Carotenoid-binding sites of the major light-harvesting complex II of higher plants.

Recombinant light-harvesting complex II (LHCII) proteins with modified carotenoid composition have been obtained by in vitro reconstitution of the Lhcb1 protein overexpressed in bacteria. The monomeric protein possesses three xanthophyll-binding sites. The L1 and L2 sites, localized by electron crystallography in the helix A/helix B cross, have the highest affinity for lutein, but also bind violaxanthin and zeaxanthin with lower affinity. The latter xanthophyll causes disruption of excitation energy transfer. The occupancy of at least one of these sites, probably L1, is essential for protein folding. Neoxanthin is bound to a distinct site (N1) that is highly selective for this species and whose occupancy is not essential for protein folding. Whereas xanthophylls in the L1 and L2 sites interact mainly with chlorophyll a, neoxanthin shows strong interaction with chlorophyll b, inducing the hyperchromic effect of the 652 nm absorption band. This observation explains the recent results of energy transfer from carotenoids to chlorophyll b obtained by femtosecond absorption spectroscopy. Whereas xanthophylls in the L1 and L2 sites are active in photoprotection through chlorophyll-triplet quenching, neoxanthin seems to act mainly in (1)O(2)(*) scavenging.     

The functional significance of the monomeric and trimeric states of the photosystem II light harvesting complexes

The main light harvesting complex of photosystem II in plants, LHCII, exists in a trimeric state. To understand the biological significance of trimerization, a comparison has been made been LHCII trimers and LHCII monomers prepared by treatment with phospholipase. The treatment used caused no loss of chlorophyll, but there was a difference in carotenoid composition, together with the previously observed alterations in absorption spectrum. It was found that, when compared to monomers, LHCII trimers showed increased thermal stability and a reduced structural flexibility as determined by the decreased rate and amplitude of fluorescence quenching in low-detergent concentration. It is suggested that LHCII should be considered as having two interacting domains: the lutein 1 domain, the site of fluorescence quenching [Wentworth et al. (2003) J. Biol. Chem. 278, 21845-21850], and the lutein 2 domain. The lutein 2 domain faces the interior of the trimer, the differences in absorption spectrum and carotenoid binding in trimers compared to monomers indicating that the trimeric state modulates the conformation of this domain. It is suggested that the lutein 2 domain controls the conformation of the lutein 1 domain, thereby providing allosteric control of fluorescence quenching in LHCII. Thus, the pigment configuration and protein conformation in trimers is adapted for efficient light harvesting and enhanced protein stability. Furthermore, trimers exhibit the optimum level of control of energy dissipation by modulating the development of the quenched state of the complex.     

Singlet and triplet state transitions of carotenoids in the antenna complexes of higher-plant photosystem I

In this work, the spectroscopic characteristics of carotenoids associated with the antenna complexes of Photosystem I have been studied. Pigment composition, absorption spectra, and laser-induced triplet-minus-singlet (T-S) spectra were determined for native LHCI from the wild type (WT) and lut2 mutant from Arabidopsis thaliana as well as for reconstituted individual Lhca WT and mutated complexes. All WT complexes bind lutein and violaxanthin, while beta-carotene was found to be associated only with the native LHCI preparation and recombinant Lhca3. In the native complexes, the main lutein absorption bands are located at 492 and 510 nm. It is shown that violaxanthin is able to occupy all lutein binding sites, but its absorption is blue-shifted to 487 and 501 nm. The "red" lutein absorbing at 510 nm was found to be associated with Lhca3 and Lhca4 which also show a second carotenoid, peaking around 490 nm. Both these xanthophylls are involved in triplet quenching and show two T-S maxima: one at 507 nm (corresponding to the 490 nm singlet absorption) and the second at 525 nm (with absorption at 510 nm). The "blue"-absorbing xanthophyll is located in site L1 and can receive triplets from chlorophylls (Chl) 1012, 1011, and possibly 1013. The red-shifted spectral component is assigned to a lutein molecule located in the L2 site. A 510 nm lutein was also observed in the trimers of LHCII but was absent in the monomers. In the case of Lhca, the 510 nm band is present in both the monomeric and dimeric complexes. We suggest that the large red shift observed for this xanthophyll is due to interaction with the neighbor Chl 1015. In the native T-S spectrum, the contribution of carotenoids associated with Lhca2 is visible while the one of Lhca1 is not. This suggests that in the Lhca2-Lhca3 heterodimeric complex energy equilibration is not complete at least on a fast time scale.     

