pH sensitivity of chlorophyll fluorescence quenching is determined by the detergent/protein ratio and the state of LHCII aggregation

Here we show how the protein environment in terms of detergent concentration/protein aggregation state, affects the sensitivity to pH of isolated, native LHCII, in terms of chlorophyll fluorescence quenching. Three detergent concentrations (200, 20 and 6 μM n-dodecyl β-d-maltoside) have been tested. It was found that at the detergent concentration of 6 μM, low pH quenching of LHCII is close to the physiological response to lumen acidification possessing pK of 5.5. The analysis has been conducted both using arbitrary PAM fluorimetry measurements and chlorophyll fluorescence lifetime component analysis. The second led to the conclusion that the 3.5 ns component lifetime corresponds to an unnatural state of LHCII, induced by the detergent used for solubilising the protein, whilst the 2 ns component is rather the most representative lifetime component of the conformational state of LHCII in the natural thylakoid membrane environment when the non-photochemical quenching (NPQ) was absent. The 2 ns component is related to a pre-aggregated LHCII that makes it more sensitive to pH than the trimeric LHCII with the dominating 3.5 ns lifetime component. The pre-aggregated LHCII displayed both a faster response to protons and a shift in the pK for quenching to higher values, from 4.2 to 4.9. We concluded that environmental factors like lipids, zeaxanthin and PsbS protein that modulate NPQ in vivo could control the state of LHCII aggregation in the dark that makes it more or less sensitive to the lumen acidification. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Higher plant photosystem II light-harvesting antenna, not the reaction center, determines the excited-state lifetime-both the maximum and the nonphotochemically quenched

The maximum chlorophyll fluorescence lifetime in isolated photosystem II (PSII) light-harvesting complex (LHCII) antenna is 4 ns; however, it is quenched to 2 ns in intact thylakoid membranes when PSII reaction centers (RCIIs) are closed (Fm). It has been proposed that the closed state of RCIIs is responsible for the quenching. We investigated this proposal using a new, to our knowledge, model system in which the concentration of RCIIs was highly reduced within the thylakoid membrane. The system was developed in Arabidopsis thaliana plants under long-term treatment with lincomycin, a chloroplast protein synthesis inhibitor. The treatment led to 1), a decreased concentration of RCIIs to 10% of the control level and, interestingly, an increased antenna component; 2), an average reduction in the yield of photochemistry to 0.2; and 3), an increased nonphotochemical chlorophyll fluorescence quenching (NPQ). Despite these changes, the average fluorescence lifetimes measured in Fm and Fm' (with NPQ) states were nearly identical to those obtained from the control. A 77 K fluorescence spectrum analysis of treated PSII membranes showed the typical features of preaggregation of LHCII, indicating that the state of LHCII antenna in the dark-adapted photosynthetic membrane is sufficient to determine the 2 ns Fm lifetime. Therefore, we conclude that the closed RCs do not cause quenching of excitation in the PSII antenna, and play no role in the formation of NPQ.     

Pigment Interactions in Light-harvesting Complex II in Different Molecular Environments

Extraction of plant light-harvesting complex II (LHCII) from the native thylakoid membrane or from aggregates by the use of surfactants brings about significant changes in the excitonic circular dichroism (CD) spectrum and fluorescence quantum yield. To elucidate the cause of these changes, e.g. trimer-trimer contacts or surfactant-induced structural perturbations, we compared the CD spectra and fluorescence kinetics of LHCII aggregates, artificial and native LHCII-lipid membranes, and LHCII solubilized in different detergents or trapped in polymer gel. By this means we were able to identify CD spectral changes specific to LHCII-LHCII interactions, at (−)-437 and (+)-484 nm, and changes specific to the interaction with the detergent n-dodecyl-β-maltoside (β-DM) or membrane lipids, at (+)-447 and (−)-494 nm. The latter change is attributed to the conformational change of the LHCII-bound carotenoid neoxanthin, by analyzing the CD spectra of neoxanthin-deficient plant thylakoid membranes. The neoxanthin-specific band at (−)-494 nm was not pronounced in LHCII in detergent-free gels or solubilized in the α isomer of DM but was present when LHCII was reconstituted in membranes composed of phosphatidylcholine or plant thylakoid lipids, indicating that the conformation of neoxanthin is sensitive to the molecular environment. Neither the aggregation-specific CD bands, nor the surfactant-specific bands were positively associated with the onset of fluorescence quenching, which could be triggered without invoking such spectral changes. Significant quenching was not active in reconstituted LHCII proteoliposomes, whereas a high degree of energetic connectivity, depending on the lipid:protein ratio, in these membranes allows for efficient light harvesting.     

