been shown to occur in a biological material. We propose that the formation of carotenoid assemblies in chromoplasts may constitute a photoprotective process during chromoplast maturation, in addition to their function in signaling processes.

Carotenoids are among the most important organic compounds present in Nature and play several essential roles in biology. Their configuration is responsible for their specific photophysical properties, which can be tailored by changes in their molecular structure and in the surrounding environment. In this review, we give a general description of the main electronic and vibrational properties of carotenoids. In the first part, we describe how the electronic and vibrational properties are related to the molecular configuration of carotenoids. We show how modifications to their configuration, as well as the addition of functional groups, can affect the length of the conjugated chain. We describe the concept of effective conjugation length, and its relationship to the S 0 → S 2 electronic transition, the decay rate of the S 1 energetic level and the frequency of the ν 1 Raman band. We then consider the dependence of these properties on extrinsic parameters such as the polarizability of their environment, and how this information (S 0 → S 2 electronic transition, ν 1 band position, effective conjugation length and polarizability of the environment) can be represented on a single graph. In the second part of the review, we use a number of specific examples to show that the relationships can be used to disentangle the different mechanisms tuning the functional properties of protein-bound carotenoids., (© 2017 The Author(s).)

Resonance Raman spectroscopy was used to evaluate pigment-binding site properties in the violaxanthin-chlorophyll-a-binding protein (VCP) from Nannochloropsis oceanica. The pigments bound to this antenna protein are chlorophyll-a, violaxanthin, and vaucheriaxanthin. The molecular structures of bound Chl-a molecules are discussed with respect to those of the plant antenna proteins LHCII and CP29, the crystal structures of which are known. We show that three populations of carotenoid molecules are bound by VCP, each of which is in an all-trans configuration. We assign the lower-energy absorption transition of each of these as follows. One violaxanthin population absorbs at 485 nm, while the second population is red-shifted and absorbs at 503 nm. The vaucheriaxanthin population absorbs at 525 nm, a position red-shifted by 2138 cm -1 as compared to isolated vaucheriaxanthin in n-hexane. The red-shifted violaxanthin is slightly less planar than the blue-absorbing one, as observed for the two central luteins in LHCII, and we suggest that these violaxanthins occupy the two equivalent binding sites in VCP at the centre of the cross-brace. The presence of a highly red-shifted vaucheriaxanthin in VCP is reminiscent of the situation of FCP, in which (even more) highly red-shifted populations of fucoxanthin are present. Tuning carotenoids to absorb in the green-yellow region of the visible spectrum appears to be a common evolutionary response to competition with other photosynthetic species in the aquatic environment.