Aggregation of LHCII Leads to a Redistribution of the Triplets over the Central Xanthophylls in LHCII

We present laser flash-induced triplet-minus-singlet (TmS(flash)) and absorbance-detected-magnetic-resonance (TmS(ADMR)) measurements on the light-harvesting chlorophyll a/b pigment-protein complex (LHCII) from pea. We investigated the influence of LHCII aggregation on xanthophyll triplet formation. The effect of aggregation was previously studied using TmS(ADMR) [van der Vos et al. (1994) Biochim. Biophys. Acta 1208, 243-250] for LHCII from spinach, and it was concluded that aggregation leads to a large increase of the amount of intertrimer triplet transfer. However, a similar study on LHCII from pea with the use of TmS(flash) measurements [Barzda et al. (1998) Biochemistry 37, 546-561] showed much smaller effects. To resolve this apparent discrepancy and to compare the results of TmS(ADMR) and TmS(flash) measurements, we used both techniques to study LHCII from pea, applying an identical aggregation procedure in both cases. It appears that aggregation does not lead to an increase of intertrimer triplet transfer as thought before but to a redistribution of the triplets over the two central xanthophylls (mainly lutein) that are present in each monomeric subunit of LHCII. Moreover, it is argued that the TmS band at 525 nm is due to lutein instead of violaxanthin as was reported in earlier studies. It is concluded that aggregation leads to a change in chlorophyll-xanthophyll interactions, which might explain the large change in excited-state lifetime of chlorophyll a in LHCII upon aggregation. This change in lifetime is possibly related to the phenomenon of nonphotochemical quenching in green plants, which is an important protective regulatory mechanism, that lowers the probability of photoinhibition.     

Time-resolved fluorescence analysis of the photosystem II antenna proteins in detergent micelles and liposomes

We have studied the time-resolved fluorescence properties of the light-harvesting complexes (Lhc) of photosystem II (Lhcb) in order to obtain information on the mechanism of energy dissipation (non-photochemical quenching) which is correlated to the conversion of violaxanthin to zeaxanthin in excess light conditions. The chlorophyll fluorescence decay of Lhcb proteins LHCII, CP29, CP26, and CP24 in detergent solution is mostly determined by two lifetime components of 1.2-1.5 and 3.6-4 ns while the contribution of the faster component is higher in CP29, CP26, and CP24 with respect to LHCII. The xanthophyll composition of Lhc proteins affects the ratio of the lifetime components: when zeaxanthin is bound into the site L2 of LHCII, the relative amplitude of the faster component is increased and, consequently, the chlorophyll fluorescence quenching is enhanced. Analysis of quenching in mutants of Arabidopsis thaliana, which incorporate either violaxanthin or zeaxanthin in their Lhc proteins, shows that the extent of quenching is enhanced in the presence of zeaxanthin. The origin of the two fluorescence lifetimes was analyzed by their temperature dependence: since lifetime heterogeneity was not affected by cooling to 77 K, it is concluded that each lifetime component corresponds to a distinct conformation of the Lhc proteins. Upon incorporation of Lhc proteins into liposomes, a quenching of chlorophyll fluorescence was observed due to shortening of all their lifetime components: this indicates that the equilibrium between the two conformations of Lhcb proteins is displaced toward the quenched conformation in lipid membranes or thylakoids with respect to detergent solution. By increasing the protein density in the liposomes, and therefore the probability of protein-protein interactions, a further decrease of fluorescence lifetimes takes place down to values typical of quenched leaves. We conclude that at least two major factors determine the quenching of chlorophyll fluorescence in Lhcb proteins, i.e., intrasubunit conformational change and intersubunit interactions within the lipid membranes, and that these processes are both important in the photoprotection mechanism of nonphotochemical quenching in vivo.     

Photoprotection in plants involves a change in lutein 1 binding domain in the major light-harvesting complex of photosystem II

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).     

