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Photoprotection in higher plants: The putative quenching site is conserved in all outer light-harvesting complexes of Photosystem II
Biochimica et Biophysica Acta 1777 (2008) 1263–1267
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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a b i o
Photoprotection in higher plants: The putative quenching site is conserved in all outer light-harvesting complexes of Photosystem II Milena Mozzo a, Francesca Passarini a, Roberto Bassi b, Herbert van Amerongen c,d, Roberta Croce a,⁎ a
Department of Biophysical Chemistry/Groningen Biotechnology and Biochemical Sciences Institute, University of Groningen, Nijenborgh 4, Groningen, 9747 AG, The Netherlands Dipartimento Scientifico e Tecnologico, Facoltà di Scienze MM.FF.NN., Università di Verona, Strada Le Grazie 15, 37134 Verona, Italy Wageningen University, Laboratory of Biophysics, PO Box 8128, 6700 ET, Wageningen, The Netherlands d MicroSpectroscopy Centre, Wageningen University, 6703 HA Wageningen, The Netherlands b c
a r t i c l e
i n f o
Article history: Received 17 March 2008 Accepted 16 April 2008 Available online 29 April 2008 Keywords: Photosynthesis Photosystem II Light-harvesting complex Photoprotection
a b s t r a c t In bright sunlight, the amount of energy harvested by plants exceeds the electron transport capacity of Photosystem II in the chloroplasts. The excess energy can lead to severe damage of the photosynthetic apparatus and to avoid this, part of the energy is thermally dissipated via a mechanism called nonphotochemical quenching (NPQ). It has been found that LHCII, the major antenna complex of Photosystem II, is involved in this mechanism and it was proposed that its quenching site is formed by the cluster of strongly interacting pigments: chlorophylls 611 and 612 and lutein 620 [A.V. Ruban, R. Berera, C. Ilioaia, I.H.M. van Stokkum, J.T.M. Kennis, A.A. Pascal, H. van Amerongen, B. Robert, P. Horton and R. van Grondelle, Identification of a mechanism of photoprotective energy dissipation in higher plants, Nature 450 (2007) 575– 578.]. In the present work we have investigated the interactions between the pigments in this cluster not only for LHCII, but also for the homologous minor antenna complexes CP24, CP26 and CP29. Use was made of wild-type and mutated reconstituted complexes that were analyzed with (low-temperature) absorption and circular-dichroism spectroscopy as well as by biochemical methods. The pigments show strong interactions that lead to highly specific spectroscopic properties that appear to be identical for LHCII, CP26 and CP29. The interactions are similar but not identical for CP24. It is concluded that if the 611/612/620 domain is responsible for the quenching in LHCII, then all these antenna complexes are prepared to act as a quencher. This can explain the finding that none of the Lhcb complexes seems to be strictly required for NPQ while, in the absence of all of them, NPQ is abolished. © 2008 Elsevier B.V. All rights reserved.
Photosynthetic antenna complexes of higher plants have a dual role: (i) They harvest sunlight, transferring excitation energy to the reaction centre, where it is used to induce charge separation that ultimately leads to the formation of chemical energy but (ii) in full sunlight they rapidly (on a seconds to minute time scale) switch into a non-photochemical quenching (NPQ) state, which safely dissipates excess energy via a mechanism known as qE, thus protecting the system from photodamage [1]. Several mechanisms were proposed to contribute to the quenching [2–4]. It has been demonstrated that the crystal structure of the main light-harvesting complex LHCII corresponds to a quenched state [5], and the pigment cluster consisting of lutein 620 (Lut 620)/Chla 611/Chla 612 (nomenclature as in [6]) (Fig. 1) was identified as a possible quenching site. Recently it was found that LHCII is in a similar quenched state when it is aggregated in vitro but more important when qE is induced in vivo [4]. In LHCII aggregates, Lut 620 was shown to be the quencher, whereas Chla 611 and Chla 612 function as a site for excitation localization, i.e. at equilibrium most of
Abbreviations: Chl, chlorophyll; Lhc, light-harvesting complex ⁎ Corresponding author. Tel.: +31 3634214; fax: +31 3634800. E-mail address: [email protected] (R. Croce). 0005-2728/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbabio.2008.04.036
the excitation energy is localized on these Chls [7,8] making them a preferential site for energy dissipation. Switching between the lightharvesting and quenched state is ascribed to a change in interaction between Lut 620 and Chla 611/Chla 612 [4,5,9,10]. Because Lut 620 is in contact with Chla 612 [6] which in turn is close to Chl 611, it was assumed that all three pigments are strongly excitonically coupled [10–13]. A change in interaction upon a conformational switch could then lead to quenching. However, experimental characterization of the mutual interactions is lacking. Although most research on NPQ focuses on LHCII, evidence exists that also the minor antenna complexes, being Lhcb4 (CP29), Lhcb5 (CP26) and Lhcb6 (CP24) might act as quenchers [14–18]. Analysis of mutant plants lacking the individual complexes Lhcb4, Lhcb5 or Lhcb1/2 has shown that none of them is strictly required for NPQ [19,20] although in the absence of all of them NPQ is nearly absent [21]. Only the mutant lacking Lhcb6 is strongly affected in NPQ [22] but this effect is ascribed to a limitation of proton pumping (De Bianchi et al., submitted). These data strongly suggest that more than one Lhcb complex contributes to NPQ, in line with the proposal that multiple quenching sites are distributed throughout Photosystem II (PSII) [23].