Cyanobacteria possess a family of one-helix high light-inducible proteins (Hlips) that are homologous to light-harvesting antenna of plants and algae. An Hlip protein, high light-inducible protein D (HliD) purified as a small complex with the Ycf39 protein is evaluated using resonance Raman spectroscopy. We show that the HliD binds two different β-carotenes, each present in two non-equivalent binding pockets with different conformations, having their (0,0) absorption maxima at 489 and 522 nm, respectively. Both populations of β-carotene molecules were in all-trans configuration and the absorption position of the farthest blue-shifted β-carotene was attributed entirely to the polarizability of the environment in its binding pocket. In contrast, the absorption maximum of the red-shifted β-carotene was attributed to two different factors: the polarizability of the environment in its binding pocket and, more importantly, to the conformation of its β-rings. This second β-carotene has highly twisted β-rings adopting a flat conformation, which implies that the effective conjugation length N is extended up to 10.5 modifying the energetic levels. This increase in N will also result in a lower S 1 energy state, which may provide a permanent energy dissipation channel. Analysis of the carbonyl stretching region for chlorophyll a excitations indicates that the HliD binds six chlorophyll a molecules in five non-equivalent binding sites, with at least one chlorophyll a presenting a slight distortion to its macrocycle. The binding modes and conformations of HliD-bound pigments are discussed with respect to the known structures of LHCII and CP29., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Resonance Raman spectroscopy was used to evaluate pigment structure in the FCP-like light-harvesting complex of Chromera velia (Chromera light-harvesting complex or CLH). This antenna protein contains chlorophyll a, violaxanthin and a new isofucoxanthin-like carotenoid (called Ifx-l). We show that Ifx-l is present in two non-equivalent binding pockets with different conformations, having their (0,0) absorption maxima at 515 and 548nm respectively. In this complex, only one violaxanthin population absorbing at 486nm is observed. All the CLH-bound carotenoid molecules are in all-trans configuration, and among the two Ifx-l carotenoid molecules, the red one is twisted, as is the red-absorbing lutein in LHCII trimers. Analysis of the carbonyl stretching region for Chl a excitations indicates CLH binds up to seven Chl a molecules in five non-equivalent binding sites, in reasonable agreement with sequence analyses which have identified eight potential coordinating residues. The binding modes and conformations of CLH-bound pigments are discussed with respect to the known structures of LHCII and FCP., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Resonance Raman spectroscopy was used to evaluate the structure of light-harvesting chlorophyll (Chl) a/b complexes of photosystem II (LHCII), reconstituted from wild-type (WT) and mutant apoproteins over-expressed in Escherichia coli. The point mutations involved residue S123, exchanged for either P (S123P) or G (S123G). In all reconstituted proteins, lutein 2 displayed a distorted conformation, as it does in purified LHCII trimers. Reconstituted WT and S123G also exhibited a conformation of bound neoxanthin (Nx) molecules identical to the native protein, while the S123P mutation was found to induce a change in Nx conformation. This structural change of neoxanthin is accompanied by a blue shift of the absorption of this carotenoid molecule. The interactions assumed by (and thus the structure of the binding sites of) the bound Chls b were found identical in all the reconstituted proteins, and only marginally perturbed as compared to purified LHCII. The interactions assumed by bound Chls a were also identical in purified LHCII and the reconstituted WT. However, the keto carbonyl group of one Chl a, originally free-from-interactions in WT LHCII, becomes involved in a strong H-bond with its environment in LHCII reconstituted from the S123P apoprotein. As the absorption in the Qy region of this protein is identical to that of the LHCII reconstituted from the WT apoprotein, we conclude that the interaction state of the keto carbonyl of Chl a does not play a significant role in tuning the binding site energy of these molecules., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Human retinal macular pigment (MP) is formed by the carotenoids lutein and zeaxanthin (including the isomer meso-zeaxanthin). MP has several functions in improving visual performance and protecting against the damaging effects of light, and MP levels are used as a proxy for macular health-specifically, to predict the likelihood of developing age-related macular degeneration. While the roles of these carotenoids in retinal health have been the object of intense study in recent years, precise mechanistic details of their protective action remain elusive. We have measured the Raman signals originating from MP carotenoids in ex vivo human retinal tissue, in order to assess their structure and conformation. We show that it is possible to distinguish between lutein and zeaxanthin, by their excitation profile (related to their absorption spectra) and the position of their ν1 Raman mode. In addition, analysis of the ν4 Raman band indicates that these carotenoids are present in a specific, constrained conformation in situ, consistent with their binding to specific proteins as postulated in the literature. We discuss how these conclusions relate to the function of these pigments in macular protection. We also address the possibilities for a more accurate, consistent measurement of MP levels by Raman spectroscopy.

Resonance Raman spectroscopy may yield precise information on the conformation of, and the interactions assumed by, the chromophores involved in the first steps of the photosynthetic process. Selectivity is achieved via resonance with the absorption transition of the chromophore of interest. Fluorescence line-narrowing spectroscopy is a complementary technique, in that it provides the same level of information (structure, conformation, interactions), but in this case for the emitting pigment(s) only (whether isolated or in an ensemble of interacting chromophores). The selectivity provided by these vibrational techniques allows for the analysis of pigment molecules not only when they are isolated in solvents, but also when embedded in soluble or membrane proteins and even, as shown recently, in vivo. They can be used, for instance, to relate the electronic properties of these pigment molecules to their structure and/or the physical properties of their environment. These techniques are even able to follow subtle changes in chromophore conformation associated with regulatory processes. After a short introduction to the physical principles that govern resonance Raman and fluorescence line-narrowing spectroscopies, the information content of the vibrational spectra of chlorophyll and carotenoid molecules is described in this article, together with the experiments which helped in determining which structural parameter(s) each vibrational band is sensitive to. A selection of applications is then presented, in order to illustrate how these techniques have been used in the field of photosynthesis, and what type of information has been obtained. This article is part of a Special Issue entitled: Vibrational spectroscopies and bioenergetic systems., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In this study, we demonstrate the selective in vivo detection of diadinoxanthin (DD) and diatoxanthin (DT) in intact Cyclotella cells using resonance Raman spectroscopy. In these cells, we were able to assess both the content of DD and DT carotenoids relative to chlorophyll and their conformation. In addition, the sensitivity and selectivity of the technique allow us to discriminate between different pools of DD on a structural basis, and to follow their fate as a function of the illumination conditions. We report that the additional DD observed when cells are grown in high-light conditions adopts a more twisted conformation than the lower levels of DD present when the cells are grown in low-light (LL) conditions. Thus, we conclude that this pool of DD is more tightly bound to a protein-binding site, which must differ from the one occupied by the DD present in LL conditions.

