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We measure the two-dimensional electronic spectra of the LHCII(M)-CP29-CP24 complex in photosystem II (PSII) and provide the first study of the ultrafast excitation energy transfer (EET) processes of an asymmetric and native light-harvesting assembly of the antenna of PSII. With comparisons to LHCII, we observe faster energy equilibrations in the intermediate levels of the LHCII(M)-CP29-CP24 complex at 662 and 670 nm. Notably, the putative "bottleneck" states in LHCII exhibit faster effective dynamics in the LHCII(M)-CP24-CP29 complex, with the average lifetime shortening from 2.5 ps in LHCII to 1.2 ps in the bigger assembly. The observations are supported by high-level structure-based calculations, and the accelerated dynamics can be attributed to the structural change of LHCII(M) in the bigger complex. This study shows that the biological functioning structures of the complexes are important to understand the overall EET dynamics of the PSII supercomplex. Ministry of Education (MOE) This work was supported by grants from the Singapore Ministry of Education Academic Research Fund (Tier 1 RG2/19 and Tier 1 RG14/20 to Howe-Siang Tan), the National Research, Development and Innovations Office, Hungary (NKFIH 2018- 1.2.1-NKP-2018-000009 to Petar H. Lambrev), and the Eötvös Loránd Research Network (KÖ -36/2021 to Petar H. Lambrev).

In cyanobacteria, phycobilisomes serve as peripheral light-harvesting complexes of the two photosystems, extending their antenna size and the wavelength range of photons available for photosynthesis. The abundance of phycobilisomes, the number of phycobiliproteins they contain, and their light-harvesting function are dynamically adjusted in response to the physiological conditions. Phycobilisomes are also thought to be involved in state transitions that maintain the excitation balance between the two photosystems. Unlike its eukaryotic counterpart, PSI is trimeric in many cyanobacterial species and the physiological significance of this is not well understood. Here we compared the composition and light-harvesting function of phycobilisomes in cells of Synechocystis sp. PCC 6803, which has primarily trimeric PSI, and the ΔpsaL mutant unable to form trimers. We also investigated a mutant additionally lacking the PsaJ and PsaF subunits of PSI. Both strains with monomeric PSI accumulated significantly more allophycocyanin per chlorophyll, indicating higher abundance of phycobilisomes. On the other hand, a higher phycocyanin:allophycocyanin ratio in WT suggests larger phycobilisomes or the presence of APC-less phycobilisomes (CpcL-type), that are not assembled in cells with monomeric PSI. Steady-state and time-resolved fluorescence spectroscopy at room temperature and 77 K revealed that PSII receives more energy from the phycobilisomes at the expense of PSI in cells with monomeric PSI, regardless of the presence of PsaF. Taken together, these results show that the oligomeric state of PSI has an impact on the excitation energy flow in Synechocystis.One-sentence summaryCyanobacterial mutants with monomeric PSI show changes in the composition and abundance of phycobilisomes and in the excitation energy transfer to PSII and PSI.

Photosystem I (PSI), found in all oxygenic photosynthetic organisms, uses solar energy to drive electron transport with nearly 100% quantum efficiency, thanks to fast energy transfer among antenna chlorophylls and charge separation in the reaction center. There is no complete consensus regarding the kinetics of the elementary steps involved in the overall trapping, especially the rate of primary charge separation. In this work, we employed two-dimensional coherent electronic spectroscopy to follow the dynamics of energy and electron transfer in a monomeric PSI complex from Synechocystis PCC 6803, containing only subunits A-E, K, and M, at 77 K. We also determined the structure of the complex to 4.3 Å resolution by cryoelectron microscopy with refinements to 2.5 Å. We applied structure-based modeling using a combined Redfield-Förster theory to compute the excitation dynamics. The absorptive 2D electronic spectra revealed fast excitonic/vibronic relaxation on time scales of 50-100 fs from the high-energy side of the absorption spectrum. Antenna excitations were funneled within 1 ps to a small pool of chlorophylls absorbing around 687 nm, thereafter decaying with 4-20 ps lifetimes, independently of excitation wavelength. Redfield-Förster energy transfer computations showed that the kinetics is limited by transfer from these red-shifted pigments. The rate of primary charge separation, upon direct excitation of the reaction center, was determined to be 1.2-1.5 ps-1. This result implies activationless electron transfer in PSI. Ministry of Education (MOE) Submitted/Accepted version This work was supported by grants from the National Research, Development and Innovations Office, Hungary (NKFIH 2018-1.2.1-NKP-2018-000009 to P.L.), Eötvös Lorand Research Network (KO ́ ̈ -37/2020 to P.L.), and the Singapore Ministry of Education Academic Research Fund (Tier 1 RG2/19 and Tier 1 RG14/20 to H.-S.T.). N.N. acknowledges support by The Israel Science Foundation (Grant No. 569/17) and by the German-Israeli Foundation for Scientific Research and Development (GIF), Grant no. G1483-207/2018.

Photosystem I is the most efficient photosynthetic enzyme with structure and composition highly conserved among all oxygenic phototrophs. Cyanobacterial Photosystem I is typically associated into trimers for reasons that are still debated. Almost universally, Photosystem I contains a number of long-wavelength-absorbing ‘red’ chlorophylls (Chls), that have a sizeable effect on the excitation energy transfer and trapping. Here we present spectroscopic comparison of trimeric Photosystem I from Synechocystis PCC 6803 with a monomeric complex from the ΔpsaL mutant and a ‘minimal’ monomeric complex ΔFIJL, containing only subunits A, B, C, D, E, K and M. The quantum yield of photochemistry at room temperature was the same in all complexes, demonstrating the functional robustness of this photosystem. The monomeric complexes had a reduced far-red absorption and emission equivalent to the loss of 1.5–2 red Chls emitting at 710–715 nm, whereas the longest-wavelength emission at 722 nm was not affected. The picosecond fluorescence kinetics at 77 K showed spectrally and kinetically distinct red Chls in all complexes and equilibration times of up to 50 ps. We found that the red Chls are not irreversible traps at 77 K but can still transfer excitations to the reaction centre, especially in the trimeric complexes. Structure-based Förster energy transfer calculations support the assignment of the lowest-energy state to the Chl pair B37/B38 and the trimer-specific red Chl emission to Chls A32/B7 located at the monomer–monomer interface. These intermediate-energy red Chls facilitate energy migration from the lowest-energy states to the reaction centre.