Assembly of the major light-harvesting complex II in lipid nanodiscs

Self-aggregation of isolated plant light-harvesting complexes (LHCs) upon detergent extraction is associated with fluorescence quenching and is used as an in vitro model to study the photophysical processes of nonphotochemical quenching (NPQ). In the NPQ state, in vivo induced under excess solar light conditions, harmful excitation energy is safely dissipated as heat. To prevent self-aggregation and probe the conformations of LHCs in a lipid environment devoid from detergent interactions, we assembled LHCII trimer complexes into lipid nanodiscs consisting of a bilayer lipid matrix surrounded by a membrane scaffold protein (MSP). The LHCII nanodiscs were characterized by fluorescence spectroscopy and found to be in an unquenched, fluorescent state. Remarkably, the absorbance spectra of LHCII in lipid nanodiscs show fine structure in the carotenoid and Q_y region that is different from unquenched, detergent-solubilized LHCII but similar to that of self-aggregated, quenched LHCII in low-detergent buffer without magnesium ions. The nanodisc data presented here suggest that 1), LHCII pigment-protein complexes undergo conformational changes upon assembly in nanodiscs that are not correlated with downregulation of its light-harvesting function; and 2), these effects can be separated from quenching and aggregation-related phenomena. This will expand our present view of the conformational flexibility of LHCII in different microenvironments.     

A novel method produces native light-harvesting complex II aggregates from the photosynthetic membrane revealing their role in nonphotochemical quenching

Nonphotochemical quenching (NPQ) is a mechanism of regulating light harvesting that protects the photosynthetic apparatus from photodamage by dissipating excess absorbed excitation energy as heat. In higher plants, the major light-harvesting antenna complex (LHCII) of photosystem (PS) II is directly involved in NPQ. The aggregation of LHCII is proposed to be involved in quenching. However, the lack of success in isolating native LHCII aggregates has limited the direct interrogation of this process. The isolation of LHCII in its native state from thylakoid membranes has been problematic because of the use of detergent, which tends to dissociate loosely bound proteins, and the abundance of pigment–protein complexes (e.g. PSI and PSII) embedded in the photosynthetic membrane, which hinders the preparation of aggregated LHCII. Here, we used a novel purification method employing detergent and amphipols to entrap LHCII in its natural states. To enrich the photosynthetic membrane with the major LHCII, we used Arabidopsis thaliana plants lacking the PSII minor antenna complexes (NoM), treated with lincomycin to inhibit the synthesis of PSI and PSII core proteins. Using sucrose density gradients, we succeeded in isolating the trimeric and aggregated forms of LHCII antenna. Violaxanthin- and zeaxanthin-enriched complexes were investigated in dark-adapted, NPQ, and dark recovery states. Zeaxanthin-enriched antenna complexes showed the greatest amount of aggregated LHCII. Notably, the amount of aggregated LHCII decreased upon relaxation of NPQ. Employing this novel preparative method, we obtained a direct evidence for the role of in vivo LHCII aggregation in NPQ.     