Predicting the complete electronic structure of carotenoid molecules remains an extremely complex problem, particularly in anisotropic media such as proteins. In this paper, we address the electronic properties of nine relatively simple carotenoids by the combined use of electronic absorption and resonance Raman spectroscopies. Linear carotenoids exhibit an excellent correlation between (i) the inverse of their conjugation chain length N, (ii) the energy of their S0 → S2 electronic transition, and (iii) the position of their ν1 Raman band (corresponding to the stretching mode of their conjugated C═C bonds). For cyclic carotenoids such as β-carotene, this correlation is also observed between the latter two parameters (S0 → S2 energy and ν1 frequency), whereas their "nominal" conjugation length N does not follow the same relationship. We conclude that β-carotene and cyclic carotenoids in general exhibit a shorter effective conjugation length than that expected from their chemical structure. In addition, the effect of solvent polarizability on these molecular parameters was investigated for four of the carotenoids used in this study. We demonstrate that resonance Raman spectroscopy can discriminate between the different effects underlying shifts in the S0 → S2 transition of carotenoid molecules.

The electronic properties of carotenoid molecules underlie their multiple functions throughout biology, and tuning of these properties by their in vivo locus is of vital importance in a number of cases. This is exemplified by photosynthetic carotenoids, which perform both light-harvesting and photoprotective roles essential to the photosynthetic process. However, despite a large number of scientific studies performed in this field, the mechanism(s) used to modulate the electronic properties of carotenoids remain elusive. We have chosen two specific cases, the two β-carotene molecules in photosystem II reaction centers and the two luteins in the major photosystem II light-harvesting complex, to investigate how such a tuning of their electronic structure may occur. Indeed, in each case, identical molecular species in the same protein are seen to exhibit different electronic properties (most notably, shifted absorption peaks). We assess which molecular parameters are responsible for this in vivo tuning process and attempt to assign it to specific molecular events imposed by their binding pockets.

The photosynthetic light-harvesting systems of purple bacteria and plants both utilize specific carotenoids as quenchers of the harmful (bacterio)chlorophyll triplet states via triplet-triplet energy transfer. Here, we explore how the binding of carotenoids to the different types of light-harvesting proteins found in plants and purple bacteria provides adaptation in this vital photoprotective function. We show that the creation of the carotenoid triplet states in the light-harvesting complexes may occur without detectable conformational changes, in contrast to that found for carotenoids in solution. However, in plant light-harvesting complexes, the triplet wavefunction is shared between the carotenoids and their adjacent chlorophylls. This is not observed for the antenna proteins of purple bacteria, where the triplet is virtually fully located on the carotenoid molecule. These results explain the faster triplet-triplet transfer times in plant light-harvesting complexes. We show that this molecular mechanism, which spreads the location of the triplet wavefunction through the pigments of plant light-harvesting complexes, results in the absence of any detectable chlorophyll triplet in these complexes upon excitation, and we propose that it emerged as a photoprotective adaptation during the evolution of oxygenic photosynthesis., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Nonphotochemical quenching (NPQ) is the fundamental process by which plants exposed to high light intensities dissipate the potentially harmful excess energy as heat. Recently, it has been shown that efficient energy dissipation can be induced in the major light-harvesting complexes of photosystem II (LHCII) in the absence of protein-protein interactions. Spectroscopic measurements on these samples (LHCII gels) in the quenched state revealed specific alterations in the absorption and circular dichroism bands assigned to neoxanthin and lutein 1 molecules. In this work, we investigate the changes in conformation of the pigments involved in NPQ using resonance Raman spectroscopy. By selective excitation we show that, as well as the twisting of neoxanthin that has been reported previously, the lutein 1 pigment also undergoes a significant change in conformation when LHCII switches to the energy dissipative state. Selective two-photon excitation of carotenoid (Car) dark states (Car S(1)) performed on LHCII gels shows that the extent of electronic interactions between Car S(1) and chlorophyll states correlates linearly with chlorophyll fluorescence quenching, as observed previously for isolated LHCII (aggregated versus trimeric) and whole plants (with versus without NPQ).