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M. Mozzo et al. / Biochimica et Biophysica Acta 1777 (2008) 1263–1267 1.2. Spectroscopy The absorption spectra at RT and 77 K were recorded using a SLM-Aminco DK2000 spectrophotometer, in 10 mM Hepes pH 7.5, 0.2 M sucrose (70% v/v glycerol at 77 K) and 0.06% n-Dodecyl-beta-D-maltoside. The wavelength-sampling step was 0.4 nm, scan rate 100 nm/min, optical pathlength 1 cm. The CD spectra were measured at 10 °C on an AVIV 62ADS or a J600 spectropolarimeter. The wavelength-sampling step was 0.5 nm with an average time of 1 s. Before subtraction, the absorption spectra were normalized to the pigment content on the basis of the area in the 630–750 nm region, using a ratio of 0.7 between the extinction coefficients of Chl b and Chl a.
2. Results and discussion
Fig. 1. Structure of the LHCII monomer [6] showing the organization of Chla 612 (dark green), Chla 611 (light green) and Lut 620 (yellow).
In aggregated LHCII the fluorescence is quenched by Lut 620 and switching to this quenched state is accompanied by the same structural change that occurs upon qE induction in vivo as can be monitored via characteristic changes in the Raman spectrum of neoxanthin [4]. However, also aggregation of the minor antenna complexes leads to fluorescence quenching and since neoxanthin is not only associated with LHCII but is also bound to Lhcb4 and Lhcb5 [24], the observed changes in vivo might also be attributed to the minor antenna complexes. Due to the high structural homology between these complexes, it might be expected that the quenching mechanism is similar in all cases. If Chla 611/Chla 612/Lut 620 would indeed form the quenching site in all complexes, this would require a highly similar pigment organization with highly similar mutual interactions. However, experimental proof for such similarity is lacking. Light-spectroscopic techniques can be very sensitive tools to probe pigment–pigment interactions and the local environment of pigments in light-harvesting complexes (Lhcs) [25]. For instance, the location of the absorption maximum of Chl a can vary from 660 to 710 nm in Lhc proteins. In particular, strong interactions between nearby chromophores play an important role in tuning the spectroscopic properties and thus the function [26]. Since it was proposed that Chla 612 interacts strongly with Chla 611 and Lut 620 it might thus be expected that a small structural change will cause major spectroscopic changes. In order to study the excitonic interactions between Lut 620, Chla 611 and Chla 612 and compare them for all complexes, we have performed mutations of the putative Chl 612 ligand in all outer antenna complexes of PSII and compared in detail the biochemical composition and the absorption and circular-dichroism (CD) spectra.
The wild-type (WT) apoprotein of Lhcb1, the main constituent of LHCII, was overexpressed and reconstituted as described before [7]. The same was done under identical conditions for the mutated apoprotein (Lhcb1-N183L), which lacks the ligand for Chla 612. As expected, a lower Chl a/Chlb ratio is found for the mutated protein (Table 1) in agreement with the loss of Chl a. In both proteins the relative amounts of neoxanthin (Neo), violaxanthin (Vio) and lutein are identical (0.8: 0.2: 2.0, Table 1), consistent with the fact that the carotenoid (Car) composition remains unaltered. This allows normalization to the carotenoid content. Taking 12 Chls for WT reconstituted protein (see [7]) it is found that the mutated complex lacks 1.4 Chl a and 0.6 Chl b, in agreement with previous results [7], where the difference was ascribed to the loss of Chls 612 and 611. The binding site for Chl 611 was concluded to have mixed occupancy, i.e. it can bind both Chl a and Chl b [7]. 77 K absorption spectra of both proteins are compared in Fig. 2A after scaling to the pigment content. The difference spectrum (Fig. 3) shows the loss of a major band at 678 nm (681 nm at room temperature (RT)) and a minor one at 660 nm for the mutated protein. These bands are due to two excitonically coupled Chls (Chl611 and Chl612) since the difference in the RT CD spectra of both proteins (Figs. 4A and 5) shows the loss of a negative CD band near 681 nm and a positive one near 660 nm. The 681 nm state is the lowest-energy state and acts as a preferential site for excitation localization. Remarkably, the loss of both bands is accompanied by a strong increase of carotenoid absorption (Fig. 3). Since Lut 620 is the only xanthophyll close to Chl 611/Chl 612, this increase is ascribed to a loss of excitonic interaction(s) between Lut 620 and Chl 611/Chl 612. Apparently, this interaction leads to substantial hypochromism (loss of absorption) for Lut 620 which must be accompanied by prominent hyperchromism (gain of absorption) for Chl a. This offers an explanation for an intriguing feature observed in triplet-minus-singlet spectra of LHCII [29,30]. The presence of a triplet on a carotenoid later identified [31] as L1 (Lut 620) leads to decreased Chl a Qy absorption. Presumably, strong excitonic interactions between Lut 620 and Chl 612/Chl 611 lead to hyperchromism in the Qy region. Upon triplet formation on Lut 620 the excitonic interaction is broken, reducing the hyperchromism and thus decreasing Chl absorption. Excitonic interaction probably also occurs between the main Lut 620 transition and the Chl Bx transition (near 437 nm). Disappearance of this interaction
1. Materials and methods 1.1. Mutagenesis and in vitro reconstitution Mature sequences of Lhcb1 and Lhcb4 from Zea mays and Lhcb5 from Arabidopsis thaliana were amplified from cDNA by PCR and cloned in pQE50 His (home modified pQE50, Qiagen); Lhcb6 from A. thaliana was cloned in pETMHis (home modified pET28 (a)+, Novagen). Mutant sequences were obtained by site directed mutagenesis of the Chl 612 ligand residues: asparagine 183 was mutated to leucine in Lhcb1, histidine 216 to phenylalanine in Lhcb4, asparagine 188 to phenylalanine in Lhcb5 and histidine 191 to phenylalanine in Lhcb6. WT and mutant apoproteins were overexpressed in SG13009 and Rosetta2 DE3 (pLysS) strains of Escherichia coli and purified as inclusion bodies. Reconstitution and purification of pigment–protein complexes were performed as described in [27] using the following Chl a/b ratios in the reconstitution mixture: 2.4 for Lhcb1, 4.0 for Lhcb4 and 3.0 for Lhcb5–6. The pigment composition was analyzed by HPLC and fitting of the absorption spectra of the acetone extracts as in [28]. Pigment composition, absorption and circular-dichroism spectra were fully reproducible for all samples obtained after all reconstitutions.
