RESUMEN
In the field of photosynthesis, only a limited number of approaches of super-resolution fluorescence microscopy can be used, as the functional architecture of the thylakoid membrane in chloroplasts is probed through the natural fluorescence of chlorophyll molecules. In this work, we have used a custom-built fluorescence microscopy method called Single Pixel Reconstruction Imaging (SPiRI) that yields a 1.4 gain in lateral and axial resolution relative to confocal fluorescence microscopy, to obtain 2D images and 3D-reconstucted volumes of isolated chloroplasts, obtained from pea (Pisum sativum), spinach (Spinacia oleracea) and Arabidopsis thaliana. In agreement with previous studies, SPiRI images exhibit larger thylakoid grana diameters when extracted from plants under low-light regimes. The three-dimensional thylakoid architecture, revealing the complete network of the thylakoid membrane in intact, non-chemically-fixed chloroplasts can be visualized from the volume reconstructions obtained at high resolution. From such reconstructions, the stromal connections between each granum can be determined and the fluorescence intensity in the stromal lamellae compared to those of neighboring grana.
RESUMEN
Reported herein is a Stark fluorescence spectroscopy study performed on photosystem II core antenna complexes CP43 and CP47 in their native and aggregated states. The systematic mathematical modeling of the Stark fluorescence spectra with the aid of conventional Liptay formalism revealed that induction of aggregation in both the core antenna complexes via detergent removal results in a single quenched species characterized by a remarkably broad and inhomogenously broadened emission lineshape peaking around 700 nm. The quenched species possesses a fairly large magnitude of charge-transfer character. From the analogy with the results from aggregated peripheral antenna complexes, the quenched species is thought to originate from the enhanced chlorophyll-chlorophyll interaction due to aggregation. However, in contrast, aggregation of both core antenna complexes did not produce a far-red emission band at â¼730 nm, which was identified in most of the aggregated peripheral antenna complexes. The 730-nm emission band of the aggregated peripheral antenna complexes was attributed to the enhanced chlorophyll-carotenoid (lutein1) interaction in the terminal emitter locus. Therefore, it is very likely that the no occurrence of the far-red band in the aggregated core antenna complexes is directly related to the absence of lutein1 in their structures. The absence of the far-red band also suggests the possibility that aggregation-induced conformational change of the core antenna complexes does not yield a chlorophyll-carotenoid interaction associated energy dissipation channel.
Asunto(s)
Clorofila , Complejo de Proteína del Fotosistema II , Carotenoides , Transferencia de Energía , Complejos de Proteína Captadores de Luz/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Espectrometría de FluorescenciaRESUMEN
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.
Asunto(s)
Clorofila A/metabolismo , Clorofila/metabolismo , Chlorophyta/metabolismo , Complejos de Proteína Captadores de Luz/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Pigmentos Biológicos/química , Xantófilas/metabolismo , Fotosíntesis , Pigmentos Biológicos/metabolismoRESUMEN
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
Cyanobacteria, the largest phylum of prokaryotes, perform oxygenic photosynthesis and are regarded as the ancestors of the plant chloroplast and the purveyors of the oxygen and biomass that shaped the biosphere. Nowadays, cyanobacteria are attracting a growing interest in being able to use solar energy, H2O, CO2 and minerals to produce biotechnologically interesting chemicals. This often requires the introduction and expression of heterologous genes encoding the enzymes that are not present in natural cyanobacteria. However, only a handful of model strains with a well-established genetic system are being studied so far, leaving the vast biodiversity of cyanobacteria poorly understood and exploited. In this study, we focused on the robust unicellular cyanobacterium Cyanothece PCC 7425 that has many interesting attributes, such as large cell size; capacity to fix atmospheric nitrogen (under anaerobiosis) and to grow not only on nitrate but also on urea (a frequent pollutant) as the sole nitrogen source; capacity to form CO2-sequestrating intracellular calcium carbonate granules and to produce various biotechnologically interesting products. We demonstrate for the first time that RSF1010-derived plasmid vectors can be used for promoter analysis, as well as constitutive or temperature-controlled overproduction of proteins and analysis of their sub-cellular localization in Cyanothece PCC 7425. These findings are important because no gene manipulation system had been developed for Cyanothece PCC 7425, yet, handicapping its potential to serve as a model host. Furthermore, using this toolbox, we engineered Cyanothece PCC 7425 to produce the high-value terpene, limonene which has applications in biofuels, bioplastics, cosmetics, food and pharmaceutical industries. This is the first report of the engineering of a Cyanothece strain for the production of a chemical and the first demonstration that terpene can be produced by an engineered cyanobacterium growing on urea as the sole nitrogen source.