In 2005, it was found that the fluorescence of crystals of the major light-harvesting complex LHCII of green plants is significantly quenched when compared to the fluorescence of isolated LHCII (A. A. Pascal et al., Nature, 2005, 436, 134-137). The Raman spectrum of crystallized LHCII was also found to be different from that of isolated LHCII but very similar to that of aggregated LHCII, which has often been considered a good model system for studying nonphotochemical quenching (NPQ), the major protection mechanism of plants against photodamage in high light. It was proposed that in the crystal LHCII adopts a similar (quenching) conformation as during NPQ and indeed similar changes in the Raman spectrum were observed during NPQ in vivo (A. V. Ruban et al., Nature, 2007, 450, 575-579). We now compared the fluorescence of various types of crystals, differing in morphology and age. Each type gave rise to its own characteristic mono-exponential fluorescence lifetime, which was 5 to 10 times shorter than that of isolated LHCII. This indicates that fluorescence is not quenched by random impurities and packing defects (as proposed recently by T. Barros et al., EMBO Journal, 2009, 28, 298-306), but that LHCII adopts a particular structure in each crystal type, that leads to fluorescence quenching. Most interestingly, the extent of quenching appears to depend on the crystal morphology, indicating that also the crystal structure depends on this crystal morphology but at the moment no data are available to correlate the crystals' structural changes to changes in fluorescence lifetime., (This journal is © the Owner Societies 2011)

To prevent photo-oxidative damage to the photosynthetic membrane in strong light, plants dissipate excess absorbed light energy as heat in a mechanism known as non-photochemical quenching (NPQ). NPQ is triggered by the trans-membrane proton gradient (ΔpH), which causes the protonation of the photosystem II light-harvesting antenna (LHCII) and the PsbS protein, as well as the de-epoxidation of the xanthophyll violaxanthin to zeaxanthin. The combination of these factors brings about formation of dissipative pigment interactions that quench the excess energy. The formation of NPQ is associated with certain absorption changes that have been suggested to reflect a conformational change in LHCII brought about by its protonation. The light-minus-dark recovery absorption difference spectrum is characterized by a series of positive and negative bands, the best known of which is ΔA(535). Light-minus-dark recovery resonance Raman difference spectra performed at the wavelength of the absorption change of interest allows identification of the pigment responsible from its unique vibrational signature. Using this technique, the origin of ΔA(535) was previously shown to be a subpopulation of red-shifted zeaxanthin molecules. In the absence of zeaxanthin (and antheraxanthin), a proportion of NPQ remains, and the ΔA(535) change is blue-shifted to 525 nm (ΔA(525)). Using resonance Raman spectroscopy, it is shown that the ΔA(525) absorption change in Arabidopsis leaves lacking zeaxanthin belongs to a red-shifted subpopulation of violaxanthin molecules formed during NPQ. The presence of the same ΔA(535) and ΔA(525) Raman signatures in vitro in aggregated LHCII, containing zeaxanthin and violaxanthin, respectively, leads to a new proposal for the origin of the xanthophyll red shifts associated with NPQ.

A recently developed technique for dilution of the naturally high protein packing density in isolated grana membranes was applied to study the dependence of the light harvesting efficiency of photosystem (PS) II on macromolecular crowding. Slight dilution of the protein packing from 80% area fraction to the value found in intact grana thylakoids (70%) leads to an improved functionality of PSII (increased antenna size, enhanced connectivity between reaction centers). Further dilution induces a functional disconnection of light-harvesting complex (LHC) II from PSII. It is concluded that efficient light harvesting by PSII requires an optimal protein packing density in grana membranes that is close to 70%. We hypothesize that the decreased efficiency in overcrowded isolated grana thylakoids is caused by excited state quenching in LHCII, which has previously been correlated with neoxanthin distortion. Resonance Raman spectroscopy confirms this increase in neoxanthin distortion in overcrowded grana as compared with intact thylakoids. Furthermore, analysis of the changes in the antenna size in highly diluted membranes indicates a lipid-induced dissociation of up to two trimeric LHCII from PSII, leaving one trimer connected. This observation supports a hierarchy of LHCII-binding sites on PSII.