Identification of the Chromophores Involved in Aggregation-dependent Energy Quenching of the Monomeric Photosystem II Antenna Protein Lhcb5

Non-photochemical quenching (NPQ) of excess absorbed light energy is a fundamental process that regulates photosynthetic light harvesting in higher plants. Among several proposed NPQ mechanisms, aggregation-dependent quenching (ADQ) and charge transfer quenching have received the most attention. In vitro spectroscopic features of both mechanisms correlate with very similar signals detected in more intact systems and in vivo, where full NPQ can be observed. A major difference between the models is the proposed quenching site, which is predominantly the major trimeric light-harvesting complex II in ADQ and exclusively monomeric Lhcb proteins in charge transfer quenching. Here, we studied ADQ in both monomeric and trimeric Lhcb proteins, investigating the activities of each antenna subunit and their dependence on zeaxanthin, a major modulator of NPQ in vivo. We found that monomeric Lhcb proteins undergo stronger quenching than light-harvesting complex II during aggregation and that this is enhanced by binding to zeaxanthin, as occurs during NPQ in vivo. Finally, the analysis of Lhcb5 mutants showed that chlorophyll 612 and 613, in close contact with lutein bound at site L1, are important facilitators of ADQ.     

A look within LHCII: differential analysis of the Lhcb1-3 complexes building the major trimeric antenna complex of higher-plant photosynthesis

The major antenna complex of higher-plant photosynthesis, LHCII, is composed by the products of three genes, namely, Lhcb1-2-3. In this paper, the biochemical and spectroscopic properties of each of the three gene products were investigated. The three complexes were obtained by overexpression of the apoproteins in bacteria and refolding in vitro with purified pigments, thus allowing detection of differences in the structure/function of the pigment-binding gene products. The analyses showed that Lhcb1 and Lhcb2 complexes have similar pigment binding properties, although not identical, while Lhcb3 is clearly different with respect to both pigment binding and spectral properties and cannot produce homotrimers in vitro. Heterotrimers containing Lhcb3 together with Lhcb1 and/or -2 proteins were obtained upon assembly with Lhcb proteins purified from thylakoids. The major functional characteristics of Lhcb3 with respect to Lhcb1 and -2 consisted in (i) a red-shift of one specific chlorophyll a chromophore, strongly affecting the red-most region of the absorption spectrum and (ii) a different specificity for xanthophylls binding to sites L2 and N1. These properties make Lhcb3 a relative sink for excitation energy in isolated heterotrimers with Lhcb1 + Lhcb2, and potentially, a preferential site of regulation of the antenna function in excess light conditions.     

Elevated ΔpH restores rapidly reversible photoprotective energy dissipation in <Emphasis Type="Italic">Arabidopsis</Emphasis> chloroplasts deficient in lutein and xanthophyll cycle activity

The xanthophylls of the light-harvesting complexes of photosystem II (LHCII), zeaxanthin, and lutein are thought to be essential for non-photochemical quenching (NPQ). NPQ is a process of photoprotective energy dissipation in photosystem II (PSII). The major rapidly reversible component of NPQ, qE, is activated by the transmembrane proton gradient, and involves the quenching of antenna chlorophyll excited states by the xanthophylls lutein and zeaxanthin. Using diaminodurene (DAD), a mediator of cyclic electron flow around photosystem I, to enhance ΔpH we demonstrate that qE can still be formed in the absence of lutein and light-induced formation of zeaxanthin in chloroplasts derived from the normally qE-deficient lut2npq1 mutant of Arabidopsis. The qE induced by high ΔpH in lut2npq1 chloroplasts quenched the level of fluorescence when all PSII reaction centers were in the open state (F _o state), protected PSII reaction centers from photoinhibition, was sensitive to the uncoupler nigericin, and was accompanied by absorption changes in the 410–565 nm region. Titrations show the ΔpH threshold for activation of qE in lut2npq1 chloroplasts lies outside the normal physiological range and is highly cooperative. Comparison of quenching in isolated trimeric (LHCII) and monomeric (CP26) light-harvesting complexes from lut2npq1 plants revealed a similarly shifted pH dependency compared with wild-type LHCII. The implications for the roles of lutein and zeaxanthin as direct quenchers of excitation energy are discussed. Furthermore, we argue that the control over the proton-antenna association constant, pK, occurs via influence of xanthophyll structure on the interconnected phenomena of light-harvesting antenna reorganization/aggregation and hydrophobicity.     

