Disentangling the low-energy states of the major light-harvesting complex of plants and their role in photoprotection

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 photosystemII 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 700 nm 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 700 nmis 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.This work was supported by the EU FP7Marie Curie Reintegration Grant (ERG 224796) (C.I.); the CEA-Eurotalents Program(PCOFUNDGA- 2008-228664) (C.I.); research and equipment grants from UK BBSRC and EPSRC (M.P.J. and A.V.R.); Grants from the Netherlands Organization for Scientific Research (700.58.305 and 700.56.014 from the Foundation of Chemical Sciences) (T.P.J.K., C.I., and R.v.G.),and the Advanced Investigator Grant (267333, PHOTPROT) from the European Research Council (ERC) (C.I., T.P.J.K., and R.v.G.).http://www.elsevier.com/locate/bbabiohb2014ai201

Energy transfer and fluorescence quenching in the lightharvesting complexes of photosystem II from higher plants

Non-photochemical quenching (NPQ) is the process by which plants exposed to high light conditions dissipate the potentially harmful excess energy as heat. It is thought to involve conformational changes in the light-harvesting complexes of photosystem II (LHCII). The work reported here involves an investigation of LHCII from various perspectives, describing energy transfer between the pigments bound as well as the role of the protein in NPQ. The 510 nm band in the 77K absorption spectra of LHCII trimers belongs to one of the luteins (lutein 2) in each monomer. The red-shift of this band may be caused by specific interaction(s) between the monomers during their association into trimers. The presence of the red-shifted lutein 2 in the unusual Lhcb3-Lhcb5 trimers from antisense Lhcb2 Arabidopsis plants is consistent with this interpretation. This lutein was found to be efficient in transferring energy to chlorophyll a. Analysis of the spectroscopic features of spinach thylakoids before and after de-epoxidation suggests the occurrence of a conformational change in the light-harvesting antenna, resulting in the remaining violaxanthin becoming more strongly involved in energy transfer to the PSII core. The quenching mechanism in LHCII was investigated. LHCII immobilised in a gel matrix showed quenching without protein aggregation, the transition to the quenched state involving a conformational change in which the neoxanthin and lutein 1 domains were affected. By monitoring the twisting of the neoxanthin molecule detected by resonance Raman spectroscopy, the same conformational change that accompanies the formation of the quenched state in vitro was observed in vivo upon NPQ induction. Transient absorption spectroscopy applied to purified LHCII in the quenched state showed the pathway for energy dissipation, involving energy transfer from chlorophyll a to the S1 excited state of lutein 1, which then decays to its ground state, dissipating the energy as heat

Energy transfer and fluorescence quenching in the lightharvesting complexes of photosystem II from higher plants

Non-photochemical quenching (NPQ) is the process by which plants exposed to high light conditions dissipate the potentially harmful excess energy as heat. It is thought to involve conformational changes in the light-harvesting complexes of photosystem II (LHCII). The work reported here involves an investigation of LHCII from various perspectives, describing energy transfer between the pigments bound as well as the role of the protein in NPQ. The 510 nm band in the 77K absorption spectra of LHCII trimers belongs to one of the luteins (lutein 2) in each monomer. The red-shift of this band may be caused by specific interaction(s) between the monomers during their association into trimers. The presence of the red-shifted lutein 2 in the unusual Lhcb3-Lhcb5 trimers from antisense Lhcb2 Arabidopsis plants is consistent with this interpretation. This lutein was found to be efficient in transferring energy to chlorophyll a. Analysis of the spectroscopic features of spinach thylakoids before and after de-epoxidation suggests the occurrence of a conformational change in the light-harvesting antenna, resulting in the remaining violaxanthin becoming more strongly involved in energy transfer to the PSII core. The quenching mechanism in LHCII was investigated. LHCII immobilised in a gel matrix showed quenching without protein aggregation, the transition to the quenched state involving a conformational change in which the neoxanthin and lutein 1 domains were affected. By monitoring the twisting of the neoxanthin molecule detected by resonance Raman spectroscopy, the same conformational change that accompanies the formation of the quenched state in vitro was observed in vivo upon NPQ induction. Transient absorption spectroscopy applied to purified LHCII in the quenched state showed the pathway for energy dissipation, involving energy transfer from chlorophyll a to the S1 excited state of lutein 1, which then decays to its ground state, dissipating the energy as heat

Apoprotein heterogeneity increases spectral disorder and a step-wise modification of the B850 fluorescence peak position

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

Pigment structure in the violaxanthin-chlorophyll-a-binding protein VCP

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

Probing the pigment binding sites in LHCII with resonance Raman spectroscopy: The effect of mutations at S123

International audienceResonance 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

Absence of far-red emission band in aggregated core antenna complexes

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 ∼730 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

Pigment structure in the light-harvesting protein of the siphonous green alga Codium fragile

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

The specificity of controlled protein disorder in the photoprotection of plants

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.The EU FP7 Marie Curie Reintegration Grant (ERG 224796) (C.I.);the CEA-Eurotalents program (PCOFUND-GA-2008-228664) (C.I.); research and equipment grants from UK BBSRC and EPSRC (M.P.J. and A.V.R.); Project Sunshine, University of Sheffield (P.H.); Grants from the Netherlands Organization for Scientific Research (700.58.305 and 700.56.014 from the Foundation of Chemical Sciences) (T.P.J.K., C.I., and R.v.G.), and the Advanced Investigator Grant (267333, PHOTPROT) from the European Research Council (ERC) (C.I., T.P.J.K., and R.v.G.).http://www.cell.com/biophysj/hb2013ai201

Conformational Switching in a Light-Harvesting Protein as Followed by Single-Molecule Spectroscopy

International audienceAmong 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