Light-harvesting State Research Articles

Discovery of Multiple Light-Harvesting States of the Photosynthetic Protein PE545.

Cryptophytes are photosynthetic microalga that flourish in a remarkable diversity of natural environments by using pigment-containing proteins with absorption maxima tuned to each ecological niche. While this diversity in the absorption has been well established, the subsequent photophysics is highly sensitive to the local protein environment and so may exhibit similar variation. Thermal fluctuations of the protein conformation are expected to introduce photophysical heterogeneity of the pigments that may have evolved important functional properties in a manner similar to that of the absorption. However, such heterogeneity is averaged out in ensemble measurements and, therefore, has not yet been probed. Here, we report single-molecule measurements of phycoerythrin 545 (PE545), the prototypical cryptophyte antenna protein, in its native dimeric form. A conformational ensemble was resolved consisting of distinct photophysical states with different light-harvesting properties. Proteins that did not quench, partially quenched, or fully quenched absorbed light were observed. Light intensity increased the quenched-state population of the dimer, potentially as a mechanism to deal with the extreme light intensities found in aqueous environments. Cross-linking, which mimics local interactions, introduces this light-dependent functionality while also suppressing other conformational dynamics. The cellular organization can, therefore, actively modulate the protein conformation and dynamics, selecting for distinct levels of light harvesting. Thus, the complex conformational equilibrium provides an additional mechanism for cryptophytes and likely other photosynthetic organisms to optimize solar energy capture and conversion.

Cryo-EM structures of LHCII in photo-active and photo-protecting states reveal allosteric regulation of light harvesting and excess energy dissipation.

The major light-harvesting complex of photosystem II (LHCII) has a dual regulatory function in a process called non-photochemical quenching to avoid the formation of reactive oxygen. LHCII undergoes reversible conformation transitions to switch between a light-harvesting state for excited-state energy transfer and an energy-quenching state for dissipating excess energy under full sunshine. Here we report cryo-electron microscopy structures of LHCII in membrane nanodiscs, which mimic in vivo LHCII, and in detergent solution at pH 7.8 and 5.4, respectively. We found that, under low pH conditions, the salt bridges at the lumenal side of LHCII are broken, accompanied by the formation of two local α-helices on the lumen side. The formation of α-helices in turn triggers allosterically global protein conformational change, resulting in a smaller crossing angle between transmembrane helices. The fluorescence decay rates corresponding to different conformational states follow the Dexter energy transfer mechanism with a characteristic transition distance of 5.6 Å between Lut1 and Chl612. The experimental observations are consistent with the computed electronic coupling strengths using multistate density function theory.

How the pH Controls Photoprotection in the Light-Harvesting Complex of Mosses.

In response to varying light conditions, light-harvesting complexes (LHCs) switch from a light-harvesting state to a quenched state to protect the photosynthetic organism from excessive light irradiation in a strategy known as nonphotochemical quenching (NPQ). NPQ is activated by an acidification of the thylakoid lumen, which is sensed directly or indirectly by the LHC, resulting in a conformational change of the complex that leads to the quenched state. The conformational changes responsible for NPQ activation and their connection to specific quenching mechanisms are still unknown. Here, we investigate the pH-triggered conformational changes in the light-harvesting complex stress-related (LHCSR) of mosses. By combining constant-pH molecular dynamics and enhanced sampling techniques, we find that the pH sensitivity of the complex is driven by the coupled protonation of three residues modulating the conformation of the short amphipathic helix placed at the lumen side of the embedding membrane. Combining these results with quantum mechanics/molecular mechanics calculations, we show that the quenching mechanism sensitive to the pH goes through a charge-transfer between a carotenoid and an excited chlorophyll, which is controlled by the protein conformation.

Structures of a phycobilisome in light-harvesting and photoprotected states.