Bryopsis corticulans is a marine green macroalga adapted to the intertidal environment. It possesses siphonaxanthin-binding light-harvesting complexes of photosystem II (LHCII) with spectroscopic properties markedly different from the LHCII in plants. By applying a phenomenological fitting procedure to the two-dimensional electronic spectra of the LHCII from B. corticulans measured at 77 K, we can extract information about the excitonic states and energy-transfer processes. The fitting method results in well-converged parameters, including excitonic energy levels with their respective transition dipole moments, spectral widths, energy-transfer rates, and coupling properties. The 2D spectra simulated from the fitted parameters concur very well with the experimental data, showing the robustness of the fitting method. An excitonic energy-transfer scheme can be constructed from the fitting parameters. It shows the rapid energy transfer from chlorophylls (Chls) b to a at subpicosecond time scales and a long-lived state in the Chl b region at around 659 nm. Three weakly connected terminal states are resolved at 671, 675, and 677 nm. The lowest state is higher in energy than that in plant LHCII, which is probably because of the fewer number of Chls a in a B. corticulans LHCII monomer. Modeling based on existing Hamiltonians for the plant LHCII structure with two Chls a switched to Chls b suggests several possible Chl a-b replacements in comparison with those of plant LHCII. The adaptive changes result in a slower energy equilibration in the complex, revealed by the longer relaxation times of several exciton states compared to those of plant LHCII. The strength of our phenomenological fitting method for obtaining excitonic energy levels and energy-transfer network is put to the test in systems such as B. corticulans LHCII, where prior knowledge on exact assignment and spatial locations of pigments are lacking. Ministry of Education (MOE) Submitted/Accepted version H.-S.T acknowledges support from the Singapore Ministry of Education Academic Research Fund (Tier 1 RG2/19 and Tier 1 RG15/18). P.H.L. acknowledges grants from the National Research, Development and Innovation Fund (Grants NN124904 and 2018-1.2.1-NKP-2018-00009). This work was also supported by a Strategic Priority Research Program (XDB17030100) of China, a Key Research Project for Frontier Science (QYZDY-SSW-SMC003) from the Chinese Academy of Sciences (CAS), China, and a bilateral project between CAS and the Hungary Academy of Sciences.

Photosystem II (PSII) uses solar energy to oxidize water and delivers electrons for life on Earth. The photochemical reaction center of PSII is known to possess two stationary states. In the open state (PSIIO), the absorption of a single photon triggers electron-transfer steps, which convert PSII into the charge-separated closed state (PSIIC). Here, by using steady-state and time-resolved spectroscopic techniques on Spinacia oleracea and Thermosynechococcus vulcanus preparations, we show that additional illumination gradually transforms PSIIC into a light-adapted charge-separated state (PSIIL). The PSIIC-to-PSIIL transition, observed at all temperatures between 80 and 308 K, is responsible for a large part of the variable chlorophyll-a fluorescence (Fv) and is associated with subtle, dark-reversible reorganizations in the core complexes, protein conformational changes at noncryogenic temperatures, and marked variations in the rates of photochemical and photophysical reactions. The build-up of PSIIL requires a series of light-induced events generating rapidly recombining primary radical pairs, spaced by sufficient waiting times between these events--pointing to the roles of local electric-field transients and dielectric relaxation processes. We show that the maximum fluorescence level, Fm, is associated with PSIIL rather than with PSIIC, and thus the Fv/Fm parameter cannot be equated with the quantum efficiency of PSII photochemistry. Our findings resolve the controversies and explain the peculiar features of chlorophyll-a fluorescence kinetics, a tool to monitor the functional activity and the structural-functional plasticity of PSII in different wild-types and mutant organisms and under stress conditions. VC American Society of Plant Biologists 2021. All rights reserved

Photosystem II (PSII) is the pigment-protein complex driving the photoinduced oxidation of water and reduction of plastoquinone in all oxygenic photosynthetic organisms. Excitations in the antenna chlorophylls are photochemically trapped in the reaction center (RC) producing the chlorophyll-pheophytin radical ion pair P+ Pheo-. When electron donation from water is inhibited, the oxidized RC chlorophyll P+ acts as an excitation quencher, but knowledge on the kinetics of quenching is limited. Here, we used femtosecond transient absorption spectroscopy to compare the excitation dynamics of PSII with neutral and oxidized RC (P+). We find that equilibration in the core antenna has a major lifetime of about 300 fs, irrespective of the RC redox state. Two-dimensional electronic spectroscopy revealed additional slower energy equilibration occurring on timescales of 3-5 ps, concurrent with excitation trapping. The kinetics of PSII with open RC can be described well with previously proposed models according to which the radical pair P+ Pheo- is populated with a main lifetime of about 40 ps, which is primarily determined by energy transfer between the core antenna and the RC chlorophylls. Yet, in PSII with oxidized RC (P+), fast excitation quenching was observed with decay lifetimes as short as 3 ps and an average decay lifetime of about 90 ps, which is shorter than the excited-state lifetime of PSII with open RC. The underlying mechanism of this extremely fast quenching prompts further investigation. Ministry of Education (MOE) Published version This work was supported by grants from the National Research, Development and Innovation Office (Grant Nos. FK-139067 to P.A., PD-138498 to G.S., and 2018-1.2.1-NKP-2018-00009 to P.H.L.), the Eötvös Loránd Research Network (Grant Nos. KÖ-37/2021 to G.G. and SA-76/2021 to P.A.), the Singapore Ministry of Education Academic Research Fund (Grant Nos. Tier 1 RG2/19 and Tier 1 RG14/20 to H.-S.T.), the National Key R & D Program of China (Grant Nos. 2017YFA0503700 and 2020YFA0907600 to G.H.), the CAS Project for Young Scientists in Basic Research (Grant No. YSBR-004 to G.H.), and a Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDA26050402 to G.H.).