Fluorescence line-narrowing and resonance Raman properties of various chlorophylls have been measured in organic solvents. Resonance Raman spectroscopy is already a well-established method for the study of photochemical reactions in the various pigment-protein complexes involved in photosynthesis, while fluorescence line-narrowing is still an emerging technique for such systems. Interpretation of these vibrational spectra requires accurate comparative data on the pure isolated pigments. By comparing three different chlorophylls, a, b, and d, which have different substituents on the porphyrin ring, the various spectral lines associated with vinyl and formyl groups on the X and Y electronic axes could be distinguished. The difference between five- and six-coordination of the central Mg atom in FT-Raman spectra was determined by varying the organic solvent used. These chlorophylls are important in photosynthesis: all three in light-harvesting and energy transfer and, in the case of a and d, also in electron transfer. The assignment of spectral bands which we provide here, along with the description of their behavior with respect to the conformation and state of interaction of the pigment molecule, constitutes an essential step if these vibrational techniques are to be exploited to their full potential.

Under conditions of excess sunlight the efficient light-harvesting antenna found in the chloroplast membranes of plants is rapidly and reversibly switched into a photoprotected quenched state in which potentially harmful absorbed energy is dissipated as heat, a process measured as the non-photochemical quenching of chlorophyll fluorescence or qE. Although the biological significance of qE is established, the molecular mechanisms involved are not. LHCII, the main light-harvesting complex, has an inbuilt capability to undergo transformation into a dissipative state by conformational change and it was suggested that this provides a molecular basis for qE, but it is not known if such events occur in vivo or how energy is dissipated in this state. The transition into the dissipative state is associated with a twist in the configuration of the LHCII-bound carotenoid neoxanthin, identified using resonance Raman spectroscopy. Applying this technique to study isolated chloroplasts and whole leaves, we show here that the same change in neoxanthin configuration occurs in vivo, to an extent consistent with the magnitude of energy dissipation. Femtosecond transient absorption spectroscopy, performed on purified LHCII in the dissipative state, shows that energy is transferred from chlorophyll a to a low-lying carotenoid excited state, identified as one of the two luteins (lutein 1) in LHCII. Hence, it is experimentally demonstrated that a change in conformation of LHCII occurs in vivo, which opens a channel for energy dissipation by transfer to a bound carotenoid. We suggest that this is the principal mechanism of photoprotection.

The xanthophyll cycle has a major role in protecting plants from photooxidative stress, although the mechanism of its action is unclear. Here, we have investigated Arabidopsis plants overexpressing a gene encoding beta-carotene hydroxylase, containing nearly three times the amount of xanthophyll cycle carotenoids present in the wild-type. In high light at low temperature wild-type plants exhibited symptoms of severe oxidative stress: lipid peroxidation, chlorophyll bleaching, and photoinhibition. In transformed plants, which accumulate over twice as much zeaxanthin as the wild-type, these symptoms were significantly ameliorated. The capacity of non-photochemical quenching is not significantly different in transformed plants compared with wild-type and therefore an enhancement of this process cannot be the cause of the stress tolerant phenotype. Rather, it is concluded that it results from the antioxidant effect of zeaxanthin. 80-90% of violaxanthin and zeaxanthin in wild-type and transformed plants was localized to an oligomeric LHCII fraction prepared from thylakoid membranes. The binding of these pigments in intact membranes was confirmed by resonance Raman spectroscopy. Based on the structural model of LHCII, we suggest that the protein/lipid interface is the active site for the antioxidant activity of zeaxanthin, which mediates stress tolerance by the protection of bound lipids.