Phycobilisome (PBS) structures are elaborate antennae in cyanobacteria and red algae1,2. These large protein complexes capture incident sunlight and transfer the energy through a network of embedded pigment molecules called bilins to the photosynthetic reaction centres. However, light harvesting must also be balanced against the risks of photodamage. A known mode of photoprotection is mediated by orange carotenoid protein (OCP), which binds to PBS when light intensities are high to mediate photoprotective, non-photochemical quenching3-6. Here we use cryogenic electron microscopy to solve four structures of the 6.2 MDa PBS, with and without OCP bound, from the model cyanobacterium Synechocystis sp. PCC 6803. The structures contain a previously undescribed linker protein that binds to the membrane-facing side of PBS.For the unquenched PBS, the structures also reveal three different conformational states of the antenna, two previously unknown. The conformational states result from positional switching of two of the rods and may constitute a new mode of regulation of light harvesting. Only one of the three PBS conformations can bind to OCP, which suggests that not every PBS is equally susceptible to non-photochemical quenching. In the OCP-PBS complex, quenching is achieved through the binding of four 34 kDa OCPs organized as two dimers. The complex reveals the structure of the active form of OCP, in which an approximately 60 Å displacement of its regulatory carboxy terminal domain occurs. Finally, by combining our structure with spectroscopic properties7, we elucidate energy transfer pathways within PBS in both the quenched and light-harvesting states. Collectively, our results provide detailed insights into the biophysical underpinnings of the control of cyanobacterial light harvesting. The data also have implications for bioengineering PBS regulation in natural and artificial light-harvesting systems.

Switchable photovoltaic window for on-demand shading and electricity generation

A next step in the development of transparent photovoltaic windows would be to introduce a switching mechanism that allows the user to switch the window between a transparent and an opaque state as desired to generate electricity and shade the building interior. In this work, we investigate a switchable photovoltaic window consisting of the combination of a semitransparent germanium solar cell and a gasochromic switchable magnesium mirror in a double glazing. We analyze the average visible transmittance (AVT) and spectral emissivity (ε) of the device in the transparent (AVT = 18%, ε = 0.27) and light harvesting states (AVT = 1%, ε = 0.06). The thermal and electrical performance of the switchable solar cell is studied under varied solar irradiance. The experimental results are used to model the annual electricity generation of a switchable photovoltaic window in different climate regions under different switching scenarios, based on datasets from the Photovoltaic Geographical Information System (PV-GIS). The model suggests that especially in Mediterranean regions, switchable transparent photovoltaic windows with an efficiency of 2% can be a promising step to combine on-demand shading with electricity generation. Moreover, it shows that to generate more than 20 kWh/m2 of electricity per year with this technology, the switching must be additionally coupled to the occupancy of the indoor spaces. Our model is applicable to other switchable PV technologies for evaluation purposes and to estimate ideal use cases.

Capturing the Quenching Mechanism of Light-Harvesting Complexes of Plants by Zooming in on the Ensemble

Summary The light-harvesting complexes (LHCs) of plants can regulate the energy flux to the reaction centers in response to fluctuating light by virtue of their vast conformational landscape. They do so by switching from a light-harvesting state to a quenched state, dissipating the excess absorbed energy as heat. However, isolated LHCs are prevalently in their light-harvesting state, which makes the identification of their photoprotective mechanism extremely challenging. Here, ensemble time-resolved fluorescence experiments show that monomeric CP29, a minor LHC of plants, exists in various emissive states with identical spectra but different lifetimes. The photoprotective mechanism active in a subpopulation of strongly quenched complexes is further investigated via ultrafast transient absorption spectroscopy, kinetic modeling, and mutational analysis. We demonstrate that the observed quenching is due to excitation energy transfer from chlorophylls to a dark state of the centrally bound lutein.

A siphonous morphology affects light-harvesting modulation in the intertidal green macroalga Bryopsis corticulans (Ulvophyceae)

Main conclusionThe macroalga Bryopsis corticulans relies on a sustained protective NPQ and a peculiar body architecture to efficiently adapt to the extreme light changes of intertidal shores.During low tides, intertidal algae experience prolonged high light stress. Efficient dissipation of excess light energy, measured as non-photochemical quenching (NPQ) of chlorophyll fluorescence, is therefore required to avoid photodamage. Light-harvesting regulation was studied in the intertidal macroalga Bryopsis corticulans, during high light and air exposure. Photosynthetic capacity and NPQ kinetics were assessed in different filament layers of the algal tufts and in intact chloroplasts to unravel the nature of NPQ in this siphonous green alga. We found that the morphology and pigment composition of the B. corticulans body provides functional segregation between surface sunlit filaments (protective state) and those that are underneath and undergo severe light attenuation (light-harvesting state). In the surface filaments, very high and sustained NPQ gradually formed. NPQ induction was triggered by the formation of transthylakoid proton gradient and independent of the xanthophyll cycle. PsbS and LHCSR proteins seem not to be active in the NPQ mechanism activated by this alga. Our results show that B. corticulans endures excess light energy pressure through a sustained protective NPQ, not related to photodamage, as revealed by the unusually quick restoration of photosystem II (PSII) function in the dark. This might suggest either the occurrence of transient PSII photoinactivation or a fast rate of PSII repair cycle.