Light-harvesting complexes of <Emphasis Type="Italic">Botryococcus braunii</Emphasis>

The colonial green alga Botryococcus braunii (BB) is a potential source of biofuel due to its natural high hydrocarbon content. Unfortunately, its slow growth limits its biotechnological potential. Understanding its photosynthetic machinery could help to identify possible growth limitations. Here, we present the first study on BB light-harvesting complexes (LHCs). We purified two LHC fractions containing the complexes in monomeric and trimeric form. Both fractions contained at least two proteins with molecular weight (MW) around 25 kDa. The chlorophyll composition is similar to that of the LHCII of plants; in contrast, the main xanthophyll is loroxanthin, which substitutes lutein in most binding sites. Circular dichroism and 77 K absorption spectra lack typical differences between monomeric and trimeric complexes, suggesting that intermonomer interactions do not play a role in BB LHCs. This is in agreement with the low stability of the BB LHCII trimers as compared to the complexes of plants, which could be related to loroxanthin binding in the central (L1 and L2) binding sites. The properties of BB LHCII are similar to those of plant LHCII, indicating a similar pigment organization. Differences are a higher content of red chlorophyll a, similar to plant Lhcb3. These differences and the different Xan composition had no effect on excitation energy transfer or fluorescence lifetimes, which were similar to plant LHCII.     

Carotenoid S(1) state in a recombinant light-harvesting complex of Photosystem II.

The carotenoid species lutein, violaxanthin, and zeaxanthin are crucial in the xanthophyll-dependent nonphotochemical quenching occurring in photosynthetic systems of higher plants, since they are involved in dissipation of excess energy and thus protect the photosynthetic machinery from irreversible inhibition. Nonetheless, important properties of the xanthophyll cycle carotenoids, such as the energy of their S(1) electronic states, are difficult to study and were only recently determined in organic solvents [Polívka, T. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 4914. Frank, H. A. (2000) Biochemistry 39, 2831]. In the present study, we have determined the S(1) energies of three carotenoid species, violaxanthin, lutein, and zeaxanthin, in their LHCII (peripheral light-harvesting complex of photosystem II) protein environment by constructing recombinant Lhcb1 (Lhc = light-harvesting complex) proteins containing single carotenoid species. Within experimental error the S(1) energy is the same for all three carotenoids in the monomeric LHCII, 13,900 +/- 300 cm(-1) (720 +/- 15 nm), thus well below the Q(y)() transitions of chlorophylls. In addition, we have found that, although the S(1) lifetimes of violaxanthin, lutein, and zeaxanthin differ substantially in solution, when incorporated into the LHCII protein, their S(1) states have in fact the same lifetime of about 11 ps. Despite the similar spectroscopic properties of the carotenoids bound to the LHCII, we observed a maximal fluorescence quenching when zeaxanthin was present in the LHCII complex. On the basis of these observations, we suggest that, rather than different photochemical properties of individual carotenoid species, changes in the protein conformation induced by binding of carotenoids with distinct molecular structures are involved in the quenching phenomena associated with Lhc proteins.     

Different roles of alpha- and beta-branch xanthophylls in photosystem assembly and photoprotection

Xanthophylls (oxygenated carotenoids) are essential components of the plant photosynthetic apparatus, where they act in photosystem assembly, light harvesting, and photoprotection. Nevertheless, the specific function of individual xanthophyll species awaits complete elucidation. In this work, we analyze the photosynthetic phenotypes of two newly isolated Arabidopsis mutants in carotenoid biosynthesis containing exclusively alpha-branch (chy1chy2lut5) or beta-branch (chy1chy2lut2) xanthophylls. Both mutants show complete lack of qE, the rapidly reversible component of nonphotochemical quenching, and high levels of photoinhibition and lipid peroxidation under photooxidative stress. Both mutants are much more photosensitive than npq1lut2, which contains high levels of viola- and neoxanthin and a higher stoichiometry of light-harvesting proteins with respect to photosystem II core complexes, suggesting that the content in light-harvesting complexes plays an important role in photoprotection. In addition, chy1chy2lut5, which has lutein as the only xanthophyll, shows unprecedented photosensitivity even in low light conditions, reduced electron transport rate, enhanced photobleaching of isolated LHCII complexes, and a selective loss of CP26 with respect to chy1chy2lut2, highlighting a specific role of beta-branch xanthophylls in photoprotection and in qE mechanism. The stronger photosystem II photoinhibition of both mutants correlates with the higher rate of singlet oxygen production from thylakoids and isolated light-harvesting complexes, whereas carotenoid composition of photosystem II core complex was not influential. In depth analysis of the mutant phenotypes suggests that alpha-branch (lutein) and beta-branch (zeaxanthin, violaxanthin, and neoxanthin) xanthophylls have distinct and complementary roles in antenna protein assembly and in the mechanisms of photoprotection.     