Table 1 Pigment composition of the complexes Sample
Chl a/b
Chl/Car
Neo
Vio
Lut
Chl tot
Chl a
Chl b
Car tot
Lhcb1-WT Lhcb1-N183L Lhcb4-WT Lhcb4-H216F Lhcb5-WT Lhcb5-N188F Lhcb6-WT Lhcb6-H191F
1.36 1.15 2.36 1.9 2.01 1.72 1.0 0.9
4.2 3.6 4.1 3.7 3.5 3.1 5.1 5.9
0.96 0.93 0.77 0.75 0.92 1.0 – –
0.18 0.16 0.42 0.44 0.18 0.06 0.96 1.05
1.71 1.65 0.86 0.81 1.73 1.86 0.99 0.38
12 10 8 7 10 9 10 9
6.9 5.4 5.6 4.6 6.7 5.7 5.0 4.3
5.1 4.6 2.4 2.4 3.3 3.3 5.0 4.7
2.9 2.8 2.0 1.9 2.8 2.9 2.0 1.5
The data are the average of 2 measurements on 2 different samples for Lhcb4 and Lhcb5 and on 4 samples for LHCII and Lhcb6. The maximum error is less than 0.1.
M. Mozzo et al. / Biochimica et Biophysica Acta 1777 (2008) 1263–1267
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Fig. 2. Absorption spectra at 77 K of WT and mutant Lhcbs. (A) Lhcb1-WT (solid) and Lhcb1-N183L (dashed); (B) Lhcb5-WT (solid) and Lhcb5-N188F (dashed); (C) Lhcb4-WT (solid) and Lhcb4-H216F (dashed); (D) Lhcb6-WT (solid) and Lhcb6-H191F (dashed). Spectra are normalized to Chl content.
causes a blue shift of lutein absorption and this explains the positive feature near 500 nm in the difference spectrum (Fig. 3). Summarizing, we have identified four fingerprints for the strong excitonic interactions in the quenching site of LHCII between Lut 620 and Chl 611/Chl 612: (i) strong hypochromism of Lut 620, (ii) redshifted absorption of Lut 620, (iii) a weak excitonic band at 660 nm and (iv) a strong excitonic band at 681 nm, accompanied by very specific features in the CD spectrum. Excitations tend to be localized in the low-energy state (681 nm) where they can be quenched by Lut 620 upon induction of qE or in the case of aggregation. In order to investigate the presence of an “LHCII-like” quenching site in the minor antenna complexes, the putative ligand for Chl 612 was also mutated in Lhcb4, Lhcb5 and Lhcb6 and the WT and mutated proteins were reconstituted under identical conditions. All mutated proteins show a lower Chl a/b ratio than the corresponding WT proteins, again indicating the loss of Chl a (Table 1). The Car composition (N:V:L) is the same for WT and mutant protein in the case of Lhcb4 and Lhcb5. Normalizing the WT and mutated proteins to the same carotenoid content, these proteins appear to lose one Chl a molecule, showing that Chl 612 is a Chl a in both complexes as was found before for Lhcb4 [32]. Lhcb6 shows a reduction of the lutein content (presumably Lut 620) upon mutation. Therefore, there is no obvious way for correct normalization of the WT and mutated protein and we assume that one Chl is lost like for Lhcb4 and Lhcb5. It should be noted however, that the conclusions presented below also hold if Lhcb6 loses two Chls. The low-temperature (LT) absorption spectra of the WT and mutated proteins are given in Fig. 2B–D and the difference spectra are shown in Figs. 3 and 6. The lowest-energy state appears to be absent for all mutants. The maximum in the RT difference spectrum is at 680.5–681 nm for Lhcb5 and Lhcb4, and at 679.6 nm for Lhcb6 which shows a broader difference band. The values differ somewhat at low temperature (LT) being 676 nm for Lhcb4 and Lhcb6, and 676.8 nm for Lhcb5. Like for LHCII, the difference spectra for Lhcb4 and Lhcb5 show the loss of a second small absorption band at 660 nm, indicating identical excitonic coupling between Chla 612 and Chla 611. This is confirmed by the CD difference spectra that are very similar to the one
observed for LHCII (Fig. 5). Below 500 nm the absorption difference spectra for Lhcb4 and Lhcb5 are dominated by similar negative bands as observed for LHCII, which are due to the increased absorption of Lut 620 in the mutated protein. Also the CD difference spectra are nearly identical to the one of LHCII with negative peaks at 500, 466 and 438 nm and positive ones at 486 and 404 nm. Finally, the absorption of Lut 620 is blue-shifted upon removal of Chla 612 as evidenced by the positive feature in the absorption difference spectrum near 500 nm (Fig. 3). Since all four fingerprints of excitonic interactions between Lut 620 and Chla 611/612 in LHCII are identical for Lhcb4 and Lhcb5 (both in absorption and CD), it is concluded that the quenching domain in all three complexes is (nearly) identical. It has been shown that induction of qE in vivo is accompanied by a conformational
Fig. 3. Difference spectra of the absorption spectra in Fig. 2 (WT–mutant) of LHCII (solid), Lhcb5 (dashed) and Lhcb4 (dotted). Spectra are normalized to the maximum (the normalization factor is 1 for Lhcb4 and Lhcb5 and 0.82 for Lhcb1).