The PsbS protein controls the organization of the photosystem II antenna in higher plant thylakoid membranes

The PsbS subunit of photosystem II (PSII) plays a key role in nonphotochemical quenching (NPQ), the major photoprotective regulatory mechanism in higher plant thylakoid membranes, but its mechanism of action is unknown. Here we describe direct evidence that PsbS controls the organization of PSII and its light harvesting system (LHCII). The changes in chlorophyll fluorescence amplitude associated with the Mg(2+)-dependent restacking of thylakoid membranes were measured in thylakoids prepared from wild-type plants, a PsbS-deficient mutant and a PsbS overexpresser. The Mg(2+) requirement and sigmoidicity of the titration curves for the fluorescence rise were negatively correlated with the level of PsbS. Using a range of PsbS mutants, this effect of PsbS was shown not to depend upon its efficacy in controlling NPQ, but to be related only to protein concentration. Electron microscopy and fluorescence spectroscopy showed that this effect was because of enhancement of the Mg(2+)-dependent re-association of PSII and LHCII by PsbS, rather than an effect on stacking per se. In the presence of PsbS the LHCII.PSII complex was also more readily removed from thylakoid membranes by detergent, and the level of PsbS protein correlated with the amplitude of the psi-type CD signal originating from features of LHCII.PSII organization. It is proposed that PsbS regulates the interaction between LHCII and PSII in the grana membranes, explaining how it acts as a pH-dependent trigger of the conformational changes within the PSII light harvesting system that result in NPQ.     

A possible molecular basis for photoprotection in the minor antenna proteins of plants

The bioenergetics of light-harvesting by photosynthetic antenna proteins in higher plants is well understood. However, investigation into the regulatory non-photochemical quenching (NPQ) mechanism, which dissipates excess energy in high light, has led to several conflicting models. It is generally accepted that the major photosystem II antenna protein, LHCII, is the site of NPQ, although the minor antenna complexes (CP24/26/29) are also proposed as alternative/additional NPQ sites. LHCII crystals were shown to exhibit the short excitation lifetime and several spectral signatures of the quenched state. Subsequent structure-based models showed that this quenching could be explained by slow energy trapping by the carotenoids, in line with one of the proposed models. Using Fluorescence Lifetime Imaging Microscopy (FLIM) we show that the crystal structure of CP29 corresponds to a strongly quenched conformation. Using a structure-based theoretical model we show that this quenching may be explained by the same slow, carotenoid-mediated quenching mechanism present in LHCII crystals.     

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.     

The causes of altered chlorophyll fluorescence quenching induction in the Arabidopsis mutant lacking all minor antenna complexes

Non-photochemical quenching (NPQ) of chlorophyll fluorescence is the process by which excess light energy is harmlessly dissipated within the photosynthetic membrane. The fastest component of NPQ, known as energy-dependent quenching (qE), occurs within minutes, but the site and mechanism of qE remain of great debate. Here, the chlorophyll fluorescence of Arabidopsis thaliana wild type (WT) plants was compared to mutants lacking all minor antenna complexes (NoM). Upon illumination, NoM exhibits altered chlorophyll fluorescence quenching induction (i.e. from the dark-adapted state) characterised by three different stages: (i) a fast quenching component, (ii) transient fluorescence recovery and (iii) a second quenching component. The initial fast quenching component originates in light harvesting complex II (LHCII) trimers and is dependent upon PsbS and the formation of a proton gradient across the thylakoid membrane (ΔpH). Transient fluorescence recovery is likely to occur in both WT and NoM plants, but it cannot be overcome in NoM due to impaired ΔpH formation and a reduced zeaxanthin synthesis rate. Moreover, an enhanced fluorescence emission peak at ~679?nm in NoM plants indicates detachment of LHCII trimers from the bulk antenna system, which could also contribute to the transient fluorescence recovery. Finally, the second quenching component is triggered by both ΔpH and PsbS and enhanced by zeaxanthin synthesis. This study indicates that minor antenna complexes are not essential for qE, but reveals their importance in electron stransport, ΔpH formation and zeaxanthin synthesis.     