Disentangling protein and lipid interactions that control a molecular switch in photosynthetic light harvesting

In the photosynthetic apparatus of plants and algae, the major Light-Harvesting Complexes (LHCII) collect excitations and funnel these to the photosynthetic reaction center where charge separation takes place. In excess light conditions, remodeling of the photosynthetic membrane and protein conformational changes produces a photoprotective state in which excitations are rapidly quenched to avoid photodamage. The quenched states are associated with protein aggregation, however the LHCII complexes are also proposed to have an intrinsic capacity to shift between light harvesting and fluorescence-quenched conformational states. To disentangle the effects of protein-protein and protein-lipid interactions on the LHCII photoprotective switch, we compared the structural and fluorescent properties of LHCII lipid nanodiscs and proteoliposomes with very low protein-to-lipid ratios. We demonstrate that LHCII proteins adapta fully fluorescent state in nanodiscs and in proteoliposomes with highly diluted protein densities. Increasing the protein density induces a transition into a mildly-quenched state that reaches a plateau at a molar protein-to-lipid ratio of 0.001 and has a fluorescence yield reminiscent of the light-harvesting state in vivo. The low onset for quenching strongly suggests that LHCII-LHCII attractive interactions occur inside membranes. The transition at low protein densities does not involve strong changes in the excitonic circular-dichroism spectrum and is distinct from a transition occurring at very high protein densities that comprises strong fluorescence quenching and circular-dichroism spectral changes involving chlorophyll 611 and 612, correlating with proposed quencher sites of the photoprotective mechanisms.

From light-harvesting to photoprotection: structural basis of the dynamic switch of the major antenna complex of plants (LHCII).

Light-Harvesting Complex II (LHCII) is largely responsible for light absorption and excitation energy transfer in plants in light-limiting conditions, while in high-light it participates in photoprotection. It is generally believed that LHCII can change its function by switching between different conformations. However, the underlying molecular picture has not been elucidated yet. The available crystal structures represent the quenched form of the complex, while solubilized LHCII has the properties of the unquenched state. To determine the structural changes involved in the switch and to identify potential quenching sites, we have explored the structural dynamics of LHCII, by performing a series of microsecond Molecular Dynamics simulations. We show that LHCII in the membrane differs substantially from the crystal and has the signatures that were experimentally associated with the light-harvesting state. Local conformational changes at the N-terminus and at the xanthophyll neoxanthin are found to strongly correlate with changes in the interactions energies of two putative quenching sites. In particular conformational disorder is observed at the terminal emitter resulting in large variations of the excitonic coupling strength of this chlorophyll pair. Our results strongly support the hypothesis that light-harvesting regulation in LHCII is coupled with structural changes.

Evidence of functional trimeric chlorophyll a/c2-peridinin proteins in the dinoflagellate Symbiodinium

The chlorophyll a-chlorophyll c2-peridinin-protein (apcPC), a major light harvesting component in peridinin-containing dinoflagellates, is an integral membrane protein complex. We isolated functional acpPC from the dinoflagellate Symbiodinium. Both SDS-PAGE and electrospray ionization mass spectrometry (ESI-MS) analysis quantified the denatured subunit polypeptide molecular weight (MW) as 18kDa. Size-exclusion chromatography (SEC) and blue native gel electrophoresis (BN-PAGE) were employed to estimate the size of native acpPC complex to be 64–66kDa. We also performed native ESI-MS, which can volatilize and ionize active biological samples in their native states. Our result demonstrated that the native acpPC complex carried 14 to 16 positive charges, and the MW of acpPC with all the associated pigments was found to be 66.5kDa. Based on these data and the pigment stoichiometry, we propose that the functional light harvesting state of acpPC is a trimer. Our bioinformatic analysis indicated that Symbiodinium acpPC shares high similarity to diatom fucoxanthin Chl a/c binding protein (FCP), which tends to form a trimer. Additionally, acpPC protein sequence variation was confirmed by de novo protein sequencing. Its sequence heterogeneity is also discussed in the context of Symbiodinium eco-physiological adaptations.

