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Increasing the absorption cross section of plants by introducing far-red absorbing chlorophylls (Chls) has been proposed as a strategy to boost crop yields. To make this strategy effective, these Chls should bind to the photosynthetic complexes without altering their functional architecture. To investigate if plant-specific antenna complexes can provide the protein scaffold to accommodate these Chls, we have reconstituted the main light-harvesting complex (LHC) of plants LHCII in vitro and in silico, with Chl d. The results demonstrate that LHCII can bind Chl d in a number of binding sites, shifting the maximum absorption μ25 nm toward the red with respect to the wild-type complex (LHCII with Chl a and b) while maintaining the native LHC architecture. Ultrafast spectroscopic measurements show that the complex is functional in light harvesting and excitation energy transfer. Overall, we here demonstrate that it is possible to obtain plant LHCs with enhanced far-red absorption and intact functional properties.

Summary Photosystem I (PSI) converts photons into electrons with a nearly 100% quantum efficiency. Its minimal energy requirement for photochemistry corresponds to a 700-nm photon, representing the well-known “red limit” of oxygenic photosynthesis. Recently, some cyanobacteria containing the red-shifted pigment chlorophyll f have been shown to harvest photons up to 800 nm. To investigate the mechanism responsible for converting such low-energy photons, we applied steady-state and time-resolved spectroscopies to the chlorophyll-f-containing PSI and chlorophyll-a-only PSI of various cyanobacterial strains. Chlorophyll-f-containing PSI displays a less optimal energetic connectivity between its pigments. Nonetheless, it consistently traps long-wavelength excitations with a surprisingly high efficiency, which can only be achieved by lowering the energy required for photochemistry, i.e., by “breaking the red limit”. We propose that charge separation occurs via a low-energy charge-transfer state to reconcile this finding with the available structural data excluding the involvement of chlorophyll f in photochemistry.

Plants and cyanobacteria use the chlorophylls embedded in their photosystems to absorb photons and perform charge separation, the first step of converting solar energy to chemical energy. While oxygenic photosynthesis is primarily based on chlorophyll a photochemistry, which is powered by red light, a few cyanobacterial species can harness less energetic photons when growing in far-red light. Acclimatization to far-red light involves the incorporation of a small number of molecules of red-shifted chlorophyll f in the photosystems, whereas the most abundant pigment remains chlorophyll a. Due to its different energetics, chlorophyll f is expected to alter the excited-state dynamics of the photosynthetic units and, ultimately, their performances. Here we combined time-resolved fluorescence measurements on intact cells and isolated complexes to show that chlorophyll f insertion slows down the overall energy trapping in both photosystems. While this marginally affects the efficiency of photosystem I, it substantially decreases that of photosystem II. Nevertheless, we show that despite the lower energy output, the insertion of red-shifted chlorophylls in the photosystems remains advantageous in environments that are enriched in far-red light and therefore represents a viable strategy for extending the photosynthetically active spectrum in other organisms, including plants. However, careful design of the new photosynthetic units will be required to preserve their efficiency.

The heterologous expression of the far-red absorbing chlorophyll (Chl) f in organisms that do not synthesize this pigment has been suggested as a viable solution to expand the solar spectrum that drives oxygenic photosynthesis. In this study, we investigate the functional binding of Chl f to the Photosystem I (PSI) of the cyanobacterium Synechococcus 7002, which has been engineered to express the Chl f synthase gene. By optimizing growth light conditions, one-to-four Chl f pigments were found in the complexes. By using a range of spectroscopic techniques, isolated PSI trimeric complexes were investigated to determine how the insertion of Chl f affects excitation energy transfer and trapping efficiency. The results show that the Chls f are functionally connected to the reaction center of the PSI complex and their presence does not change the overall pigment organization of the complex. Chl f substitutes Chl a (but not the Chl a red forms) while maintaining efficient energy transfer within the PSI complex. At the same time, the introduction of Chl f extends the photosynthetically active radiation of the new hybrid PSI complexes up to 750 nm, which is advantageous in far-red light enriched environments. These conclusions provide insights to engineer the photosynthetic machinery of crops to include Chl f and therefore increase the light-harvesting capability of photosynthesis.

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes capable of oxidizing crystalline cellulose and the enzyme has large practical application in the process of refining biomass. The LPMO catalytic mechanism still remains debated despite several proposed reaction mechanisms. Here, we report a long-lived intermediate (t½= 6 – 8 minutes) observed in an LPMO fromThermoascus aurantiacus(TaLPMO9A). The intermediate with a strong absorption around 420 nm is formed when reduced LPMO-Cu(I) reacts with H2O2. UV-vis absorption spectroscopy, electron paramagnetic resonance (EPR), and stopped-flow spectroscopy indicate that the observed long-lived intermediate involves the copper center and a nearby tyrosine (Tyr175). We propose that the reaction with H2O2first forms a highly reactive short-lived Cu(III)-intermediate which is subsequently transformed into the observed long-lived copper-dependent intermediate. Since sub-equimolar amount of H2O2to LPMO boosts oxidation of phosphoric acid swollen cellulose (PASC) suggests that the long-lived copper-dependent intermediate is part of the catalytic mechanism for LPMOs. The proposed mechanism offers new perspectives in the oxidative reaction mechanism of copper enzymes and hence for the biomass oxidation and the reactivity of copper in biological systems.