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Fig. 4. CD spectra of WT and mutant Lhcbs. (A) LHCII-WT (solid) and LHCII-N183L (dashed); (B) Lhcb5-WT (solid) and Lhcb5-N188F (dashed); (C) Lhcb4-WT (solid) and Lhcb4-H216F (dashed); (D) Lhcb6-WT (solid) and Lhcb6-H191F (dashed). Spectra are normalized to the absorption.
change of the neoxanthin [4]. Although most of the neoxanthin is associated to LHCII, it was recently shown [24] that Lhcb4 and Lhcb5 bind neoxanthin in the same site as LHCII. Therefore, the change in neoxanthin conformation that is observed in vivo might also partly be ascribed to neoxanthin present in Lhcb4 and Lhcb5. For Lhcb6 the situation is somewhat different. Whereas the lowenergy band of the Chl pair is at a similar position, the second band is at 670 instead of 660 nm and its intensity is similar to that of the redmost band (Fig. 6), indicating that the interacting Chls have different mutual orientations/positions [26]. The CD difference spectrum confirms the excitonic nature of both bands (Figs. 4D and 6). Although the high-energy band is at a different position than for the other complexes, the overall appearance of the CD difference spectrum is
similar. Because a lutein is lost in the mutated protein, it is not possible to identify the coupling between Chls 611/612 and Lut 620 nm. However, the absorption spectrum of the WT protein also shows redshifted absorption around 500 nm that is lost in the mutated protein (Fig. 6). Although the quenching cluster seems to be present in Lhcb6, its nature is not identical to that of the other three complexes. So far the molecular origin of the quenching is unknown. It was realized before that strong interactions between carotenoids and Chls
Fig. 5. Difference spectra of the CD spectra in Fig. 3 (WT–mutant) of LHCII (solid), Lhcb5 (dashed) and Lhcb4 (dotted). Spectra are normalized to the minimum in the Qy region.
Fig. 6. Difference spectra of Lhcb6 obtained from the spectra in Figs. 2 and 4 (WT– mutant). (A) Absorption; (B) CD.
M. Mozzo et al. / Biochimica et Biophysica Acta 1777 (2008) 1263–1267
in LHCII might lead to a shortening of the Chl fluorescence lifetime [11,33] and it was proposed that excitonic interactions between lutein and Chl a would lead to mixing of their excited states [11]. Since the excited state of lutein is much shorter-lived (~ 10 ps) this will shorten the Chl excited-state lifetime. Here it is shown that strong excitonic coupling indeed occurs, in particular between Lut 620 and the Chls 611/612 pair. An increase of the interaction strength due to a small conformational change could thus account for a decrease of the Chl excited-state lifetime, thereby explaining qE induction. Although the molecular origin of the quenching mechanism still needs further substantiation, it is now clear that excitonic interactions in the quenching cluster of LHCII are identical for Lhcb4 and Lhcb5, whereas they seem to be similar for Lhcb6. We conclude that if Lut 620, Chl 612 and Chl 611 form the quenching cluster in vivo, as was recently found [4], then the minor complexes are most likely also involved in qE via the same molecular mechanism as in LHCII. This can explain a series of data obtained on knock-out and antisense mutants of Lhcbs, which demonstrate that none of these complexes per se is strictly required for quenching, but that removal of all of them leads to the disappearance of NPQ. Acknowledgements This work was supported by the Netherlands Organization for Scientific Research (NWO) – Earth and Life Science (ALW) through a VIDI grant (to RC), by the European Union (grant MRTN-CT-2003-505069, Intro2) (to RC) and the Provincia of Trento (grant SAMBAx2) (to RC and RB). References [1] K.K. Niyogi, Photoprotection revisited: genetic and molecular approaches, Annu. Rev. Plant Physiol. Plant Mol. Biol. 50 (1999) 333–359. [2] X.P. Li, O. Bjorkman, C. Shih, A.R. Grossman, M. Rosenquist, S. Jansson, K.K. Niyogi, A pigment-binding protein essential for regulation of photosynthetic light harvesting, Nature 403 (2000) 391–395. [3] N.E. Holt, D. Zigmantas, L. Valkunas, X.P. Li, K.K. Niyogi, G.R. Fleming, Carotenoid cation formation and the regulation of photosynthetic light harvesting, Science 307 (2005) 433–436. [4] A.V. Ruban, R. Berera, C. Ilioaia, I.H.M. van Stokkum, J.T.M. Kennis, A.A. Pascal, H. van Amerongen, B. Robert, P. Horton, R. van Grondelle, Identification of a mechanism of photoprotective energy dissipation in higher plants, Nature 450 (2007) 575–578. [5] A.A. Pascal, Z.F. Liu, K. Broess, B. van Oort, H. van Amerongen, C. Wang, P. Horton, B. Robert, W.R. Chang, A. Ruban, Molecular basis of photoprotection and control of photosynthetic light-harvesting, Nature 436 (2005) 134–137. [6] Z. Liu, H. Yan, K. Wang, T. Kuang, J. Zhang, L. Gui, X. An, W. Chang, Crystal structure of spinach major light-harvesting complex at 2.72 Å resolution, Nature 428 (2004) 287–292. [7] R. Remelli, C. Varotto, D. Sandona, R. Croce, R. Bassi, Chlorophyll binding to monomeric light-harvesting complex. A mutation analysis of chromophorebinding residues, J. Biol. Chem. 274 (1999) 33510–33521. [8] V.I. Novoderezhkin, M.A. Palacios, H. van Amerongen, R. van Grondelle, Excitation dynamics in the LHCII complex of higher plants: modeling based on the 2.72 angstrom crystal structure, J. Phys. Chem. B 109 (2005) 10493–10504. [9] M. Wentworth, A.V. Ruban, P. Horton, Thermodynamic investigation into the mechanism of the chlorophyll fluorescence quenching in isolated photosystem II light-harvesting complexes, J. Biol. Chem. 278 (2003) 21845–21850. [10] H.C. Yan, P.F. Zhang, C. Wang, Z.F. Liu, W.R. Chang, Two lutein molecules in LHCII have different conformations and functions: insights into the molecular mechanism of thermal dissipation in plants, Biochem. Biophys. Res. Commun. 355 (2007) 457–463.