Light-harvesting complex II (LHCII) is the major antenna complex in higher plants and green algae. It has been suggested that a major part of the excited state energy dissipation in the so-called "non-photochemical quenching" (NPQ) is located in this antenna complex. We have performed an ultrafast kinetics study of the low-energy fluorescent states related to quenching in LHCII in both aggregated and the crystalline form. In both sample types the chlorophyll (Chl) excited states of LHCII are strongly quenched in a similar fashion. Quenching is accompanied by the appearance of new far-red (FR) fluorescence bands from energetically low-lying Chl excited states. The kinetics of quenching, its temperature dependence down to 4 K, and the properties of the FR-emitting states are very similar both in LHCII aggregates and in the crystal. No such FR-emitting states are found in unquenched trimeric LHCII. We conclude that these states represent weakly emitting Chl-Chl charge-transfer (CT) states, whose formation is part of the quenching process. Quantum chemical calculations of the lowest energy exciton and CT states, explicitly including the coupling to the specific protein environment, provide detailed insight into the chemical nature of the CT states and the mechanism of CT quenching. The experimental data combined with the results of the calculations strongly suggest that the quenching mechanism consists of a sequence of two proton-coupled electron transfer steps involving the three quenching center Chls 610/611/612. The FR-emitting CT states are reaction intermediates in this sequence. The polarity-controlled internal reprotonation of the E175/K179 aa pair is suggested as the switch controlling quenching. A unified model is proposed that is able to explain all known conditions of quenching or non-quenching of LHCII, depending on the environment without invoking any major conformational changes of the protein.

Context. Homochirality is a generic and unique property of life on Earth and is considered a universal and agnostic biosignature. Homochirality induces fractional circular polarization in the incident light that it reflects. Because this circularly polarized light can be sensed remotely, it can be one of the most compelling candidate biosignatures in life detection missions. While there are also other sources of circular polarization, these result in spectrally flat signals with lower magnitude. Additionally, circular polarization can be a valuable tool in Earth remote sensing because the circular polarization signal directly relates to vegetation physiology. Aims. While high-quality circular polarization measurements can be obtained in the laboratory and under semi-static conditions in the field, there has been a significant gap to more realistic remote sensing conditions. Methods. In this study, we present sensitive circular spectropolarimetric measurements of various landscape elements taken from a fast-moving helicopter. Results. We demonstrate that during flight, within mere seconds of measurements, we can differentiate (S/N>5) between grass fields, forests, and abiotic urban areas. Importantly, we show that with only nonzero circular polarization as a discriminant, photosynthetic organisms can even be measured in lakes. Conclusions. Circular spectropolarimetry can be a powerful technique to detect life beyond Earth, and we emphasize the potential of utilizing circular spectropolarimetry as a remote sensing tool to characterize and monitor in detail the vegetation physiology and terrain features of Earth itself., 7 pages, 6 figures

Incorporation of membrane proteins into reconstituted lipid membranes is a common approach for studying their structure and function relationship in a native-like environment. In this work, we investigated fluorescence properties of liposome-reconstituted major light-harvesting complexes of plants (LHCII). By utilizing liposome labelling with the fluorescent dye molecules and single-molecule microscopy techniques, we were able to study truly liposome-reconstituted LHCII and compare them with bulk measurements and liposome-free LHCII aggregates bound to the surface. Our results showed that fluorescence lifetime obtained in bulk and in single liposome measurements were correlated. The fluorescence lifetimes of LHCII were shorter for liposome-free LHCII than for reconstituted LHCII. In the case of liposome-reconstituted LHCII, fluorescence lifetime showed dependence on the protein density reminiscent to concentration quenching. The dependence of fluorescence lifetime of LHCII on the liposome size was not significant. Our results demonstrated that fluorescence quenching can be induced by LHCII - LHCII interactions in reconstituted membranes, most likely occurring via the same mechanism as photoprotective non-photochemical quenching in vivo.

Incorporation of membrane proteins into reconstituted lipid membranes is a common approach for studying their structure and function relationship in a native-like environment. In this work, we investigated fluorescence properties of liposome-reconstituted LHCII. By utilizing liposome labelling with the fluorescent dye molecules and single-molecule microscopy techniques, we were able to study truly liposome-reconstituted LHCII and compare them with bulk measurements and liposome-free LHCII aggregates on bound surface. Our results showed that fluorescence lifetime in bulk and of that for single liposome measurements were correlated. The fluorescence lifetimes of LHCII were shorter for liposome-free LHCII than for reconstituted LHCII. In the case of liposome-reconstituted LHCII, fluorescence lifetime showed dependence on the protein density reminiscent to concentration quenching. The dependence of fluorescence lifetime of LHCII on the liposome size was not significant. Our results demonstrated that fluorescence quenching can be induced by LHCII-LHCII interactions in reconstituted membranes, most likely occurring via the same mechanism as photoprotective non-photochemical quenching in vivo.