In order to maximize their use of light energy in photosynthesis, plants have molecules that act as light-harvesting antennae, which collect light quanta and deliver them to the reaction centres, where energy conversion into a chemical form takes place. The functioning of the antenna responds to the extreme changes in the intensity of sunlight encountered in nature. In shade, light is efficiently harvested in photosynthesis. However, in full sunlight, much of the energy absorbed is not needed and there are vitally important switches to specific antenna states, which safely dissipate the excess energy as heat. This is essential for plant survival, because it provides protection against the potential photo-damage of the photosynthetic membrane. But whereas the features that establish high photosynthetic efficiency have been highlighted, almost nothing is known about the molecular nature of the dissipative states. Recently, the atomic structure of the major plant light-harvesting antenna protein, LHCII, has been determined by X-ray crystallography. Here we demonstrate that this is the structure of a dissipative state of LHCII. We present a spectroscopic analysis of this crystal form, and identify the specific changes in configuration of its pigment population that give LHCII the intrinsic capability to regulate energy flow. This provides a molecular basis for understanding the control of photosynthetic light-harvesting.

To understand physiological processes at the molecular level, new techniques are needed to determine the details of protein structure and dynamics in intact systems. We describe a specific example of such an approach, involving differential analysis of the carotenoid resonance Raman signal in the plant photosynthetic membrane. Carotenoids play important roles in the photosynthetic membrane and are particularly vital to photoprotective regulatory mechanisms. Our methodology selectively revealed the details of associations between specific carotenoid molecules and specific protein binding sites. Changes in the molecular configuration of these cofactors associated with alterations in the physiological state of the photosynthetic system were observed. This approach can be applied to a wide range of complex biological systems, whenever a protein with a light-absorbing cofactor is involved.

By dynamic changes in protein structure and function, the photosynthetic membranes of plants are able to regulate the partitioning of absorbed light energy between utilization in photosynthesis and photoprotective non-radiative dissipation of the excess energy. This process is controlled by features of the intact membrane, the transmembrane pH gradient, the organization of the photosystem II antenna proteins and the reversible binding of a specific carotenoid, zeaxanthin. Resonance Raman spectroscopy has been applied for the first time to wild type and mutant Arabidopsis leaves and to intact thylakoid membranes to investigate the nature of the absorption changes obligatorily associated with the energy dissipation process. The observed changes in the carotenoid Resonance Raman spectrum proved that zeaxanthin was involved and indicated a dramatic change in zeaxanthin environment that specifically alters the pigment configuration and red-shifts the absorption spectrum. This activation of zeaxanthin is a key event in the regulation of light harvesting.

Resonance Raman excitation spectroscopy combined with ultra low temperature absorption spectral analysis of the major xanthophylls of higher plants in isolated antenna and intact thylakoid membranes was used to identify carotenoid absorption regions and study their molecular configuration. The major electronic transitions of the light-harvesting complex of photosystem II (LHCIIb) xanthophylls have been identified for both the monomeric and trimeric states of the complex. One long wavelength state of lutein with a 0-0 transition at 510 nm was detected in LHCIIb trimers. The short wavelength 0-0 transitions of lutein and neoxanthin were located at 495 and 486 nm, respectively. In monomeric LHCIIb, both luteins absorb around 495 nm, but slight differences in their protein environments give rise to a broadening of this band. The resonance Raman spectra of violaxanthin and zeaxanthin in intact thylakoid membranes was determined. The broad 0-0 absorption transition for zeaxanthin was found to be located in the 503-511 nm region. Violaxanthin exhibited heterogeneity, having two populations with one absorbing at 497 nm (0-0), 460 nm (0-1), and 429 nm (0-2), and the other major pool absorbing at 488 nm (0-0), 452 nm (0-1), and 423 nm (0-2). The origin of this heterogeneity is discussed. The configuration of zeaxanthin and violaxanthin in thylakoid membranes was different from that of free pigments, and both xanthophylls (notably, zeaxanthin) were found to be well coordinated within the antenna proteins in vivo, arguing against the possibility of their free diffusion in the membrane and supporting our recent biochemical evidence of their association with intact oligomeric light-harvesting complexes (Ruban, A. V., Lee, P. J., Wentworth, M., Young, A. J., and Horton, P. (1999) J. Biol. Chem. 274, 10458-10465).