Membrane-dependent heterogeneity of LHCII characterized using single-molecule spectroscopy

In green plants, light harvesting complex of Photosystem II (LHCII) absorbs and transports excitation energy toward the photosynthetic reaction centers and serves as a site for energy-dependent nonphotochemical quenching (qE), the photoprotective dissipation of energy as heat. LHCII is thought to activate dissipation through conformational changes that change the photophysical behaviors. Understanding this balance requires a characterization of how the conformations of LHCII, and thus its photophysics, are influenced by individual factors within the membrane environment. Here, we used ensemble and single-molecule fluorescence to characterize the excited-state lifetimes and switching kinetics of LHCII embedded in nanodisc- and liposome-based model membranes of various sizes and lipid compositions. As the membrane area decreased, the quenched population and the rate of conformational dynamics both increased because of interactions with other proteins, the aqueous solution, and/or disordered lipids. Although the conformational states and dynamics were similar in both thylakoid and asolectin lipids, photodegradation increased with thylakoid lipids, likely because of their charge and pressure properties. Collectively, these findings demonstrate the ability of membrane environments to tune the conformations and photophysics of LHCII.     

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).     

Importance of trimer-trimer interactions for the native state of the plant light-harvesting complex II

Aggregates and solubilized trimers of LHCII were characterized by circular dichroism (CD), linear dichroism and time-resolved fluorescence spectroscopy and compared with thylakoid membranes in order to evaluate the native state of LHCII in vivo. It was found that the CD spectra of lamellar aggregates closely resemble those of unstacked thylakoid membranes whereas the spectra of trimers solubilized in n-dodecyl-beta,D-maltoside, n-octyl-beta,D-glucopyranoside, or Triton X-100 were drastically different in the Soret region. Thylakoid membranes or LHCII aggregates solubilized with detergent exhibited CD spectra similar to the isolated trimers. Solubilization of LHCII was accompanied by profound changes in the linear dichroism and increase in fluorescence lifetime. These data support the notion that lamellar aggregates of LHCII retain the native organization of LHCII in the thylakoid membranes. The results indicate that the supramolecular organization of LHCII, most likely due to specific trimer-trimer contacts, has significant impact on the pigment interactions in the complexes.     

In pea stipules a functional photosynthetic electron flow occurs despite a reduced dynamicity of LHCII association with photosystems

The flexible association of the light harvesting complex II (LHCII) to photosystem (PS) I and PSII to balance their excitation is a major short-term acclimation process of the thylakoid membrane, together with the thermal dissipation of excess absorbed energy, reflected in non-photochemical quenching of chlorophyll fluorescence (NPQ). In Pisum sativum, the leaf includes two main photosynthetic parts, the basal stipules and the leaflets. Since the stipules are less efficient in carbon fixation than leaflets, the adjustments of the thylakoid system, which safeguard the photosynthetic membrane against photodamage, were analysed. As compared to leaflets, the stipules experienced a decay in PSII photochemical activity. The supramolecular organization of photosystems in stipules showed a more conspicuous accumulation of large PSII-LHCII supercomplexes in the grana, but also a tendency to retain the PSI-LHCI-LHCII state transition complex and the PSI-LHCI-PSII-LHCII megacomplexes probably located at the interface between appressed and stroma-exposed membranes. As a consequence, stipules had a lower capacity to perform state transitions and the overall thylakoid architecture was less structurally flexible and ordered than in leaflets. Yet, stipules proved to be quite efficient in regulating the redox state of the electron transport chain and more capable of inducing NPQ than leaflets. It is proposed that, in spite of a relatively static thylakoid arrangement, LHCII interaction with both photosystems in megacomplexes can contribute to a regulated electron flow.     

Importance of trimer-trimer interactions for the native state of the plant light-harvesting complex II

Aggregates and solubilized trimers of LHCII were characterized by circular dichroism (CD), linear dichroism and time-resolved fluorescence spectroscopy and compared with thylakoid membranes in order to evaluate the native state of LHCII in vivo. It was found that the CD spectra of lamellar aggregates closely resemble those of unstacked thylakoid membranes whereas the spectra of trimers solubilized in n-dodecyl-β,d-maltoside, n-octyl-β,d-glucopyranoside, or Triton X-100 were drastically different in the Soret region. Thylakoid membranes or LHCII aggregates solubilized with detergent exhibited CD spectra similar to the isolated trimers. Solubilization of LHCII was accompanied by profound changes in the linear dichroism and increase in fluorescence lifetime. These data support the notion that lamellar aggregates of LHCII retain the native organization of LHCII in the thylakoid membranes. The results indicate that the supramolecular organization of LHCII, most likely due to specific trimer-trimer contacts, has significant impact on the pigment interactions in the complexes.     