The phycobilisome (PBS) serves as the major light-harvesting system, funnelling excitation energy to both photosystems (PS) in cyanobacteria and red algae. The picosecond kinetics involving the excitation energy transfer has been studied within the isolated systems and intact filaments of the cyanobacterium Anabaena variabilis PCC 7120. A target model is proposed which resolves the dynamics of the different chromophore groups. The energy transfer rate of 8.5 ± 1.0/ns from the rod to the core is the rate-limiting step, both in vivo and in vitro. The PBS-PSI-PSII supercomplex reveals efficient excitation energy migration from the low-energy allophycocyanin, which is the terminal emitter, in the PBS core to the chlorophyll a in the photosystems. The terminal emitter of the phycobilisome transfers energy to both PSI and PSII with a rate of 50 ± 10/ns, equally distributing the solar energy to both photosystems. Finally, the excitation energy is trapped by charge separation in the photosystems with trapping rates estimated to be 56 ± 6/ns in PSI and 14 ± 2/ns in PSII. Electronic supplementary material The online version of this article (10.1007/s11120-020-00723-0) contains supplementary material, which is available to authorized users.

We simulated two-dimensional electronic spectra (2DES) of the chlorophyll a manifold of light-harvesting complex II (LHCII) at various temperatures (77, 110, 150, 190, 230, 273, and 293 K) using the hierarchical equations of the motion-phase matching approach. We confirm the main excitation energy transfer pathways assignments within the chlorophyll a manifold of LHCII measured in a recent work (J. Phys. Chem. B 2019, 123, 6765-6775). The calculated transfer rates are also in general agreement with the measured rates. We also provided theoretical confirmation for the experimental assignments, as uphill and downhill energy transfer processes, of 2D spectral features that were reported in recent experimental reports. These temperature-dependent features were also ascertained to follow the detailed-balance principle.

Light-harvesting complex II (LHCII) from the marine green macroalga Bryopsis corticulans is spectroscopically characterized to understand the structural and functional changes resulting from adaptation to intertidal environment. LHCII is homologous to its counterpart in land plants but has a different carotenoid and chlorophyll (Chl) composition. This is reflected in the steady-state absorption, fluorescence, linear dichroism, circular dichroism and anisotropic circular dichroism spectra. Time-resolved fluorescence and two-dimensional electronic spectroscopy were used to investigate the consequences of this adaptive change in the pigment composition on the excited-state dynamics. The complex contains additional Chl b spectral forms - absorbing at around 650 nm and 658 nm - and lacks the red-most Chl a forms compared with higher-plant LHCII. Similar to plant LHCII, energy transfer between Chls occurs on timescales from under hundred fs (mainly from Chl b to Chl a) to several picoseconds (mainly between Chl a pools). However, the presence of long-lived, weakly coupled Chl b and Chl a states leads to slower exciton equilibration in LHCII from B. corticulans. The finding demonstrates a trade-off between the enhanced absorption of blue-green light and the excitation migration time. However, the adaptive change does not result in a significant drop in the overall photochemical efficiency of Photosystem II. These results show that LHCII is a robust adaptable system whose spectral properties can be tuned to the environment for optimal light harvesting. Ministry of Education (MOE) Published version The work is supported by grants from the Hungarian Ministry of Finance (GINOP-2.3.2-15-2016-00001), the National Research, Development and Innovation Fund (NKFI NN 124904; 2018-1.2.1-NKP-2018-00009), the Singapore Ministry of Education Academic Research Fund (MOE2015-T2-1-039), the National Key Research and Development Program of China (2017YFA0503700), a CAS Key Research Program for Frontier Science (QYZDY-SSW-SMC003), and the National Natural Science Foundation of China (31600191). CD measurements at the B23 beamline of the Diamond Light Source Ltd. were supported by the project CALIPSOplus under Grant Agreement 730872 from the EU Framework Programme for Research and Innovation HORIZON 2020.

Excitation energy transfer (EET) and trapping in Anabaena variabilis (PCC 7120) intact cells, isolated phycobilisomes (PBS) and photosystem I (PSI) complexes have been studied by picosecond time-resolved fluorescence spectroscopy at room temperature. Global analysis of the time-resolved fluorescence kinetics revealed two lifetimes of spectral equilibration in the isolated PBS, 30–35 ps and 110–130 ps, assigned primarily to energy transfer within the rods and between the rods and the allophycocyanin core, respectively. An additional intrinsic kinetic component with a lifetime of 500–700 ps was found, representing non-radiative decay or energy transfer in the core. Isolated tetrameric PSI complexes exhibited biexponential fluorescence decay kinetics with lifetimes of about 10 ps and 40 ps, representing equilibration between the bulk antenna chlorophylls with low-energy “red” states and trapping of the equilibrated excitations, respectively. The cascade of EET in the PBS and in PSI could be resolved in intact filaments as well. Virtually all energy absorbed by the PBS was transferred to the photosystems on a timescale of 180–190 ps. Electronic supplementary material The online version of this article (10.1007/s11120-020-00719-w) contains supplementary material, which is available to authorized users.

The principal function of the thylakoid membrane depends on the integrity of the lipid bilayer, yet almost half of the thylakoid lipids are of non-bilayer-forming type, whose exact functions are not fully understood. Non-bilayer lipids can be extruded from the membrane in the presence of high concentrations of co-solutes. We applied 2 M sucrose to induce lipid phase separation in isolated thylakoid membranes, following consequent structural and physiological effects. Circular dichroism spectroscopy indicated significant changes in the chiral macro-arrangement of the pigment–protein complexes, which were reversed after washing out the co-solute. Similarly, merocyanine-540 fluorescence suggested reversible changes in the lipid phases. The PSII function, as tested by chlorophyll fluorescence induction transients and time-resolved fluorescence, was almost unaffected. However, the presence of sucrose dramatically increased the PSII thermostability, which can partly be explained by a direct osmolyte effect and partly by the lipid phase separation stabilizing the stacked membrane.