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Contents lists available at ScienceDirect
Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a b i o
Photoprotection in higher plants: The putative quenching site is conserved in all outer light-harvesting complexes of Photosystem II Milena Mozzo a, Francesca Passarini a, Roberto Bassi b, Herbert van Amerongen c,d, Roberta Croce a,⁎ a
Department of Biophysical Chemistry/Groningen Biotechnology and Biochemical Sciences Institute, University of Groningen, Nijenborgh 4, Groningen, 9747 AG, The Netherlands Dipartimento Scientifico e Tecnologico, Facoltà di Scienze MM.FF.NN., Università di Verona, Strada Le Grazie 15, 37134 Verona, Italy Wageningen University, Laboratory of Biophysics, PO Box 8128, 6700 ET, Wageningen, The Netherlands d MicroSpectroscopy Centre, Wageningen University, 6703 HA Wageningen, The Netherlands b c
a r t i c l e
i n f o
Article history: Received 17 March 2008 Accepted 16 April 2008 Available online 29 April 2008 Keywords: Photosynthesis Photosystem II Light-harvesting complex Photoprotection
a b s t r a c t In bright sunlight, the amount of energy harvested by plants exceeds the electron transport capacity of Photosystem II in the chloroplasts. The excess energy can lead to severe damage of the photosynthetic apparatus and to avoid this, part of the energy is thermally dissipated via a mechanism called nonphotochemical quenching (NPQ). It has been found that LHCII, the major antenna complex of Photosystem II, is involved in this mechanism and it was proposed that its quenching site is formed by the cluster of strongly interacting pigments: chlorophylls 611 and 612 and lutein 620 [A.V. Ruban, R. Berera, C. Ilioaia, I.H.M. van Stokkum, J.T.M. Kennis, A.A. Pascal, H. van Amerongen, B. Robert, P. Horton and R. van Grondelle, Identification of a mechanism of photoprotective energy dissipation in higher plants, Nature 450 (2007) 575– 578.]. In the present work we have investigated the interactions between the pigments in this cluster not only for LHCII, but also for the homologous minor antenna complexes CP24, CP26 and CP29. Use was made of wild-type and mutated reconstituted complexes that were analyzed with (low-temperature) absorption and circular-dichroism spectroscopy as well as by biochemical methods. The pigments show strong interactions that lead to highly specific spectroscopic properties that appear to be identical for LHCII, CP26 and CP29. The interactions are similar but not identical for CP24. It is concluded that if the 611/612/620 domain is responsible for the quenching in LHCII, then all these antenna complexes are prepared to act as a quencher. This can explain the finding that none of the Lhcb complexes seems to be strictly required for NPQ while, in the absence of all of them, NPQ is abolished. © 2008 Elsevier B.V. All rights reserved.
Photosynthetic antenna complexes of higher plants have a dual role: (i) They harvest sunlight, transferring excitation energy to the reaction centre, where it is used to induce charge separation that ultimately leads to the formation of chemical energy but (ii) in full sunlight they rapidly (on a seconds to minute time scale) switch into a non-photochemical quenching (NPQ) state, which safely dissipates excess energy via a mechanism known as qE, thus protecting the system from photodamage [1]. Several mechanisms were proposed to contribute to the quenching [2–4]. It has been demonstrated that the crystal structure of the main light-harvesting complex LHCII corresponds to a quenched state [5], and the pigment cluster consisting of lutein 620 (Lut 620)/Chla 611/Chla 612 (nomenclature as in [6]) (Fig. 1) was identified as a possible quenching site. Recently it was found that LHCII is in a similar quenched state when it is aggregated in vitro but more important when qE is induced in vivo [4]. In LHCII aggregates, Lut 620 was shown to be the quencher, whereas Chla 611 and Chla 612 function as a site for excitation localization, i.e. at equilibrium most of
Abbreviations: Chl, chlorophyll; Lhc, light-harvesting complex ⁎ Corresponding author. Tel.: +31 3634214; fax: +31 3634800. E-mail address: [email protected] (R. Croce). 0005-2728/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbabio.2008.04.036
the excitation energy is localized on these Chls [7,8] making them a preferential site for energy dissipation. Switching between the lightharvesting and quenched state is ascribed to a change in interaction between Lut 620 and Chla 611/Chla 612 [4,5,9,10]. Because Lut 620 is in contact with Chla 612 [6] which in turn is close to Chl 611, it was assumed that all three pigments are strongly excitonically coupled [10–13]. A change in interaction upon a conformational switch could then lead to quenching. However, experimental characterization of the mutual interactions is lacking. Although most research on NPQ focuses on LHCII, evidence exists that also the minor antenna complexes, being Lhcb4 (CP29), Lhcb5 (CP26) and Lhcb6 (CP24) might act as quenchers [14–18]. Analysis of mutant plants lacking the individual complexes Lhcb4, Lhcb5 or Lhcb1/2 has shown that none of them is strictly required for NPQ [19,20] although in the absence of all of them NPQ is nearly absent [21]. Only the mutant lacking Lhcb6 is strongly affected in NPQ [22] but this effect is ascribed to a limitation of proton pumping (De Bianchi et al., submitted). These data strongly suggest that more than one Lhcb complex contributes to NPQ, in line with the proposal that multiple quenching sites are distributed throughout Photosystem II (PSII) [23].