The inter-monomer interface of the major light-harvesting chlorophyll a/b complexes of photosystem II (LHCII) influences the chlorophyll triplet distribution

Under strong light conditions, long-lived chlorophyll triplets (³Chls) are formed, which can sensitize singlet oxygen, a species harmful to the photosynthetic apparatus of plants. Plants have developed multiple photoprotective mechanisms to quench ³Chl and scavenge singlet oxygen in order to sustain the photosynthetic activities. The lumenal loop of light-harvesting chlorophyll a/b complex of photosystem II (LHCII) plays important roles in regulating the pigment conformation and energy dissipation. In this study, site-directed mutagenesis analysis was applied to investigate triplet–triplet energy transfer and quenching of ³Chl in LHCII. We mutated the amino acid at site 123 located in this region to Gly, Pro, Gln, Thr and Tyr, respectively, and recorded fluorescence excitation spectra, triplet-minus-singlet (TmS) spectra and kinetics of carotenoid triplet decay for wild type and all the mutants. A red-shift was evident in the TmS spectra of the mutants S123T and S123P, and all of the mutants except S123Y showed a decrease in the triplet energy transfer efficiency. We propose, on the basis of the available structural information, that these phenomena are related to the involvement, due to conformational changes in the lumenal region, of a long-wavelength lutein (Lut2) involved in quenching ³Chl.     

The Zeaxanthin-Independent and Zeaxanthin-Dependent qE Components of Nonphotochemical Quenching Involve Common Conformational Changes within the Photosystem II Antenna in Arabidopsis