Different response of photosystem II to short and long‐term drought stress in Arabidopsis thaliana

Short‐ and long‐term drought stress on photosystem II (PSII) and oxidative stress were studied in Arabidopsis thaliana. Under drought stress, chlorophyll (Chl) content, Chl fluorescence, relative water content and oxygen evolution capacity gradually decreased, and the thylakoid structure was gradually damaged. Short‐term drought stress caused a rapid disassembly of the light‐harvesting complex II (LHCII). However, PSII dimers kept stable under the short‐term drought stress and significantly decreased only after 15 days of drought stress. Immunoblotting analysis of the thylakoid membrane proteins showed that most of the photosystem proteins decreased after the stress, especially for Lhcb5, Lhcb6 and PsbQ proteins. However, surprisingly, PsbS significantly increased after the long‐term drought stress, which is consistent with the substantially increased non‐photochemical quenching (NPQ) after the stress. Our results suggest that the PSII–LHCII supercomplexes and LHCII assemblies play an important role in preventing photo‐damages to PSII under drought stress.     

Control of the light harvesting function of chloroplast membranes: the LHCII-aggregation model for non-photochemical quenching

Dissipation of excess excitation energy within the photosystem II light-harvesting antenna (LHCII) by non-photochemical quenching (NPQ) is an important photoprotective process in plants. An update to a hypothesis for the mechanism of NPQ [FEBS Letters 292, 1991] is presented. The impact of recent advances in understanding the structure, organisation and photophysics of LHCII is assessed. We show possible locations of the predicted regulatory and quenching pigment-binding sites in the structural model of the major LHCII. We suggest that NPQ is a highly regulated concerted response of the organised thylakoid macrostructure, which can include different mechanisms and sites at different times.     

Functional domain size in aggregates of light-harvesting complex II and thylakoid membranes

The functional domain size for efficient excited singlet state quenching was studied in artificial aggregates of the main light-harvesting complex II (LHCIIb) from spinach and in native thylakoid membranes by picosecond time-resolved fluorescence spectroscopy and quantum yield measurements. The domain size was estimated from the efficiency of added exogenous singlet excitation quenchers-phenyl-p-benzoquinone (PPQ) and dinitrobenzene (DNB). The mean fluorescence lifetimes τ(av) were quantified for a range of quencher concentrations. Applying the Stern-Volmer formalism, apparent quenching rate constants k(q) were determined from the dependencies on quencher concentration of the ratio τ(0)(av)/τ(av), where τ(0)(av) is the average fluorescence lifetime of the sample without addition of an exogenous quencher. The functional domain size was gathered from the ratio k(q)'/k(q), i.e., the apparent quenching rate constants determined in aggregates (or membranes), k(q)', and in detergent-solubilised LHCII trimers, k(q), respectively. In LHCII macroaggregates, the resulting values for the domain size were 15-30 LHCII trimers. In native thylakoid membranes the domain size was equivalent to 12-24 LHCII trimers, corresponding to 500-1000 chlorophylls. Virtually the same results were obtained when membranes were suspended in buffers promoting either membrane stacking or destacking. These domain sizes are orders of magnitude smaller than the number of physically connected pigment-protein complexes. Therefore our results imply that the physical size of an antenna system beyond the numbers of a functional domain size has little or no effect on improving the light-harvesting efficiency.     

Zeaxanthin independence of photophysics in light-harvesting complex II in a membrane environment