RESUMEN
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 ß-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 Qy absorption transitions, inducing red-shifts that are proportional to the strength of the hydrogen bond involved. Chls-a with non-H-bonded keto C131 groups exhibit the blue-most absorption bands, while both transitions are progressively red-shifted with increasing hydrogen-bonding strength - by up 382 & 605â¯cm-1 in the Qy 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.
Asunto(s)
Clorofila A/metabolismo , Complejos de Proteína Captadores de Luz/metabolismo , Clorofila A/química , Enlace de Hidrógeno , Espectrometría RamanRESUMEN
It has already been established that the quaternary structure of the main light-harvesting complex (LH2) from the photosynthetic bacterium Rhodopseudomonas palustris is a nonameric 'ring' of PucAB heterodimers and under low-light culturing conditions an increased diversity of PucB synthesis occurs. In this work, single molecule fluorescence emission studies show that different classes of LH2 'rings' are present in "low-light" adapted cells and that an unknown chaperon process creates multiple sub-types of 'rings' with more conformational sub-states and configurations. This increase in spectral disorder significantly augments the cross-section for photon absorption and subsequent energy flow to the reaction centre trap when photon availability is a limiting factor. This work highlights yet another variant used by phototrophs to gather energy for cellular development.
Asunto(s)
Apoproteínas/química , Proteínas Bacterianas/química , Complejos de Proteína Captadores de Luz/química , Rhodopseudomonas/química , Espectrometría de FluorescenciaRESUMEN
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 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
Among the ultimate goals of protein physics, the complete, experimental description of the energy paths leading to protein conformational changes remains a challenge. Single protein fluorescence spectroscopy constitutes an approach of choice for addressing protein dynamics, and, among naturally fluorescing proteins, light-harvesting (LH) proteins from purple bacteria constitute an ideal object for such a study. LHs bind bacteriochlorophyll a molecules, which confer on them a high intrinsic fluorescence yield. Moreover, the electronic properties of these pigment-proteins result from the strong excitonic coupling between their bound bacteriochlorophyll a molecules in combination with the large energetic disorder due to slow fluctuations in their structure. As a result, the position and probability of their fluorescence transition delicately depends on the precise realization of the disorder of the set of bound pigments, which is governed by the LH protein dynamics. Analysis of these parameters using time-resolved single-molecule fluorescence spectroscopy thus yields direct access to the protein dynamics. Applying this technique to the LH2 protein from Rhodovulum (Rdv.) sulfidophilum, the structure-and consequently the fluorescence properties-of which depends on pH, allowed us to follow a single protein, pH-induced, reversible, conformational transition. Hence, for the first time, to our knowledge, a protein transition can be visualized through changes in the electronic structure of the intrinsic cofactors, at a level of a single LH protein, which opens a new, to our knowledge, route for understanding the changes in energy landscape that underlie protein function and adaptation to the needs of living organisms.
Asunto(s)
Complejos de Proteína Captadores de Luz/química , Concentración de Iones de Hidrógeno , Modelos Moleculares , Conformación Proteica , Rhodovulum/enzimología , Espectrometría de FluorescenciaRESUMEN
The ability to dissipate large fractions of their absorbed light energy as heat is a vital photoprotective function of the peripheral light-harvesting pigment-protein complexes in photosystem II of plants. The major component of this process, known as qE, is characterised by the appearance of low-energy (red-shifted) absorption and fluorescence bands. Although the appearance of these red states has been established, the molecular mechanism, their site and particularly their involvement in qE are strongly debated. Here, room-temperature single-molecule fluorescence spectroscopy was used to study the red emission states of the major plant light-harvesting complex (LHCII) in different environments, in particular conditions mimicking qE. It was found that most states correspond to peak emission at around 700nm and are unrelated to energy dissipative states, though their frequency of occurrence increased under conditions that mimicked qE. Longer-wavelength emission appeared to be directly related to energy dissipative states, in particular emission beyond 770nm. The ensemble average of the red emission bands shares many properties with those obtained from previous bulk in vitro and in vivo studies. We propose the existence of at least three excitation energy dissipating mechanisms in LHCII, each of which is associated with a different spectral signature and whose contribution to qE is determined by environmental control of protein conformational disorder. Emission at 700nm is attributed to a conformational change in the Lut 2 domain, which is facilitated by the conformational change associated with the primary quenching mechanism involving Lut 1.