The light-harvesting antenna of higher plant photosystem II (LHCII) has the intrinsic capacity to dissipate excess light energy as heat in a process termed nonphotochemical quenching (NPQ). Recent studies suggest that zeaxanthin and lutein both contribute to the rapidly relaxing component of NPQ, qE, possibly acting in the minor monomeric antenna complexes and the major trimeric LHCII, respectively. To distinguish whether zeaxanthin and lutein act independently as quenchers at separate sites, or alternatively whether zeaxanthin fulfills an allosteric role regulating lutein-mediated quenching, the kinetics of qE and the qE-related conformational changes (ΔA₅₃₅) were compared in Arabidopsis (Arabidopsis thaliana) mutant/antisense plants with altered contents of minor antenna (kolhcb6, aslhcb4), trimeric LHCII (aslhcb2), lutein (lut2, lut2npq1, lut2npq2), and zeaxanthin (npq1, npq2). The kinetics of the two components of NPQ induction arising from zeaxanthin-independent and zeaxanthin-dependent qE were both sensitive to changes in the protein composition of the photosystem II antenna. The replacement of lutein by zeaxanthin or violaxanthin in the internal Lhcb protein-binding sites affected the kinetics and relative amplitude of each component as well as the absolute chlorophyll fluorescence lifetime. Both components of qE were characterized by a conformational change leading to nearly identical absorption changes in the Soret region that indicated the involvement of the LHCII lutein 1 domain. Based on these observations, we suggest that both components of qE arise from a common quenching mechanism based upon a conformational change within the photosystem II antenna, optimized by Lhcb subunit-subunit interactions and tuned by the synergistic effects of external and internally bound xanthophylls.The chlorophyll a/b-binding light-harvesting antenna of photosystem II (PSII of higher plants is responsible for the efficient collection and transfer of excitation energy to the reaction center. The PSII antenna comprises the main trimeric light-harvesting complex, LHCII, which is composed of the Lhcb1 to -3 polypeptides, and the minor light-harvesting complexes, CP29, CP26, and CP24, composed of Lhcb4, -5, and -6, respectively. In Arabidopsis (Arabidopsis thaliana), four LHCII trimers associate with two copies each of CP24, CP26, and CP29 and a core dimer of PSII (CP43/D1/D2/CP47) to form the C₂S₂M₂ LHCII-PSII supercomplex (Dekker and Boekema, 2005). In addition, depending upon the growth conditions, two or three extra LHCII trimers per PSII may be present in LHCII-only regions of the grana, providing additional light-harvesting capacity.The PSII antenna is a highly dynamic system that is able to tune the amount of excitation delivered to the PSII reaction center to match physiological need (Horton et al., 1996). The regulation of energy flow occurs by control of the thermal dissipation of excess excitation within the PSII antenna, a process termed nonphotochemical quenching (NPQ). NPQ is heterogeneous, comprising a slowly reversible qI component and a rapidly reversible qE component (Horton et al., 1996). The trigger for qE is the buildup of the transmembrane proton gradient or ΔpH (Briantais et al., 1979). The ΔpH is sensed by the PsbS protein (Li et al., 2004), without which the rapidly reversible behavior of NPQ is lost (Li et al., 2000). Full expression of qE in vivo is associated with the enzymatic deepoxidation of the epoxy-xanthophyll violaxanthin to zeaxanthin, via the action of the xanthophyll cycle (Demmig-Adams, 1990). The majority of the photoconvertible xanthophyll cycle pool is associated with trimeric LHCII, bound at the external V1 binding site (Ruban et al., 1999, 2002a; Caffarri et al., 2001; Liu et al., 2004). Trimeric LHCII binds two other types of xanthophylls internally: two all-trans-luteins at the L1 and L2 sites associated with the central membrane-spanning α-helices; and a 9-cis-neoxanthin at the N1 site associated with the C-helix chlorophyll b domain (Liu et al., 2004). The minor monomeric complexes CP24, CP26, and CP29 all bind lutein at the L1 site. In addition, CP29 binds two xanthophyll cycle carotenoids and one-half to one neoxanthin, CP24 binds two xanthophyll cycle carotenoids, while CP26 binds one xanthophyll cycle carotenoid and one neoxanthin (Peter and Thornber, 1991; Bassi et al., 1993; Ruban et al., 1994, 1999; Morosinotto et al., 2002).Although there is strong evidence that qE occurs in the PSII antenna light-harvesting proteins and that xanthophylls are involved, the mechanism of energy dissipation remains unclear. There is evidence for two distinct quenching mechanisms, one involving zeaxanthin (type I) and the other lutein (type II). In the type I mechanism, it is proposed that qE obligatorily depends upon zeaxanthin acting as a quencher of excited chlorophyll via the formation of a charge transfer state. Evidence for type I is the formation of a carotenoid radical cation absorbing at approximately 1,000 nm that correlates with the extent of qE (Holt et al., 2005). Recently, evidence was obtained that formation of the zeaxanthin radical cation occurs exclusively at the L2 binding site of the minor antenna complexes (Ahn et al., 2008; Avenson et al., 2008), quenching therefore requiring reversible insertion of zeaxanthin into this internal site. Because the effect of this cation on the excited-state lifetime of the minor antenna complexes was found to be very small, it was suggested that in vivo, under the influence of the ΔpH, a large population of complexes would adopt a conformation in which this species could form (Avenson et al., 2008). Evidence was also obtained that a zeaxanthin radical cation may form in trimeric LHCII (Amarie et al., 2007). Again, the effect on the chlorophyll excited-state lifetime was very small, leading these authors to conclude that the type I mechanism could not be responsible for qE (Amarie et al., 2007; Dreuw and Wormit, 2008).In the type II mechanism, qE is an inbuilt property of LHCII proteins; a protein conformational change alters the configuration of bound pigments and results in the xanthophyll bound at the L1 site (normally lutein) becoming an effective quencher of chlorophyll excited states (Ruban et al., 2007; Ilioaia et al., 2008). Evidence for a type II mechanism came from studies of trimeric LHCII aggregates (Ruban et al., 2007). Here, it was concluded that energy dissipation occurs by energy transfer from chlorophyll a to the S₁ state (2Ag¹) of lutein bound at the L1 site. Notably, this quenching mechanism decreases the chlorophyll excited-state lifetime by a magnitude sufficient to fully account for qE in vivo. A change in the conformation of another LHCII-bound xanthophyll (neoxanthin) correlates with the extent of quenching. This conformational change takes place in vivo with an amplitude that correlates with the amount of qE. In the model for type II quenching proposed by Horton and coworkers (1991, 2005), zeaxanthin acts not as a quencher but as an allosteric modulator of the ΔpH sensitivity of this intrinsic LHCII quenching process.Although the type I and type II mechanisms involve different xanthophylls operating at different sites, there are similarities: in particular, both are proposed to involve a ΔpH-triggered, PsbS-mediated conformational change (Ruban et al., 2007; Ahn et al., 2008). Indeed, it is possible that both mechanisms contribute to in vivo qE, since the process occurs in both the presence and absence of zeaxanthin (Adams et al., 1990; Crouchman et al., 2006). The crucial question is whether zeaxanthin-dependent and zeaxanthin-independent qE arise from the same mechanism (type II) or from two different ones (types I and II, respectively). The kinetics of NPQ formation upon the illumination of dark-adapted leaves comprise two components: the first forms rapidly and is zeaxanthin independent; the second, slower component correlates with violaxanthin deepoxidation and therefore is described as zeaxanthin dependent (Adams et al., 1990; Ruban and Horton, 1999). The two components of NPQ formation are of the qE type: both relax rapidly upon darkening (Adams et al., 1990); both are dependent upon PsbS (Li et al., 2000); and both are enhanced by PsbS overexpression (Li et al., 2002; Crouchman et al., 2006). Investigation of these kinetics provides an opportunity to determine whether a single mechanism can account for qE and to give clues to which type of mechanism is involved. Here, we test the hypothesis that the two components arise from different mechanisms: the zeaxanthin-dependent component arising in the minor monomeric antenna by a type I mechanism (Gilmore et al., 1998; Ahn et al., 2008; Avenson et al., 2008), and the zeaxanthin-independent component arising in the major trimeric LHCII by the type II mechanism. An alternative explanation for zeaxanthin-independent qE, at least under low-light conditions, when qE forms transiently, is that it is caused by quenching in the PSII reaction center (Finazzi et al., 2004). Several predictions emerge from this hypothesis. First, the removal of certain Lhcb proteins by mutation would differentially affect the two components of qE. Second, because the two components would be additive and could not compensate for the loss of one another (Niyogi et al., 1998; Pogson et al., 1998), they should each contribute a discrete component to the kinetics of qE formation and relaxation. Third, in mutants lacking lutein, the capacity of the type II mechanism would be reduced, while the zeaxanthin-dependent component would be unaffected. Finally, the two components may be expected to be characterized by different absorption changes in the Soret region, which reflect changes in the absorption spectra of bound pigments brought about by conformational changes within the PSII antenna upon qE formation (Ruban et al., 1993a, 1993b, 2002b; Bilger and Björkman, 1994). We tested this hypothesis by analysis of qE kinetics, fluorescence lifetimes, and qE-related absorption difference spectra. Contrary to the above predictions, the data indicated that both steady-state and transient qE arise from a common mechanism within the PSII antenna, in both the presence and absence of zeaxanthin.     