Structural Basis for the Non-Photochemical Quenching Switch of the Green Alga Chlamydomonas Reinhardtii

Feedback mechanisms that dissipate excess photo-excitations in Light-Harvesting Complexes (LHCs) are necessary to avoid detrimental oxidative stress in most photosynthetic eukaryotes. Here we demonstrate the unique ability of LHCSR, a stress related LHC from the model organism Chlamydomonas reinhardtii, to sense pH variations, tuning its conformation from a light-harvesting state to a dissipative one. This conformational change is induced exclusively by the acidification of the environment. We show that this ability to respond to different environments is missing in the related major Light Harvesting Complex II (LHCII) despite the high structural homology. Via mutagenesis and spectroscopy characterization we show that LHCSR uniqueness relies on its peculiar C-terminus subdomain, which acts as a sensor of the lumenal pH, able to tune the quenching level of the complex.

Regulation of Light Harvesting in the Green Alga Chlamydomonas reinhardtii: The C-Terminus of LHCSR Is the Knob of a Dimmer Switch

Feedback mechanisms that dissipate excess photoexcitations in light-harvesting complexes (LHCs) are necessary to avoid detrimental oxidative stress in most photosynthetic eukaryotes. Here we demonstrate the unique ability of LHCSR, a stress-related LHC from the model organism Chlamydomonas reinhardtii, to sense pH variations, reversibly tuning its conformation from a light-harvesting state to a dissipative one. This conformational change is induced exclusively by the acidification of the environment, and the magnitude of quenching is correlated to the degree of acidification of the environment. We show that this ability to respond to different pH values is missing in the related major LHCII, despite high structural homology. Via mutagenesis and spectroscopic characterization, we show that LHCSR's uniqueness relies on its peculiar C-terminus subdomain, which acts as a sensor of the lumenal pH, able to tune the quenching level of the complex.

High Excitation Energy Quenching in Fucoxanthin Chlorophyll a/c-Binding Protein Complexes from the Diatom Chaetoceros gracilis

The fucoxanthin chlorophyll (Chl) a/c-binding protein (FCP) is responsible for excellent light-harvesting strategies that enable survival in fluctuating light conditions. Here, we report the light-harvesting and quenching states of two FCP complexes, FCP-A and FCP-B/C, isolated from the diatom Chaetoceros gracilis. Pigment analysis revealed that FCP-A is enriched in Chl c, whereas FCP-B/C is enriched in diadinoxanthin, reflecting differences in low-temperature steady-state absorption and fluorescence spectra of each FCP complex. Time-resolved fluorescence spectra were measured at 77 K, and the characteristic lifetimes were determined using global fitting analysis of the spectra. Tens of picosecond (ps) components revealed energy transfer to low-energy Chl a from Chls a and c, whereas the other components showed only fluorescence decay components with no concomitant rise components. The normalized amplitudes of hundreds of picosecond components were relatively 30% in the total fluorescence, whereas those of longest-lived components were 60%. The hundreds of picosecond components were assigned as excitation energy quenching, whereas the longest-lived components were assigned as fluorescence from the final energy traps. These results suggest that 30% of FCP complex forming quenching state and the other 60% of FCP complex forming light-harvesting state exist heterogeneously in each FCP fraction under continuous low-light condition.

D–D−π–A-Type Organic Dyes for Dye-Sensitized Solar Cells with a Potential for Direct Electron Injection and a High Extinction Coefficient: Synthesis, Characterization, and Theoretical Investigation

A series of organic sensitizers with the direct electron injection mechanism and a high molar extinction coefficient comprising double donors, a π-spacer, and anchoring acceptor groups (D–D−π–A type) were synthesized and characterized by experimental and theoretical methods for dye-sensitized solar cells. (E)-2-Cyano-3-(5″-(4-((4-(3,6-di-tert-butylcarbazol-9-yl)phenyl)dodecylamino)phenyl)-[2,2′:5′,2″-terthiophene]-5-yl)acrylic acid showed performance with a maximal incident photon to electron conversion efficiency of 83%, Jsc value of 10.89 mA cm–2, Voc value of 0.70 V, and fill factor of 0.67, which correspond to an overall conversion efficiency of 5.12% under AM 1.5G illumination. The molecular geometry, electronic structure, and excited states were investigated with density functional theory, time-dependent density functional theory, and the symmetry-adapted cluster-configuration interaction method. The double donor moieties not only contribute to enhancement of the electron-donating ability, but also inhibit aggregation between dye molecules and prevent iodide/triiodide in the electrolyte from recombining with injected electrons in TiO2. Detailed assignments of the UV–vis spectra below the ionization threshold are given. The low-lying light-harvesting state has intramolecular charge transfer character with a high molar extinction coefficient because of the long π-spacer. Our experimental and theoretical findings support the potential of direct electron injection from the dye to TiO2 in one step with electronic excitation for the present D–D−π–A sensitizers. The direct electron injection, inhibited aggregation, and high molar extinction coefficient may be the origin of the observed high efficiency. This type of D–D−π–A structure with direct electron injection would simplify the strategy for designing organic sensitizers.