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M. Mozzo et al. / Biochimica et Biophysica Acta 1777 (2008) 1263–1267 1.2. Spectroscopy The absorption spectra at RT and 77 K were recorded using a SLM-Aminco DK2000 spectrophotometer, in 10 mM Hepes pH 7.5, 0.2 M sucrose (70% v/v glycerol at 77 K) and 0.06% n-Dodecyl-beta-D-maltoside. The wavelength-sampling step was 0.4 nm, scan rate 100 nm/min, optical pathlength 1 cm. The CD spectra were measured at 10 °C on an AVIV 62ADS or a J600 spectropolarimeter. The wavelength-sampling step was 0.5 nm with an average time of 1 s. Before subtraction, the absorption spectra were normalized to the pigment content on the basis of the area in the 630–750 nm region, using a ratio of 0.7 between the extinction coefficients of Chl b and Chl a.
2. Results and discussion
Fig. 1. Structure of the LHCII monomer [6] showing the organization of Chla 612 (dark green), Chla 611 (light green) and Lut 620 (yellow).
In aggregated LHCII the fluorescence is quenched by Lut 620 and switching to this quenched state is accompanied by the same structural change that occurs upon qE induction in vivo as can be monitored via characteristic changes in the Raman spectrum of neoxanthin [4]. However, also aggregation of the minor antenna complexes leads to fluorescence quenching and since neoxanthin is not only associated with LHCII but is also bound to Lhcb4 and Lhcb5 [24], the observed changes in vivo might also be attributed to the minor antenna complexes. Due to the high structural homology between these complexes, it might be expected that the quenching mechanism is similar in all cases. If Chla 611/Chla 612/Lut 620 would indeed form the quenching site in all complexes, this would require a highly similar pigment organization with highly similar mutual interactions. However, experimental proof for such similarity is lacking. Light-spectroscopic techniques can be very sensitive tools to probe pigment–pigment interactions and the local environment of pigments in light-harvesting complexes (Lhcs) [25]. For instance, the location of the absorption maximum of Chl a can vary from 660 to 710 nm in Lhc proteins. In particular, strong interactions between nearby chromophores play an important role in tuning the spectroscopic properties and thus the function [26]. Since it was proposed that Chla 612 interacts strongly with Chla 611 and Lut 620 it might thus be expected that a small structural change will cause major spectroscopic changes. In order to study the excitonic interactions between Lut 620, Chla 611 and Chla 612 and compare them for all complexes, we have performed mutations of the putative Chl 612 ligand in all outer antenna complexes of PSII and compared in detail the biochemical composition and the absorption and circular-dichroism (CD) spectra.
The wild-type (WT) apoprotein of Lhcb1, the main constituent of LHCII, was overexpressed and reconstituted as described before [7]. The same was done under identical conditions for the mutated apoprotein (Lhcb1-N183L), which lacks the ligand for Chla 612. As expected, a lower Chl a/Chlb ratio is found for the mutated protein (Table 1) in agreement with the loss of Chl a. In both proteins the relative amounts of neoxanthin (Neo), violaxanthin (Vio) and lutein are identical (0.8: 0.2: 2.0, Table 1), consistent with the fact that the carotenoid (Car) composition remains unaltered. This allows normalization to the carotenoid content. Taking 12 Chls for WT reconstituted protein (see [7]) it is found that the mutated complex lacks 1.4 Chl a and 0.6 Chl b, in agreement with previous results [7], where the difference was ascribed to the loss of Chls 612 and 611. The binding site for Chl 611 was concluded to have mixed occupancy, i.e. it can bind both Chl a and Chl b [7]. 77 K absorption spectra of both proteins are compared in Fig. 2A after scaling to the pigment content. The difference spectrum (Fig. 3) shows the loss of a major band at 678 nm (681 nm at room temperature (RT)) and a minor one at 660 nm for the mutated protein. These bands are due to two excitonically coupled Chls (Chl611 and Chl612) since the difference in the RT CD spectra of both proteins (Figs. 4A and 5) shows the loss of a negative CD band near 681 nm and a positive one near 660 nm. The 681 nm state is the lowest-energy state and acts as a preferential site for excitation localization. Remarkably, the loss of both bands is accompanied by a strong increase of carotenoid absorption (Fig. 3). Since Lut 620 is the only xanthophyll close to Chl 611/Chl 612, this increase is ascribed to a loss of excitonic interaction(s) between Lut 620 and Chl 611/Chl 612. Apparently, this interaction leads to substantial hypochromism (loss of absorption) for Lut 620 which must be accompanied by prominent hyperchromism (gain of absorption) for Chl a. This offers an explanation for an intriguing feature observed in triplet-minus-singlet spectra of LHCII [29,30]. The presence of a triplet on a carotenoid later identified [31] as L1 (Lut 620) leads to decreased Chl a Qy absorption. Presumably, strong excitonic interactions between Lut 620 and Chl 612/Chl 611 lead to hyperchromism in the Qy region. Upon triplet formation on Lut 620 the excitonic interaction is broken, reducing the hyperchromism and thus decreasing Chl absorption. Excitonic interaction probably also occurs between the main Lut 620 transition and the Chl Bx transition (near 437 nm). Disappearance of this interaction
1. Materials and methods 1.1. Mutagenesis and in vitro reconstitution Mature sequences of Lhcb1 and Lhcb4 from Zea mays and Lhcb5 from Arabidopsis thaliana were amplified from cDNA by PCR and cloned in pQE50 His (home modified pQE50, Qiagen); Lhcb6 from A. thaliana was cloned in pETMHis (home modified pET28 (a)+, Novagen). Mutant sequences were obtained by site directed mutagenesis of the Chl 612 ligand residues: asparagine 183 was mutated to leucine in Lhcb1, histidine 216 to phenylalanine in Lhcb4, asparagine 188 to phenylalanine in Lhcb5 and histidine 191 to phenylalanine in Lhcb6. WT and mutant apoproteins were overexpressed in SG13009 and Rosetta2 DE3 (pLysS) strains of Escherichia coli and purified as inclusion bodies. Reconstitution and purification of pigment–protein complexes were performed as described in [27] using the following Chl a/b ratios in the reconstitution mixture: 2.4 for Lhcb1, 4.0 for Lhcb4 and 3.0 for Lhcb5–6. The pigment composition was analyzed by HPLC and fitting of the absorption spectra of the acetone extracts as in [28]. Pigment composition, absorption and circular-dichroism spectra were fully reproducible for all samples obtained after all reconstitutions.