Photosystem II (PSII) catalyses the photoinduced oxygen evolution and, by producing reducing equivalents drives, in concert with PSI, the conversion of carbon dioxide to sugars. Our knowledge about the architecture of the reaction centre (RC) complex and the mechanisms of charge separation and stabilisation is well advanced. However, our understanding of the processes associated with the functioning of RC is incomplete: the photochemical activity of PSII is routinely monitored by chlorophyll-a fluorescence induction but the presently available data are not free of controversy. In this work, we examined the nature of gradual fluorescence rise of PSII elicited by trains of single-turnover saturating flashes (STSFs) in the presence of a PSII inhibitor, permitting only one stable charge separation. We show that a substantial part of the fluorescence rise originates from light-induced processes that occur after the stabilisation of charge separation, induced by the first STSF; the temperature-dependent relaxation characteristics suggest the involvement of conformational changes in the additional rise. In experiments using double flashes with variable waiting times (∆τ) between them, we found that no rise could be induced with zero or short ∆τ, the value of which depended on the temperature - revealing a previously unknown rate-limiting step in PSII.

Photosystem I is a robust and highly efficient biological solar engine. Its capacity to utilize virtually every absorbed photon’s energy in a photochemical reaction generates great interest in the kinetics and mechanisms of excitation energy transfer and charge separation. In this work, we have employed room-temperature coherent two-dimensional electronic spectroscopy and time-resolved fluorescence spectroscopy to follow exciton equilibration and excitation trapping in intact Photosystem I complexes as well as core complexes isolated from Pisum sativum. We performed two-dimensional electronic spectroscopy measurements with low excitation pulse energies to record excited-state kinetics free from singlet–singlet annihilation. Global lifetime analysis resolved energy transfer and trapping lifetimes closely matches the time-correlated single-photon counting data. Exciton energy equilibration in the core antenna occurred on a timescale of 0.5 ps. We further observed spectral equilibration component in the core complex with a 3–4 ps lifetime between the bulk Chl states and a state absorbing at 700 nm. Trapping in the core complex occurred with a 20 ps lifetime, which in the supercomplex split into two lifetimes, 16 ps and 67–75 ps. The experimental data could be modelled with two alternative models resulting in equally good fits—a transfer-to-trap-limited model and a trap-limited model. However, the former model is only possible if the 3–4 ps component is ascribed to equilibration with a “red” core antenna pool absorbing at 700 nm. Conversely, if these low-energy states are identified with the P700 reaction centre, the transfer-to-trap-model is ruled out in favour of a trap-limited model. MOE (Min. of Education, S’pore) Accepted version

The light reactions of photosynthesis are hosted and regulated by the chloroplast thylakoid membrane (TM) — the central structural component of the photosynthetic apparatus of plants and algae. The two-dimensional and three-dimensional arrangement of the lipid–protein assemblies, aka macroorganisation, and its dynamic responses to the fluctuating physiological environment, aka flexibility, are the subject of this review. An emphasis is given on the information obtainable by spectroscopic approaches, especially circular dichroism (CD). We briefly summarise the current knowledge of the composition and three-dimensional architecture of the granal TMs in plants and the supramolecular organisation of Photosystem II and light-harvesting complex II therein. We next acquaint the non-specialist reader with the fundamentals of CD spectroscopy, recent advances such as anisotropic CD, and applications for studying the structure and macroorganisation of photosynthetic complexes and membranes. Special attention is given to the structural and functional flexibility of light-harvesting complex II in vitro as revealed by CD and fluorescence spectroscopy. We give an account of the dynamic changes in membrane macroorganisation associated with the light-adaptation of the photosynthetic apparatus and the regulation of the excitation energy flow by state transitions and non-photochemical quenching.

We measured two-dimensional electronic spectra of light-harvesting complex II (LHCII) at various temperatures (77, 110, 150, 230, and 295 K) under conditions free from singlet-singlet annihilation. We elucidated the temperature-dependent excitation energy transfer dynamics in the Chl a manifold of LHCII. Global analysis revealed that the dynamics can be summarized in distinct time scales from 200 fs up to 15 ps. While the fastest dynamics with a decay time of similar to 0.2-0.3 ps are relatively temperature-independent, the lifetimes and relative contributions of slower components showed considerable temperature dependence. The slowest time scale of equilibration with the lowest-energy Chl a increased from similar to 5 ps at 295 K to similar to 15 ps at 77 K. The final excited state is independent of initial excitation at 230 K and above, whereas static energy disorder is apparent at lower temperatures. A clear temperature dependence of uphill energy transfer processes was also discerned, which is consistent with the detailed-balance condition.

During the past two decades, two-dimensional electronic spectroscopy (2DES) and related techniques have emerged as a potent experimental toolset to study the ultrafast elementary steps of photosynthesis. Apart from the highly engaging albeit controversial analysis of the role of quantum coherences in the photosynthetic processes, 2DES has been applied to resolve the dynamics and pathways of energy and electron transport in various light-harvesting antenna systems and reaction centres, providing unsurpassed level of detail. In this paper we discuss the main technical approaches and their applicability for solving specific problems in photosynthesis. We then recount applications of 2DES to study the exciton dynamics in plant and photosynthetic light-harvesting complexes, especially light-harvesting complex II (LHCII) and the fucoxanthin-chlorophyll proteins of diatoms, with emphasis on the types of unique information about such systems that 2DES is capable to deliver. This article is part of a Special Issue entitled Light harvesting, edited by Dr. Roberta Croce.