Green plants protect against photodamage by dissipating excess energy in a process called non-photochemical quenching (NPQ). In vivo, NPQ is activated by a drop in the luminal pH of the thylakoid membrane that triggers conformational changes of the antenna complexes, which activate quenching channels. The drop in pH also triggers de-epoxidation of violaxanthin, one of the carotenoids bound within the antenna complexes, into zeaxanthin, and so the amplitude of NPQ in vivo has been shown to increase in the presence of zeaxanthin. In vitro studies on light-harvesting complex II (LHCII), the major antenna complex in plants, compared different solubilization environments, which give rise to different levels of quenching and so partially mimic NPQ in vivo. However, in these studies both completely zeaxanthin-independent and zeaxanthin-dependent quenching have been reported, potentially due to the multiplicity of solubilization environments. Here, we characterize the zeaxanthin dependence of the photophysics in LHCII in a near-physiological membrane environment, which produces slightly enhanced quenching relative to detergent solubilization, the typical in vitro environment. The photophysical pathways of dark-adapted and in vitro de-epoxidized LHCIIs are compared, representative of the low-light and high-light conditions in vivo, respectively. The amplitude of quenching as well as the dissipative photophysics are unaffected by zeaxanthin at the level of individual LHCIIs, suggesting that zeaxanthin-dependent quenching is independent of the channels induced by the membrane. Furthermore, our results demonstrate that additional factors beyond zeaxanthin incorporation in LHCII are required for full development of NPQ.     

Comparison of the Thermodynamic Landscapes of Unfolding and Formation of the Energy Dissipative State in the Isolated Light Harvesting Complex II

In biochemistry and cell biology, understanding the molecular mechanisms by which physiological processes are regulated is regarded as an ultimate goal. In higher plants, one of the most widely investigated regulatory processes occurs in the light harvesting complexes (LHCII) of the chloroplast thylakoid membranes. Under limiting photon flux densities, LHCII harvests sunlight with high efficiency. When the intensity of incident radiation reaches levels close to the saturation of the photosynthesis, the efficiency of light harvesting is decreased by a process referred to as nonphotochemical quenching (NPQ), which enhances the singlet-excited state deactivation via nonradiative dissipative processes. Conformational rearrangements in LHCII are known to be crucial in promoting and controlling NPQ in vitro and in vivo. In this article, we address the thermodynamic nature of the conformational rearrangements promoting and controlling NPQ in isolated LHCII. A combined, linear reaction scheme in which the folded, quenched state represents a stable intermediate on the unfolding pathway was employed to describe the temperature dependence of the spectroscopic signatures associated with the chlorophyll fluorescence quenching and the loss of secondary structure motifs in LHCII. The thermodynamic model requires considering the temperature dependence of Gibbs free energy difference between the quenched and the unquenched states, as well as the unfolded and quenched states, of LHCII. Even though the same reaction scheme is adequate to describe the quenching and the unfolding processes in LHCII monomers and trimers, their thermodynamic characteristics were found to be markedly different. The results of the thermodynamic analysis shed light on the physiological importance of the trimeric state of LHCII in stabilizing the efficient light harvesting mode as well as preventing the quenched conformation of the protein from unfolding. Moreover, the transition to the quenched conformation in trimers reveals a larger degree of cooperativity than in monomers, explained by a small characteristic entropy (ΔH_q = 85 ± 3 kJ mol⁻¹ compared to 125 ± 5 kJ mol⁻¹ in monomers), which enables the fine-tuning of nonphotochemical quenching in vivo.     

Crystal structure of plant light‐harvesting complex shows the active,energy‐transmitting state

Plants dissipate excess excitation energy as heat by non‐photochemical quenching (NPQ). NPQ has been thought to resemble in vitro aggregation quenching of the major antenna complex, light harvesting complex of photosystem II (LHC‐II). Both processes are widely believed to involve a conformational change that creates a quenching centre of two neighbouring pigments within the complex. Using recombinant LHC‐II lacking the pigments implicated in quenching, we show that they have no particular role. Single crystals of LHC‐II emit strong, orientation‐dependent fluorescence with an emission maximum at 680 nm. The average lifetime of the main 680 nm crystal emission at 100 K is 1.31 ns, but only 0.39 ns for LHC‐II aggregates under identical conditions. The strong emission and comparatively long fluorescence lifetimes of single LHC‐II crystals indicate that the complex is unquenched, and that therefore the crystal structure shows the active, energy‐transmitting state of LHC‐II. We conclude that quenching of excitation energy in the light‐harvesting antenna is due to the molecular interaction with external pigments in vitro or other pigment–protein complexes such as PsbS in vivo, and does not require a conformational change within the complex.