Table 1 Pigment composition of the complexes Sample
Chl a/b
Chl/Car
Neo
Vio
Lut
Chl tot
Chl a
Chl b
Car tot
Lhcb1-WT Lhcb1-N183L Lhcb4-WT Lhcb4-H216F Lhcb5-WT Lhcb5-N188F Lhcb6-WT Lhcb6-H191F
1.36 1.15 2.36 1.9 2.01 1.72 1.0 0.9
4.2 3.6 4.1 3.7 3.5 3.1 5.1 5.9
0.96 0.93 0.77 0.75 0.92 1.0 – –
0.18 0.16 0.42 0.44 0.18 0.06 0.96 1.05
1.71 1.65 0.86 0.81 1.73 1.86 0.99 0.38
12 10 8 7 10 9 10 9
6.9 5.4 5.6 4.6 6.7 5.7 5.0 4.3
5.1 4.6 2.4 2.4 3.3 3.3 5.0 4.7
2.9 2.8 2.0 1.9 2.8 2.9 2.0 1.5
The data are the average of 2 measurements on 2 different samples for Lhcb4 and Lhcb5 and on 4 samples for LHCII and Lhcb6. The maximum error is less than 0.1.
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Fig. 2. Absorption spectra at 77 K of WT and mutant Lhcbs. (A) Lhcb1-WT (solid) and Lhcb1-N183L (dashed); (B) Lhcb5-WT (solid) and Lhcb5-N188F (dashed); (C) Lhcb4-WT (solid) and Lhcb4-H216F (dashed); (D) Lhcb6-WT (solid) and Lhcb6-H191F (dashed). Spectra are normalized to Chl content.
causes a blue shift of lutein absorption and this explains the positive feature near 500 nm in the difference spectrum (Fig. 3). Summarizing, we have identified four fingerprints for the strong excitonic interactions in the quenching site of LHCII between Lut 620 and Chl 611/Chl 612: (i) strong hypochromism of Lut 620, (ii) redshifted absorption of Lut 620, (iii) a weak excitonic band at 660 nm and (iv) a strong excitonic band at 681 nm, accompanied by very specific features in the CD spectrum. Excitations tend to be localized in the low-energy state (681 nm) where they can be quenched by Lut 620 upon induction of qE or in the case of aggregation. In order to investigate the presence of an “LHCII-like” quenching site in the minor antenna complexes, the putative ligand for Chl 612 was also mutated in Lhcb4, Lhcb5 and Lhcb6 and the WT and mutated proteins were reconstituted under identical conditions. All mutated proteins show a lower Chl a/b ratio than the corresponding WT proteins, again indicating the loss of Chl a (Table 1). The Car composition (N:V:L) is the same for WT and mutant protein in the case of Lhcb4 and Lhcb5. Normalizing the WT and mutated proteins to the same carotenoid content, these proteins appear to lose one Chl a molecule, showing that Chl 612 is a Chl a in both complexes as was found before for Lhcb4 [32]. Lhcb6 shows a reduction of the lutein content (presumably Lut 620) upon mutation. Therefore, there is no obvious way for correct normalization of the WT and mutated protein and we assume that one Chl is lost like for Lhcb4 and Lhcb5. It should be noted however, that the conclusions presented below also hold if Lhcb6 loses two Chls. The low-temperature (LT) absorption spectra of the WT and mutated proteins are given in Fig. 2B–D and the difference spectra are shown in Figs. 3 and 6. The lowest-energy state appears to be absent for all mutants. The maximum in the RT difference spectrum is at 680.5–681 nm for Lhcb5 and Lhcb4, and at 679.6 nm for Lhcb6 which shows a broader difference band. The values differ somewhat at low temperature (LT) being 676 nm for Lhcb4 and Lhcb6, and 676.8 nm for Lhcb5. Like for LHCII, the difference spectra for Lhcb4 and Lhcb5 show the loss of a second small absorption band at 660 nm, indicating identical excitonic coupling between Chla 612 and Chla 611. This is confirmed by the CD difference spectra that are very similar to the one
observed for LHCII (Fig. 5). Below 500 nm the absorption difference spectra for Lhcb4 and Lhcb5 are dominated by similar negative bands as observed for LHCII, which are due to the increased absorption of Lut 620 in the mutated protein. Also the CD difference spectra are nearly identical to the one of LHCII with negative peaks at 500, 466 and 438 nm and positive ones at 486 and 404 nm. Finally, the absorption of Lut 620 is blue-shifted upon removal of Chla 612 as evidenced by the positive feature in the absorption difference spectrum near 500 nm (Fig. 3). Since all four fingerprints of excitonic interactions between Lut 620 and Chla 611/612 in LHCII are identical for Lhcb4 and Lhcb5 (both in absorption and CD), it is concluded that the quenching domain in all three complexes is (nearly) identical. It has been shown that induction of qE in vivo is accompanied by a conformational
Fig. 3. Difference spectra of the absorption spectra in Fig. 2 (WT–mutant) of LHCII (solid), Lhcb5 (dashed) and Lhcb4 (dotted). Spectra are normalized to the maximum (the normalization factor is 1 for Lhcb4 and Lhcb5 and 0.82 for Lhcb1).