Anisotropic circular dichroism (ACD) spectroscopy of macroscopically aligned molecules reveals additional information about their excited states that is lost in the CD of randomly oriented solutions. ACD spectra of light-harvesting complex II (LHCII)-the main peripheral antenna of photosystem II in plants-in oriented lipid bilayers were recorded from the far-UV to the visible wavelength region. ACD spectra show a drastically enhanced magnitude and level of detail compared to the isotropic CD spectra, resolving a greater number of bands and weak optical transitions. Exciton calculations show that the spectral features in the chlorophyll Q y region are well-reproduced by an existing Hamiltonian for LHCII, providing further evidence for the identity of energy sinks at chlorophylls a603 and a610 in the stromal layer and chlorophylls a604 and a613 in the luminal layer. We propose ACD spectroscopy to be a valuable tool linking the three-dimensional structure and the photophysical properties of pigment-protein complexes.

International audience; Recent chlorophyll-a fluorescence yield measurements, using single-turnover saturating flashes (STSFs), have revealed the involvement of a rate-limiting step in the reactions following the charge separation induced by the first flash (Magyar et al. 2018). As also shown here, in diuron-inhibited PSII core complexes isolated from Thermosynechococcus vulcanus the fluorescence maximum could only be reached by a train of STSFs. In order to elucidate the origin of the fluorescence yield increments in STSF series, we performed transient absorption measurements at 819 nm, reflecting the photooxidation and re-reduction kinetics of the primary electron donor P680. Upon single flash excitation of the dark-adapted sample, the decay kinetics could be described with lifetimes of 17 ns (~50%) and 167 ns (~30%), and a longer-lived component (~20%). This kinetics are attributed to re-reduction of P680•+ by the donor side of PSII. In contrast, upon second-flash (with Δt between 5 μs and 100 ms) or repetitive excitation, the 819 nm absorption changes decayed with lifetimes of about 2 ns (~60%) and 10 ns (~30%), attributed to recombination of the primary radical pair P680•+ Pheo•- , and a small longer-lived component (~10%). These data confirm that only the first STSF is capable of generating stable charge separation - leading to the reduction of QA ; and thus, the fluorescence yield increments elicited by the consecutive flashes must have a different physical origin. Our double-flash experiments indicate that the rate-limiting steps, detected by chlorophyll-a fluorescence, are not correlated with the turnover of P680. This article is protected by copyright. All rights reserved.

The quenching of chlorophyll-a fluorescence was investigated in plant light-harvesting complex II (LHCII) embedded in reconstituted membranes containing thylakoid lipids and a lipid label. The proteoliposomes were further separated by density and the protein and lipid contents of the fractions were quantified spectrophotometrically, allowing tighter control over the L/P ratios in a wide range of values. Using time-resolved fluorescence, we found a strong correlation between the fluorescence quenching and the L/P ratio, in line with other studies reporting progressive quenching at low L/P ratios, presumably triggered by self-clustering in the membrane. The average fluorescence lifetimes decreased to 0.3 ns at L/P ratios below 50:1; these values are comparable to the quenching observed in plants under excess-light conditions and are accompanied by a similar far-red fluorescence signature. It is hypothesized that plants can exploit the intrinsic quenching propensity of LHCII-only membrane domains to safely store extra antenna units.

Light-harvesting complex II (LHCII) – the light-harvesting antenna of Photosystem II – is a naturally abundant system that plays an important role in photosynthesis. In this study, we present a phenomenological analysis of the excitonic energy transfer in LHCII using ultrafast two-dimensional electronic spectroscopy, that we find compares well with previous theoretical and experimental results.

Reconstitution of transmembrane proteins into liposomes is a widely used method to study their behavior under conditions closely resembling the natural ones. However, this approach does not allow precise control of the liposome size, reconstitution efficiency, and the actual protein-to-lipid ratio in the formed proteoliposomes, which might be critical for some applications and/or interpretation of data acquired during the spectroscopic measurements. Here, we present a novel strategy employing methods of proteoliposome preparation, fluorescent labeling, purification, and surface immobilization that allow us to quantify these properties using fluorescence microscopy at the single-liposome level and for the first time apply it to study photosynthetic pigment-protein complexes LHCII. We show that LHCII proteoliposome samples, even after purification with a density gradient, always contain a fraction of nonreconstituted protein and are extremely heterogeneous in both protein density and liposome sizes. This strategy enables quantitative analysis of the reconstitution efficiency of different protocols and precise fluorescence spectroscopic study of various transmembrane proteins in a controlled nativelike environment.

In higher-plant Photosystem I (PSI), the majority of “red” chlorophylls (absorbing at longer wavelengths than the reaction centre P700) are located in the peripheral antenna, but contradicting reports are given about red forms in the core complex. Here we attempt to clarify the spectroscopic characteristics and quantify the red forms in the PSI core complex, which have profound implication on understanding the energy transfer and charge separation dynamics. To this end we compare the steady-state absorption and fluorescence spectra and picosecond time-resolved fluorescence kinetics of isolated PSI core complex and PSI–LHCI supercomplex from Pisum sativum recorded at 77 K. Gaussian decomposition of the absorption spectra revealed a broad band at 705 nm in the core complex with an oscillator strength of three chlorophylls. Additional absorption at 703 nm and 711 nm in PSI–LHCI indicated up to five red chlorophylls in the peripheral antenna. Analysis of fluorescence emission spectra resolved states emitting at 705, 715 and 722 nm in the core and additional states around 705–710 nm and 733 nm in PSI–LHCI. The red states compete with P700 in trapping excitations in the bulk antenna, which occurs on a timescale of ~20 ps. The three red forms in the core have distinct decay kinetics, probably in part determined by the rate of quenching by the oxidized P700. These results affirm that the red chlorophylls in the core complex must not be neglected when interpreting kinetic experimental results of PSI.