The electronic transitions of lutein and neoxanthin in the major light-harvesting complex, LHCIIb, have been identified for the first time. It was found that 0-0, 0-1 and 0-2 transitions of neoxanthin were located around 486, 457 and 430 nm, whilst those for lutein were dependent on the oligomerisation state. For the monomer, the absorption bands of lutein were found at 495, 466 and 437 nm. Trimerisation caused a decrease in lutein absorption and the parallel appearance of an additional absorption band around 510 nm, which was identified by resonance Raman excitation spectra to originate from lutein. Circular dichroism measurements together with analysis of the nu(4) resonance Raman region of xanthophylls suggested that this lutein molecule is distorted in the trimer. This feature is not predicted by the LHCIIb atomic model of Kühlbrandt and co-workers [Kühlbrandt, W., Wang, D.N. and Fugiyoshi, Y. (1994) Nature 367, 614-621] and is an important step in understanding pigment dynamics of the complex. Oligomerisation of trimers led to a specific distortion of the neoxanthin molecule. These observations suggest that the xanthophylls of LHCIIb sense the protein conformation and which may reflect their special role in the assembly and function of the light-harvesting antenna of higher plants.

Resonance Raman spectroscopy of an antenna protein from the brown alga Laminaria saccharina has been used to investigate the molecular structure of this light-harvesting complex (LHC) at the level of its bound pigments, chlorophylls (chl) a and c and the xanthophyll fucoxanthin. Evidence has been obtained for the conservation of pigment structure during the isolation procedure used. Six chl a and two chl c molecules are indicated from the positions and relative contributions of stretching modes of their keto-carbonyl groups. Of special interest is the presence of a population of chls a having a protein-binding conformation highly similar to that seen in antenna proteins from higher plants, possibly indicating a common structural motif within this extended gene family. The eight fucoxanthin molecules evidenced are all in the all-trans conformation; however, one or two have a highly twisted configuration. The results are discussed in terms of common and varying structural features of LHCs in higher plants and algae.

The energy transfer process in the minor light-harvesting antenna complex CP29 of green plants was probed in multicolor transient absorption experiments at 77 K using selective subpicosecond excitation pulses at 640 and 650 nm. Energy flow from each of the chlorophyll (Chl) b molecules of the complex could thus be studied separately. The analysis of our data showed that the "blue" Chl b (absorption around 640 nm) transfers excitation to a "red" Chl a with a time constant of 350 +/- 100 fs, while the 'red' Chl b (absorption at 650 nm) transfers on a picosecond time scale (2.2 +/- 0.5 ps) toward a "blue" Chl a. Furthermore, both fast (280 +/- 50 fs) and slow (10-13 ps) equilibration processes among the Chl a molecules were observed, with rates and associated spectra very similar to those of the major antenna complex, LHC-II. Based on the protein sequence homology between CP29 and LHC-II, a basic modelling of the observed kinetics was performed using the LHC-II structure and the Förster theory of energy transfer. Thus, an assignment for the spectral properties and orientation of the two Chl's b, as well as for their closest Chl a neighbors, is put forward, and a comparison is made with the previous assignments and models for LHC-II and CP29.

The xanthophyll composition of the light-harvesting chlorophyll a/b proteins of photosystem II (LHCII) has been determined for spinach (Spinacia oleracea L.) leaves after dark adaptation and following illumination under conditions optimized for conversion of violaxanthin into zeaxanthin. Each of the four LHCII components was found to have a unique xanthophyll composition. The major carotenoid was lutein, comprising 60% of carotenoid in the bulk LHCIIb and 35 to 50% in the minor LHCII components LHCIIa, LHCIIc, and LHCIId. The percent of carotenoid found in the xanthophyll cycle pigments was approximately 10 to 15% in LHCIIb and 30 to 40% in LHCIIa, LHCIIc, and LHCIId. The xanthophyll cycle was active for the pigments bound to all of the LHCII components. The extent of deepoxidation for complexes prepared from light-treated leaves was 27, 65, 69, and 43% for LHCIIa, -b, -c, and -d, respectively. These levels of conversion of violaxanthin to zeaxanthin were found in LHCII prepared by three different isolation procedures. It was estimated that approximately 50% of the zeaxanthin associated with photosystem II is in LHCIIb and 30% is associated with the minor LHCII components.

A new hypothesis is presented to explain the major molecular process that regulates the efficiency of light harvesting by chloroplast membranes. It is proposed that in excess light the decrease in the thylakoid lumen pH causes an increase in aggregation of the light harvesting complexes of photosystem II resulting in formation of an efficient pathway for non-radiative dissipation of excitation energy. The aggregation is potentiated by the conversion of violaxanthin to zeaxanthin. This hypothesis is based upon (i) similarity between the spectroscopic changes associated with energy dissipation and those observed upon aggregation of isolated light harvesting complex; and (ii) the link between changes in light scattering and increased energy dissipation.