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Fig. 4. CD spectra of WT and mutant Lhcbs. (A) LHCII-WT (solid) and LHCII-N183L (dashed); (B) Lhcb5-WT (solid) and Lhcb5-N188F (dashed); (C) Lhcb4-WT (solid) and Lhcb4-H216F (dashed); (D) Lhcb6-WT (solid) and Lhcb6-H191F (dashed). Spectra are normalized to the absorption.
change of the neoxanthin [4]. Although most of the neoxanthin is associated to LHCII, it was recently shown [24] that Lhcb4 and Lhcb5 bind neoxanthin in the same site as LHCII. Therefore, the change in neoxanthin conformation that is observed in vivo might also partly be ascribed to neoxanthin present in Lhcb4 and Lhcb5. For Lhcb6 the situation is somewhat different. Whereas the lowenergy band of the Chl pair is at a similar position, the second band is at 670 instead of 660 nm and its intensity is similar to that of the redmost band (Fig. 6), indicating that the interacting Chls have different mutual orientations/positions [26]. The CD difference spectrum confirms the excitonic nature of both bands (Figs. 4D and 6). Although the high-energy band is at a different position than for the other complexes, the overall appearance of the CD difference spectrum is
similar. Because a lutein is lost in the mutated protein, it is not possible to identify the coupling between Chls 611/612 and Lut 620 nm. However, the absorption spectrum of the WT protein also shows redshifted absorption around 500 nm that is lost in the mutated protein (Fig. 6). Although the quenching cluster seems to be present in Lhcb6, its nature is not identical to that of the other three complexes. So far the molecular origin of the quenching is unknown. It was realized before that strong interactions between carotenoids and Chls
Fig. 5. Difference spectra of the CD spectra in Fig. 3 (WT–mutant) of LHCII (solid), Lhcb5 (dashed) and Lhcb4 (dotted). Spectra are normalized to the minimum in the Qy region.
Fig. 6. Difference spectra of Lhcb6 obtained from the spectra in Figs. 2 and 4 (WT– mutant). (A) Absorption; (B) CD.
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in LHCII might lead to a shortening of the Chl fluorescence lifetime [11,33] and it was proposed that excitonic interactions between lutein and Chl a would lead to mixing of their excited states [11]. Since the excited state of lutein is much shorter-lived (~ 10 ps) this will shorten the Chl excited-state lifetime. Here it is shown that strong excitonic coupling indeed occurs, in particular between Lut 620 and the Chls 611/612 pair. An increase of the interaction strength due to a small conformational change could thus account for a decrease of the Chl excited-state lifetime, thereby explaining qE induction. Although the molecular origin of the quenching mechanism still needs further substantiation, it is now clear that excitonic interactions in the quenching cluster of LHCII are identical for Lhcb4 and Lhcb5, whereas they seem to be similar for Lhcb6. We conclude that if Lut 620, Chl 612 and Chl 611 form the quenching cluster in vivo, as was recently found [4], then the minor complexes are most likely also involved in qE via the same molecular mechanism as in LHCII. This can explain a series of data obtained on knock-out and antisense mutants of Lhcbs, which demonstrate that none of these complexes per se is strictly required for quenching, but that removal of all of them leads to the disappearance of NPQ. Acknowledgements This work was supported by the Netherlands Organization for Scientific Research (NWO) – Earth and Life Science (ALW) through a VIDI grant (to RC), by the European Union (grant MRTN-CT-2003-505069, Intro2) (to RC) and the Provincia of Trento (grant SAMBAx2) (to RC and RB). References [1] K.K. Niyogi, Photoprotection revisited: genetic and molecular approaches, Annu. Rev. Plant Physiol. Plant Mol. Biol. 50 (1999) 333–359. [2] X.P. Li, O. Bjorkman, C. Shih, A.R. Grossman, M. Rosenquist, S. Jansson, K.K. Niyogi, A pigment-binding protein essential for regulation of photosynthetic light harvesting, Nature 403 (2000) 391–395. [3] N.E. Holt, D. Zigmantas, L. Valkunas, X.P. Li, K.K. Niyogi, G.R. Fleming, Carotenoid cation formation and the regulation of photosynthetic light harvesting, Science 307 (2005) 433–436. [4] A.V. Ruban, R. Berera, C. Ilioaia, I.H.M. van Stokkum, J.T.M. Kennis, A.A. Pascal, H. van Amerongen, B. Robert, P. Horton, R. van Grondelle, Identification of a mechanism of photoprotective energy dissipation in higher plants, Nature 450 (2007) 575–578. [5] A.A. Pascal, Z.F. Liu, K. Broess, B. van Oort, H. van Amerongen, C. Wang, P. Horton, B. Robert, W.R. Chang, A. Ruban, Molecular basis of photoprotection and control of photosynthetic light-harvesting, Nature 436 (2005) 134–137. [6] Z. Liu, H. Yan, K. Wang, T. Kuang, J. Zhang, L. Gui, X. An, W. Chang, Crystal structure of spinach major light-harvesting complex at 2.72 Å resolution, Nature 428 (2004) 287–292. [7] R. Remelli, C. Varotto, D. Sandona, R. Croce, R. Bassi, Chlorophyll binding to monomeric light-harvesting complex. A mutation analysis of chromophorebinding residues, J. Biol. Chem. 274 (1999) 33510–33521. [8] V.I. Novoderezhkin, M.A. Palacios, H. van Amerongen, R. van Grondelle, Excitation dynamics in the LHCII complex of higher plants: modeling based on the 2.72 angstrom crystal structure, J. Phys. Chem. B 109 (2005) 10493–10504. [9] M. Wentworth, A.V. Ruban, P. Horton, Thermodynamic investigation into the mechanism of the chlorophyll fluorescence quenching in isolated photosystem II light-harvesting complexes, J. Biol. Chem. 278 (2003) 21845–21850. [10] H.C. Yan, P.F. Zhang, C. Wang, Z.F. Liu, W.R. Chang, Two lutein molecules in LHCII have different conformations and functions: insights into the molecular mechanism of thermal dissipation in plants, Biochem. Biophys. Res. Commun. 355 (2007) 457–463.
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