Diatoms possess special light-harvesting proteins involved in the photoprotection mechanism called non-photochemical quenching (NPQ). These Lhcx proteins were shown to be subunits of trimeric fucoxanthin-chlorophyll complexes (FCPa) in centric diatoms, but their mode of action is still unclear. Here we investigated the influence of Fcp6, an orthologue to Lhcx1 of Thalassiosira pseudonana in the diatom Cyclotella meneghiniana, by reducing its amount using an antisense approach. Whereas the pigment interactions inside FCPa were not influenced by the presence or absence of Fcp6, as demonstrated by unaltered spectra of circular dichroism, changes could be observed on the level of thylakoids and cells in the mutants compared to WT. This fits to recent models of NPQ in diatoms, where FCP aggregation or supramolecular reorganisation is thought to be a major feature. Thus, Fcp6 (Lhcx1) appears to alter pigment-pigment interactions inside the aggregates, but not inside (un-aggregated) FCPa itself.

Energy equilibration in light-harvesting antenna systems normally occurs before energy is transferred to a reaction center. The equilibration mechanism is a characteristic of the excitation energy transfer (EET) network of the antenna. Characterizing this network is crucial in understanding the first step of photosynthesis. We present our phenomenology-based analysis procedure and results in obtaining the excitonic energy levels, spectral linewidths, and transfer-rate matrix of Light-Harvesting Complex II directly from its 2D electronic spectra recorded at 77 K with waiting times between 100 fs to 100 ps. Due to the restriction of the models and complexity of the system, a unique EET network cannot be constructed. Nevertheless, a recurring pattern of energy transfer with very similar overall time scales between spectral components (excitons) is consistently obtained. The models identify a "bottleneck" state in the 664-668 nm region although with a relatively shorter lifetime (∼4-6 ps) of this state compared to previous studies. The model also determines three terminal exciton states at 675, 677-678, and 680-681 nm that are weakly coupled to each other. The excitation energy equilibration between the three termini is found to be independent of the initial excitation conditions, which is a crucial design for the light-harvesting complexes to ensure the energy flow under different light conditions and avoid excitation trapping. We proposed two EET schemes with tentative pigment assignments based on the interpretation of the modeling results together with previous structure-based calculations and spectroscopic observables. Published version The authors acknowledge support from the Hungarian Ministry of Innovation and Technology, National Research, Development and Innovation Office (Grant Nos. NN-124904 and 2018-1.2.1-NKP-2018-00009 to P.H.L.) and the Singapore Ministry of Education, Academic Research Fund (Tier 2 Grant No. MOE2015-T2-1-039 and Tier 1 Grant No. RG15/18 to H.-S.T.). The ELI-ALPS Project (No. GINOP-2.3.6-15-2015-00001) is supported by the European Union and co-financed by the European Regional Development Fund.

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.

Chloroplast thylakoid membranes contain virtually all components of the energy-converting photosynthetic machinery. Their energized state, driving ATP synthesis, is enabled by the bilayer organization of the membrane. However, their most abundant lipid species is a non-bilayer-forming lipid, monogalactosyl-diacylglycerol; the role of lipid polymorphism in these membranes is poorly understood. Earlier 31P-NMR experiments revealed the coexistence of a bilayer and a non-bilayer, isotropic lipid phase in spinach thylakoids. Packing of lipid molecules, tested by fluorescence spectroscopy of the lipophilic dye, merocyanine-540 (MC540), also displayed heterogeneity. Now, our 31P-NMR experiments on spinach thylakoids uncover the presence of a bilayer and three non-bilayer lipid phases; time-resolved fluorescence spectroscopy of MC540 also reveals the presence of multiple lipidic environments. It is also shown by 31P-NMR that: (i) some lipid phases are sensitive to the osmolarity and ionic strength of the medium, (ii) a lipid phase can be modulated by catalytic hydrogenation of fatty acids and (iii) a marked increase of one of the non-bilayer phases upon lowering the pH of the medium is observed. These data provide additional experimental evidence for the polymorphism of lipid phases in thylakoids and suggest that non-bilayer phases play an active role in the structural dynamics of thylakoid membranes.

The multisubunit pigment-protein complex of photosystem I (PSI) consists of a core and peripheral light-harvesting antenna (LHCI). PSI is thought to be a rather rigid system and very little is known about its structural and functional flexibility. Recent data, however, suggest LHCI detachment from the PSI supercomplex upon heat and light treatments. Furthermore, it was suggested that the splitting off of LHCI acts as a safety valve for PSI core upon photoinhibition (Alboresi et al., 2009). In this work we analyzed the heat- and light-induced reorganizations in isolated PSI vesicles (stroma membrane vesicles enriched in PSI). Using differential scanning calorimetry we revealed a stepwise disassembly of PSI supercomplex above 50°C. Circular dichroism, sucrose gradient centrifugation and 77K fluorescence experiments identified the sequence of events of PSI destabilization: 3min heating at 60°C or 40min white light illumination at 25°C resulted in pronounced Lhca1/4 detachment from the PSI supercomplex, which is then followed by the degradation of Lhca2/3. The similarity of the main structural effects due to heat and light treatments supports the notion that thermo-optic mechanism, structural changes induced by ultrafast local thermal transients, which has earlier been shown to be responsible for structural changes in the antenna system of photosystem II, can also regulate the assembly and functioning of PSI antenna.

