RESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/enzimología , Complejos de Proteína Captadores de Luz/química , Complejo de Proteína del Fotosistema II/químicaRESUMEN
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 CâC double bond into the conjugated CâC 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 CâO 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 CâC antisymmetric stretching mode, induced by the presence of the allene group in the carotenoid CâC chain.
Asunto(s)
Alcadienos , Carotenoides , Carotenoides/química , Electrónica , Espectrometría RamanRESUMEN
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.
Asunto(s)
Corteza Cerebral/química , Canales de Potasio Éter-A-Go-Go/química , Hemo/química , Neuronas/química , Corteza Cerebral/metabolismo , Canales de Potasio Éter-A-Go-Go/metabolismo , Hemo/metabolismo , Humanos , Neuronas/metabolismo , Unión Proteica , Dominios ProteicosRESUMEN
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.
Asunto(s)
Biofortificación , Carotenoides , Cloroplastos , Hojas de la Planta , PlastidiosRESUMEN
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.
Asunto(s)
Colorantes Fluorescentes/química , Luteína/química , Dimerización , Cinética , Conformación Molecular , Procesos Fotoquímicos , Plastidios/química , Espectrometría de Fluorescencia , Xantófilas/químicaRESUMEN
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.
RESUMEN
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 ß-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 ß-carotene molecules were in all-trans configuration and the absorption position of the farthest blue-shifted ß-carotene was attributed entirely to the polarizability of the environment in its binding pocket. In contrast, the absorption maximum of the red-shifted ß-carotene was attributed to two different factors: the polarizability of the environment in its binding pocket and, more importantly, to the conformation of its ß-rings. This second ß-carotene has highly twisted ß-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 S1 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.
Asunto(s)
Proteínas Bacterianas/química , Complejos de Proteína Captadores de Luz/química , Synechocystis/química , beta Caroteno/química , Proteínas Bacterianas/genética , Complejos de Proteína Captadores de Luz/genética , Dominios Proteicos , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Synechocystis/genética , beta Caroteno/genéticaRESUMEN
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.
Asunto(s)
Complejos de Proteína Captadores de Luz/química , Estramenopilos/química , Carotenoides/química , Cromatografía Líquida de Alta Presión , Conformación Proteica , Espectrometría RamanRESUMEN
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 ß-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 ß-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 ß-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 ß-carotenes.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Clorofila/metabolismo , Synechocystis/metabolismo , beta Caroteno/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Complejos de Proteína Captadores de Luz/genética , Complejos de Proteína Captadores de Luz/metabolismo , Multimerización de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Espectrometría Raman , Synechocystis/genética , Synechocystis/fisiologíaRESUMEN
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.
Asunto(s)
Complejos de Proteína Captadores de Luz/química , Complejo de Proteína del Fotosistema II/química , Espectrometría Raman/métodos , Sitios de Unión , Clorofila/química , Clorofila A , Luteína/química , Mutación , Xantófilas/químicaRESUMEN
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.
Asunto(s)
Alveolados/química , Complejos de Proteína Captadores de Luz/química , Xantófilas/química , Alveolados/metabolismo , Sitios de Unión , Complejos de Proteína Captadores de Luz/metabolismo , Unión Proteica , Xantófilas/metabolismoRESUMEN
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.
Asunto(s)
Carotenoides/química , Proteínas Portadoras/química , Clorofila/química , Complejos de Proteína Captadores de Luz/química , Espectrometría Raman , Xantófilas/químicaRESUMEN
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.
Asunto(s)
Fotosíntesis/fisiología , Espectrometría de Fluorescencia/métodos , Espectrometría Raman/métodos , Carotenoides/química , Carotenoides/metabolismo , Clorofila/química , Clorofila/metabolismo , Pigmentos Biológicos/química , Pigmentos Biológicos/metabolismo , VibraciónRESUMEN
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 ß-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.
Asunto(s)
Complejos de Proteína Captadores de Luz/química , Luteína/química , Complejo de Proteína del Fotosistema II/química , beta Caroteno/química , Luz , Fotosíntesis , Unión Proteica , Conformación Proteica , Solventes , Espectrofotometría Ultravioleta , Espectrometría Raman , Spinacia oleracea/enzimología , TemperaturaRESUMEN
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.
Asunto(s)
Carotenoides/análisis , Diatomeas/metabolismo , Espectrometría Raman/métodos , Sitios de Unión , Carotenoides/metabolismo , Cromatografía Líquida de Alta Presión , Diatomeas/crecimiento & desarrollo , Luz , Xantófilas/análisis , Xantófilas/metabolismoRESUMEN
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.
RESUMEN
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.
Asunto(s)
Arabidopsis/citología , Calor , Complejos de Proteína Captadores de Luz/metabolismo , Luz , Spinacia oleracea/metabolismo , Cloroplastos/metabolismo , Cloroplastos/efectos de la radiación , Complejos de Proteína Captadores de Luz/química , Complejos de Proteína Captadores de Luz/aislamiento & purificación , Modelos Moleculares , Complejo de Proteína del Fotosistema II/aislamiento & purificación , Complejo de Proteína del Fotosistema II/metabolismo , Hojas de la Planta/metabolismo , Hojas de la Planta/efectos de la radiación , Espectrometría Raman , Factores de Tiempo , Xantófilas/química , Xantófilas/metabolismoRESUMEN
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).
Asunto(s)
Arabidopsis/fisiología , Luteína/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Arabidopsis/metabolismo , Sitios de Unión , Cromatografía Líquida de Alta Presión , Espectrometría RamanRESUMEN
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.