Excitation dynamics and relaxation in the major antenna of a marine green alga Bryopsis corticulans

The light-harvesting complexes II (LHCIIs) of spinach and Bryopsis corticulans as a green alga are similar in structure, but differ in carotenoid (Car) and chlorophyll (Chl) compositions. Carbonyl Cars siphonein (Spn) and siphonaxanthin (Spx) bind to B. corticulans LHCII likely in the sites as a pair of lutein (Lut) molecules bind to spinach LHCII in the central domain. To understand the light-harvesting and photoprotective properties of the algal LHCII, we compared its excitation dynamics and relaxation to those of spinach LHCII been well documented. It was found that B. corticulans LHCII exhibited a substantially longer chlorophyll (Chl) fluorescence lifetime (4.9 ns vs 4.1 ns) and a 60% increase of the fluorescence quantum yield. Photoexcitation populated ³Car* equally between Spn and Spx in B. corticulans LHCII, whereas predominantly at Lut620 in spinach LHCII. These results prove the functional differences of the LHCIIs with different Car pairs and Chl a/b ratios: B. corticulans LHCII shows the enhanced blue-green light absorption, the alleviated quenching of ¹Chl*, and the dual sites of quenching ³Chl*, which may facilitate its light-harvesting and photoprotection functions. Moreover, for both types of LHCIIs, the triplet excitation profiles revealed the involvement of extra ³Car* formation mechanisms besides the conventional Chl-to-Car triplet transfer, which are discussed in relation to the ultrafast processes of ¹Chl* quenching. Our experimental findings will be helpful in deepening the understanding of the light harvesting and photoprotection functions of B. corticulans living in the intertidal zone with dramatically changing light condition.     

Triplet–triplet energy transfer in fucoxanthin-chlorophyll protein from diatom Cyclotella meneghiniana: Insights into the structure of the complex

Although the major light harvesting complexes of diatoms, called FCPs (fucoxanthin chlorophyll a/c binding proteins), are related to the cab proteins of higher plants, the structures of these light harvesting protein complexes are much less characterized. Here, a structural/functional model for the “core” of FCP, based on the sequence homology with LHCII, in which two fucoxanthins replace the central luteins and act as quenchers of the Chl a triplet states, is proposed. Combining the information obtained by time-resolved EPR spectroscopy on the triplet states populated under illumination, with quantum mechanical calculations, we discuss the chlorophyll triplet quenching in terms of the geometry of the chlorophyll–carotenoid pairs participating to the process. The results show that local structural rearrangements occur in FCP, with respect to LHCII, in the photoprotective site.     