We present here the first room-temperature 2D electronic spectroscopy study of energy transfer in the plant light-harvesting complex II, LHCII. Two-dimensional electronic spectroscopy has been used to study energy transfer dynamics in LHCII trimers from the chlorophyll b Qy band to the chlorophyll a Qy band. Observing cross-peak regions corresponding to couplings between different excitonic states reveals partially resolved fine structure at the exciton level that cannot be isolated by pump–probe or linear spectroscopy measurements alone. Global analysis of the data has been performed to identify the pathways and time constants of energy transfer. The measured waiting time (Tw) dependent 2D spectra are found to be composed of 2D decay-associated spectra with three timescales (0.3 ps, 2.3 ps and >20 ps). Direct and multistep cascading pathways from the high-energy chlorophyll b states to the lowest-energy chlorophyll a states have been resolved occurring on time scales of hundreds of femtoseconds to picoseconds. ASTAR (Agency for Sci., Tech. and Research, S’pore) Published version

Excited-state relaxation dynamics and energy-transfer processes in the chlorophyll a (Chl a) manifold of the light-harvesting complex II (LHCII) were examined at physiological temperature using femtosecond two-dimensional electronic spectroscopy (2DES). The experiments were done under conditions free from singlet–singlet annihilation and anisotropic decay. Energy transfer between the different domains of the Chl a manifold was found to proceed on time scales from hundreds of femtoseconds to five picoseconds, before reaching equilibration. No component slower than 10 ps was observed in the spectral equilibration dynamics. We clearly observe the bidirectional (uphill and downhill) energy transfer of the equilibration process between excited states. This bidirectional energy flow, although implicit in the modeling and simulation of the EET processes, has not been observed in any prior transient absorption studies. Furthermore, we identified the spectral forms associated with the different energy transfer lifetimes in the equilibration process. MOE (Min. of Education, S’pore) Accepted version

Photosynthetic antenna systems enable organisms harvesting light and transfer the energy to the photosynthetic reaction centre, where the conversion to chemical energy takes place. One of the most complex antenna systems, the chlorosome, found in the photosynthetic green sulfur bacterium Chlorobaculum (Cba.) tepidum contains a baseplate, which is a scaffolding super-structure, formed by the protein CsmA and bacteriochlorophyll a. Here we present the first high-resolution structure of the CsmA baseplate using intact fully functional, light-harvesting organelles from Cba. tepidum, following a hybrid approach combining five complementary methods: solid-state NMR spectroscopy, cryo-electron microscopy, isotropic and anisotropic circular dichroism and linear dichroism. The structure calculation was facilitated through development of new software, GASyCS for efficient geometry optimization of highly symmetric oligomeric structures. We show that the baseplate is composed of rods of repeated dimers of the strongly amphipathic CsmA with pigments sandwiched within the dimer at the hydrophobic side of the helix., The chlorosome of the photosynthetic bacterium C. tepidum harvests light and transfers the energy to the photosynthetic reaction centre. Here the authors determine the structure of the baseplate, a scaffolding super-structure, to show that the baseplate consists of rods of repeated CsmA dimers containing pigment molecules.

Light-harvesting complex II (LHCII), the major peripheral antenna of Photosystem II in plants, participates in several concerted mechanisms for regulation of the excitation energy and electron fluxes in thylakoid membranes. In part, these include interaction of LHCII with Photosystem I (PSI) enhancing the latter's absorption cross-section – for example in the well-known state 1 – state 2 transitions or as a long-term acclimation to high light. In this work we examined the capability of LHCII to deliver excitations to PSI in reconstituted membranes in vitro. Proteoliposomes with native plant thylakoid membrane lipids and different stoichiometric ratios of LHCII:PSI were reconstituted and studied by steady-state and time-resolved fluorescence spectroscopy. Fluorescence emission from LHCII was strongly decreased in PSI–LHCII membranes due to trapping of excitations by PSI. Kinetic modelling of the time-resolved fluorescence data revealed the existence of separate pools of LHCII distinguished by the time scale of energy transfer. A strongly coupled pool, equivalent to one LHCII trimer per PSI, transferred excitations to PSI with near-unity efficiency on a time scale of less than 10 ps but extra LHCIIs also contributed significantly to the effective antenna size of PSI, which could be increased by up to 47% in membranes containing 3 LHCII trimers per PSI. The results demonstrate a remarkable competence of LHCII to increase the absorption cross-section of PSI, given the opportunity that the two types of complexes interact in the membrane.

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.

In photosynthesis research, circular dichroism (CD) spectroscopy is an indispensable tool to probe molecular architecture at virtually all levels of structural complexity. At the molecular level, the chirality of the molecule results in intrinsic CD; pigment-pigment interactions in protein complexes and small aggregates can give rise to excitonic CD bands, while "psi-type" CD signals originate from large, densely packed chiral aggregates. It has been well established that anisotropic CD (ACD), measured on samples with defined non-random orientation relative to the propagation of the measuring beam, carries specific information on the architecture of molecules or molecular macroassemblies. However, ACD is usually combined with linear dichroism and can be distorted by instrumental imperfections, which given the strong anisotropic nature of photosynthetic membranes and complexes, might be the reason why ACD is rarely studied in photosynthesis research. In this study, we present ACD spectra, corrected for linear dichroism, of isolated intact thylakoid membranes of granal chloroplasts, washed unstacked thylakoid membranes, photosystem II (PSII) membranes (BBY particles), grana patches, and tightly stacked lamellar macroaggregates of the main light-harvesting complex of PSII (LHCII). We show that the ACD spectra of face- and edge-aligned stacked thylakoid membranes and LHCII lamellae exhibit profound differences in their psi-type CD bands. Marked differences are also seen in the excitonic CD of BBY and washed thylakoid membranes. Magnetic CD (MCD) spectra on random and aligned samples, and the largely invariable nature of the MCD spectra, despite dramatic variations in the measured isotropic and anisotropic CD, testify that ACD can be measured without substantial distortions and thus employed to extract detailed information on the (supra)molecular organization of photosynthetic complexes. An example is provided showing the ability of CD data to indicate such an organization, leading to the discovery of a novel crystalline structure in macroaggregates of LHCII.