Insights into the region responding to ΔpH change in major light harvesting complex

Nonradioactive dissipation of excitation energy is the most important photoprotective mechanism in plants. In high light, the trans-thylakoid ΔpH can activate the xanthophyll cycle and protonate the PsbS protein, which are important in regulating the process of non-photochemical quenching. Despite it has been proven that the ΔpH can directly switch the major light harvesting complex (LHCIIb) from the light harvesting state to the energy dissipating state both in vitro and in vivo, the region responding to ΔpH change in LHCIIb remains obscure. In the present study, we proposed that Asp211 and Asp215 should be the amino acids that respond to the change of the trans-thylakoid ΔpH on the basis of pKa calculations of LHCIIb. Furthermore, Asp215 is the outmost acidic amino acid in trimeric LHCIIb, and its pKa value is sensitive to the protein environment, suggesting that the regulation of the protonation state of Asp215 may be important for the aggregation of LHCIIb occurred in the process of qE in vivo and in vitro.

Photosynthetic acclimation: Does the dynamic structure and macro‐organisation of photosystem II in higher plant grana membranes regulate light harvesting states?

The efficiency of light harvesting in higher plant photosynthesis is regulated in response to external environmental conditions. Under conditions of excess light, the normally highly efficient light-harvesting system of photosystem II is switched into a state in which unwanted, potentially harmful, energy is dissipated as heat. This process, known as nonphotochemical quenching, occurs by the creation of energy quenchers following conformational change in the light-harvesting complexes, which is initiated by the build up of the thylakoid pH gradient and controlled by the xanthophyll cycle. In the present study, the evidence to support the notion that this regulatory mechanism is dependent upon the organization of the different antenna subunits in the stacked grana membranes is reviewed. We postulate that nonphotochemical quenching occurs within a structural locus comprising the PsbS subunit and components of the light-harvesting antenna, CP26, CP24, CP29 and LHCIIb (the major trimeric light-harvesting complex), formed in response to protonation and controlled by the xanthophyll cycle carotenoids.

The importance of a highly active and ΔpH-regulated diatoxanthin epoxidase for the regulation of the PS II antenna function in diadinoxanthin cycle containing algae

The present study focuses on the regulation of diatoxanthin (Dtx) epoxidation in the diadinoxanthin (Ddx) cycle containing algae Phaeodactylum tricornutum, Thalassiosira pseudonana, Cyclotella meneghiniana and Prymnesium parvum and its significance for the control of the photosystem II (PS II) antenna function. Our data show that Dtx epoxidase can exhibit extremely high activities when algal cells are transferred from high light (HL) to low light (LL). Under HL conditions, Dtx epoxidation is strongly inhibited by the light-driven proton gradient. Uncoupling of the cells during HL illumination restores the high epoxidation rates observed during LL. In Ddx cycle containing algae, non-photochemical quenching of chlorophyll fluorescence (NPQ) is directly correlated with the Dtx concentration and independent of the presence of the proton gradient. This means that a fast conversion of PS II from the heat dissipating state back to the light-harvesting state can only be realized by an efficient removal of the quenching pigment Dtx. It is proposed that the high Dtx epoxidation rates during LL illumination serve exactly this purpose. The inhibition of Dtx epoxidation by the DeltapH, on the other hand, ensures rapid increases in the Dtx concentration when photoprotection under conditions of HL illumination is required. The regulation of the PS II antenna function in Ddx cycle containing algae is different to that in violaxanthin (Vx) cycle containing plants, where for the zeaxanthin (Zx)-dependent NPQ the presence of a proton gradient is mandatory. In the green alga Chlorella vulgaris conversion of PS II from the heat dissipating state back to the light-harvesting state is controlled by the DeltapH, whose relaxation after a transition from HL to darkness or LL rapidly abolishes the thermal dissipation of excitation energy, including the Zx-dependent NPQ. Due to the inability of Zx to quench fluorescence in the absence of the DeltapH a fast epoxidation of Zx to Vx in LL is not needed and is missing in Chlorella vulgaris.