Asunto(s)
Complejos de Proteína Captadores de Luz/metabolismo , Procesos Fotoquímicos , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Fluorescencia , Complejos de Proteína Captadores de Luz/química , Datos de Secuencia Molecular , Proteínas de Plantas/química , Estructura Terciaria de Proteína , Spinacia oleracea/metabolismo , TermodinámicaRESUMEN
Light-harvesting pigment-protein complexes of photosystem II of plants have a dual function: they efficiently use absorbed energy for photosynthesis at limiting sunlight intensity and dissipate the excess energy at saturating intensity for photoprotection. Recent single-molecule spectroscopy studies on the trimeric LHCII complex showed that environmental control of the intrinsic protein disorder could in principle explain the switch between their light-harvesting and photoprotective conformations in vivo. However, the validity of this proposal depends strongly on the specificity of the protein dynamics. Here, a similar study has been performed on the minor monomeric antenna complexes of photosystem II (CP29, CP26, and CP24). Despite their high structural homology, similar pigment content and organization compared to LHCII trimers, the environmental response of these proteins was found to be rather distinct. A much larger proportion of the minor antenna complexes were present in permanently weakly fluorescent states under most conditions used; however, unlike LHCII trimers the distribution of the single-molecule population between the strongly and weakly fluorescent states showed no significant sensitivity to low pH, zeaxanthin, or low detergent conditions. The results support a unique role for LHCII trimers in the regulation of light harvesting by controlled fluorescence blinking and suggest that any contribution of the minor antenna complexes to photoprotection would probably involve a distinct mechanism.
Asunto(s)
Complejos de Proteína Captadores de Luz/química , Complejos de Proteína Captadores de Luz/metabolismo , Luz , Plantas/metabolismo , Plantas/efectos de la radiación , Ambiente , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Espectrometría de FluorescenciaRESUMEN
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
Energy transfer pathways between photosystem II (PSII) antenna complexes in intact thylakoid membranes have been studied using low-temperature (77 K) excitation fluorescence spectroscopy. The focus of this study was to see whether de-epoxidation of violaxanthin into zeaxanthin causes any alterations in the energetic couplings between the core antenna complexes CP43 and CP47 and the peripheral light-harvesting antenna (LHCII). It was discovered that the appearance of zeaxanthin caused characteristic alterations in the PSII excitation fluorescence spectra in the Soret-band region. While in the dark violaxanthin was found to be largely uncoupled from any emitting chlorophylls, its intensive de-epoxidation resulted in the appearance of two additional bands at 509 and 492 nm. The former was attributed to weak coupling of zeaxanthin to emitters in the CP43 and LHCII complexes and the latter to efficient coupling of violaxanthin of the CP29 complex to emitters in the CP43, CP47, and LHCII complexes. The role of CP29-bound violaxanthin is discussed in light of both its efficient energetic coupling and strong physical binding to this complex. The finding that zeaxanthin is energetically coupled to chlorophyll a emitters of the PSII antenna is discussed with respect to its suggested role as a quencher involved in photoprotective energy dissipation, or non-photochemical quenching (NPQ), in the photosynthetic membrane.
Asunto(s)
Transferencia de Energía , Complejo de Proteína del Fotosistema II/metabolismo , Espectrometría de Fluorescencia , Spinacia oleracea/citología , Spinacia oleracea/enzimología , Temperatura , Tilacoides/metabolismo , Xantófilas/metabolismoRESUMEN
The light-harvesting antenna of photosystem II (PSII) has the ability to switch rapidly between a state of efficient light use and one in which excess excitation energy is harmlessly dissipated as heat, a process known as qE. We investigated the single-molecule fluorescence intermittency of the main component of the PSII antenna (LHCII) under conditions that mimic efficient use of light or qE, and we demonstrate that weakly fluorescing states are stabilized under qE conditions. Thus, we propose that qE is explained by biological control over the intrinsic dynamic disorder in the complex-the frequencies of switching establish whether the population of complexes is unquenched or quenched. Furthermore, the quenched states were accompanied by two distinct spectral signatures, suggesting more than one mechanism for energy dissipation in LHCII.