Determination of the stoichiometry and strength of binding of xanthophylls to the photosystem II light harvesting complexes

Xanthophylls have a crucial role in the structure and function of the light harvesting complexes of photosystem II (LHCII) in plants. The binding of xanthophylls to LHCII has been investigated, particularly with respect to the xanthophyll cycle carotenoids violaxanthin and zeaxanthin. It was found that most of the violaxanthin pool was loosely bound to the major complex and could be removed by mild detergent treatment. Gentle solubilization of photosystem II particles and thylakoids allowed the isolation of complexes, including a newly described oligomeric preparation, enriched in trimers, that retained all of the in vivo violaxanthin pool. It was estimated that each LHCII monomer can bind at least one violaxanthin. The extent to which different pigments can be removed from LHCII indicated that the relative strength of binding was chlorophyll b > neoxanthin > chlorophyll a > lutein > zeaxanthin > violaxanthin. The xanthophyll binding sites are of two types: internal sites binding lutein and peripheral sites binding neoxanthin and violaxanthin. In CP29, a minor LHCII, both a lutein site and the neoxanthin site can be occupied by violaxanthin. Upon activation of the violaxanthin de-epoxidase, the highest de-epoxidation state was found for the main LHCII component and the lowest for CP29, suggesting that only violaxanthin loosely bound to LHCII is available for de-epoxidation.     

Short- and long-term operation of the lutein-epoxide cycle in light-harvesting antenna complexes

The lutein-5,6-epoxide (Lx) cycle operates in some plants between lutein (L) and its monoepoxide, Lx. Whereas recent studies have established the photoprotective roles of the analogous violaxanthin cycle, physiological functions of the Lx cycle are still unknown. In this article, we investigated the operation of the Lx cycle in light-harvesting antenna complexes (Lhcs) of Inga sapindoides Willd, a tropical tree legume accumulating substantial Lx in shade leaves, to identify the xanthophyll-binding sites involved in short- and long-term responses of the Lx cycle and to analyze the effects on light-harvesting efficiency. In shade leaves, Lx was converted into L upon light exposure, which then replaced Lx in the peripheral V1 site in trimeric Lhcs and the internal L2 site in both monomeric and trimeric Lhcs, leading to xanthophyll composition resembling sun-type Lhcs. Similar to the violaxanthin cycle, the Lx cycle was operating in both photosystems, yet the light-induced Lx --> L conversion was not reversible overnight. Interestingly, the experiments using recombinant Lhcb5 reconstituted with different Lx and/or L levels showed that reconstitution with Lx results in a significantly higher fluorescence yield due to higher energy transfer efficiencies among chlorophyll (Chl) a molecules, as well as from xanthophylls to Chl a. Furthermore, the spectroscopic analyses of photosystem I-LHCI from I. sapindoides revealed prominent red-most Chl forms, having the lowest energy level thus far reported for higher plants, along with reduced energy transfer efficiency from antenna pigments to Chl a. These results are discussed in the context of photoacclimation and shade adaptation.     

Chlorophyll a and carotenoid triplet states in light-harvesting complex II of higher plants.

Laser-flash-induced transient absorption measurements were performed on trimeric light-harvesting complex II to study carotenoid (Car) and chlorophyll (Chl) triplet states as a function of temperature. In these complexes efficient transfer of triplets from Chl to Car occurs as a protection mechanism against singlet oxygen formation. It appears that at room temperature all triplets are being transferred from Chl to Car; at lower temperatures (77 K and below) the transfer is less efficient and chlorophyll triplets can be observed. In the presence of oxygen at room temperature the Car triplets are partly quenched by oxygen and two different Car triplet spectral species can be distinguished because of a difference in quenching rate. One of these spectral species is replaced by another one upon cooling to 4 Ki demonstrating that at least three carotenoids are in close contact with chlorophylls. The triplet minus singlet absorption (T-S) spectra show maxima at 504-506 nm and 517-523 nm, respectively. In the Chl Qy region absorption changes can be observed that are caused by Car triplets. The T-S spectra in the Chl region show an interesting temperature dependence which indicates that various Car's are in contact with different Chl a molecules. The results are discussed in terms of the crystal structure of light-harvesting complex II.