Asunto(s)
Complejos de Proteína Captadores de Luz/metabolismo , Procesos Fotoquímicos , Complejo de Proteína del Fotosistema II/metabolismo , Spinacia oleracea/metabolismo , Centrifugación por Gradiente de Densidad , Fluorescencia , Complejos de Proteína Captadores de Luz/química , Modelos Moleculares , Complejo de Proteína del Fotosistema II/química , Pigmentos Biológicos/metabolismo , Multimerización de Proteína , Estructura Secundaria de Proteína , Espectrometría de Fluorescencia , TermodinámicaRESUMEN
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
We present a simple method to resolve discrete intensity shifts from time-resolved single-molecule emission data. This new method uses multiples of the standard deviation of the measured intensities that are integrated into short time bins. By applying the technique to trimeric units of the main light-harvesting complex (LHCII) of plants, it is shown that the amount of information that can be extracted from an intensity time trace increases considerably, thereby enlarging the possibility to reveal new phenomena. It is demonstrated how shot noise can lead to substantial deviations and misleading interpretations when the conventional two-state kinetic model for intensity fluctuations is applied. By first resolving the accessed intensity levels, the artifactual effect of shot noise is sufficiently reduced. The technique is particularly applicable to the analysis of fluorescence intermittency from multichromophoric systems.
Asunto(s)
Complejos de Proteína Captadores de Luz/química , Proteínas de Plantas/química , Algoritmos , Colorantes Fluorescentes/química , Complejos de Proteína Captadores de Luz/metabolismo , Proteínas de Plantas/metabolismo , Puntos Cuánticos , Spinacia oleracea/enzimologíaRESUMEN
The time-resolved fluorescence intensity fluctuations from single, immobilized complexes of the main light-harvesting complex (LHCII) of plants were investigated in different pH environments close to room temperature and under different light conditions. The efficiency of light harvesting, which was represented by complexes typically residing for long periods in strongly fluorescing states, was significantly reduced by decreasing the pH or increasing the incident photon flux. The same environmental changes significantly increased the switching frequency between strongly and weakly fluorescing states. The environmental dependence became more evident when the various accessed intensity levels were first resolved, a technique that significantly reduced the obscuring effect of shot noise. The strong environmental sensitivity suggests that the immediate environment of an LHCII complex can modulate the amount of energy dissipation. A simple model illustrates how this may be achieved: the dynamic equilibrium between the strongly and weakly fluorescing states can be shifted by environmentally controlling the conformational diffusion on the potential energy surface of LHCII.
Asunto(s)
Complejos de Proteína Captadores de Luz/química , Proteínas de Plantas/química , Transferencia de Energía , Concentración de Iones de Hidrógeno , Complejos de Proteína Captadores de Luz/metabolismo , Fotones , Proteínas de Plantas/metabolismo , Spinacia oleracea/enzimologíaRESUMEN
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.
Asunto(s)
Arabidopsis/metabolismo , Arabidopsis/efectos de la radiación , Luz , Xantófilas/deficiencia , Absorción , Arabidopsis/enzimología , Sitios de Unión , Cinética , Complejos de Proteína Captadores de Luz , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Multimerización de Proteína/efectos de la radiación , Estructura Cuaternaria de Proteína , Espectrometría Raman , Xantófilas/metabolismo , ZeaxantinasRESUMEN
Single-molecule spectroscopy was employed to elucidate the fluorescence spectral heterogeneity and dynamics of individual, immobilized trimeric complexes of the main light-harvesting complex of plants in solution near room temperature. Rapid reversible spectral shifts between various emitting states, each of which was quasi-stable for seconds to tens of seconds, were observed for a fraction of the complexes. Most deviating states were characterized by the appearance of an additional, red-shifted emission band. Reversible shifts of up to 75 nm were detected. By combining modified Redfield theory with a disordered exciton model, fluorescence spectra with peaks between 670 nm and 705 nm could be explained by changes in the realization of the static disorder of the pigment-site energies. Spectral bands beyond this wavelength window suggest the presence of special protein conformations. We attribute the large red shifts to the mixing of an excitonic state with a charge-transfer state in two or more strongly coupled chlorophylls. Spectral bluing is explained by the formation of an energy trap before excitation energy equilibration is completed.