Light-harvesting complex II (LHCII) is a pigment-protein complex present in higher plants and green algae. LHCII represents the main site of light absorption, and its role is to transfer the excitation energy toward the photosynthetic reaction centers, where primary energy conversion reactions take place. The optical properties of LHCII are known to depend on protein conformation. However, the relation between the structural and spectroscopic properties of the pigments is not fully understood yet. In this respect, previous classical molecular dynamics simulations of LHCII in a model membrane [Sci. Rep. 2015, 5, 1-10] have shown that the configuration and excitonic coupling of a chlorophyll (Chl) dimer functioning as the main terminal emitter of the complex are particularly sensitive to conformational changes. Here, we use quantum chemistry calculations to investigate in greater detail the effect of pigment-pigment interactions on the excited-state landscape. While most previous studies have used a local picture in which electrons are localized on single pigments, here we achieve a more accurate description of the Chl dimer by adopting a supramolecular picture where time-dependent density functional theory is applied to the whole system at once. Our results show that specific dimer configurations characterized by shorter inter-pigment distances can result in a sizable intensity decrease (up to 36%) of the Chl absorption bands in the visible spectral region. Such a decrease can be predicted only when accounting for Chl-Chl charge-transfer excitations, which is possible using the above-mentioned supramolecular approach. The charge-transfer character of the excitations is quantified by two types of analyses: one focusing on the composition of the excitations and the other directly on the observable total absorption intensities.

Cyanobacteria carry out photosynthetic light-energy conversion using phycobiliproteins for light harvesting and the chlorophyll-rich photosystems for photochemistry. While most cyanobacteria only absorb visible photons, some of them can acclimate to harvest far-red light (FRL, 700-800 nm) by integrating chlorophyll f and d in their photosystems and producing red-shifted allophycocyanin. Chlorophyll f insertion enables the photosystems to use FRL but slows down charge separation, reducing photosynthetic efficiency. Here we demonstrate with time-resolved fluorescence spectroscopy that charge separation in chlorophyll-f-containing Photosystem II becomes faster in the presence of red-shifted allophycocyanin antennas. This is different from all known photosynthetic systems, where additional light-harvesting complexes slow down charge separation. Based on the available structural information, we propose a model for the connectivity between the phycobiliproteins and Photosystem II that qualitatively accounts for our spectroscopic data. This unique design is probably important for these cyanobacteria to efficiently switch between visible and far-red light.

Photosynthesis starts when a pigment in the photosynthetic antennae absorbs a photon. The electronic excitation energy is then transferred through the network of light-harvesting pigments to special chlorophyll (Chl) molecules in the reaction centres, where electron transfer is initiated. Energy transfer and primary electron transfer processes take place on timescales ranging from femtoseconds to nanoseconds, and can be monitored in real time via time-resolved fluorescence spectroscopy. This method is widely used for measurements on unicellular photosynthetic organisms, isolated photosynthetic membranes, and individual complexes. Measurements on intact leaves remain a challenge due to their high structural heterogeneity, high scattering, and high optical density, which can lead to optical artefacts. However, detailed information on the dynamics of these early steps, and the underlying structure–function relationships, is highly informative and urgently required in order to get deeper insights into the physiological regulation mechanisms of primary photosynthesis. Here, we describe a current methodology of time-resolved fluorescence measurements on intact leaves in the picosecond to nanosecond time range. Principles of fluorescence measurements on intact leaves, possible sources of alterations of fluorescence kinetics and the ways to overcome them are addressed. We also describe how our understanding of the organisation and function of photosynthetic proteins and energy flow dynamics in intact leaves can be enriched through the application of time-resolved fluorescence spectroscopy on leaves. For that, an example of a measurement on Zea mays leaves is presented. Electronic supplementary material The online version of this article (10.1007/s11120-018-0607-8) contains supplementary material, which is available to authorized users.

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes which promote the degradation of recalcitrant polysaccharides like cellulose or chitin. Here, we have investigated the thermostability of an LPMO from Thermoascus aurantiacus (TaLPMO9A). TaLPMO9A was found to retain most of its initial activity after incubating at 100 °C while its apparent melting temperature (Tm) is 69 °C at neutral pH. Interestingly, our studies show that holoTaLPMO9A, apoTaLPMO9A and deglycosylated TaLPMO9A can fold back to their original conformation upon lowering the temperature. In the presence of β-mercaptoethanol the protein does not refold. Activity of TaLPMO9A and refolded TaLPMO9A was studied by an Amplex® Red assay as well as by TaLPMO9A catalysed oxidation of phosphoric acid swollen cellulose (PASC). These studies confirm the functional regain of TaLPMO9A activity upon going through one cycle of unfolding and refolding. The thermal unfolding and refolding of TaLPMO9A was measured spectroscopically. Utilizing the two-state model, detailed thermodynamic parameters were obtained for holoTaLPMO. Furthermore, we have investigated the kinetics of TaLPMO9A unfolding and refolding. Our results have implications in understanding LPMO stability, which is crucial for the efficient application of LPMOs as biocatalysts during biomass degradation.

Photosynthetic reactions in plants, algae, and cyanobacteria are driven by photosystem I and photosystem II complexes, which specifically reduce or oxidize partner redox biomolecules. Photosynthetic complexes can also bind synthetic organic molecules, which inhibit their photoactivity and can be used both to study the electron transport chain and as herbicides and algicides. Thus, their development, characterization, and sensing bears fundamental and applied interest. Substantial efforts have been devoted to developing photosensors based on photosystem II to detect compounds that bind to the plastoquinone sites of this complex. In comparison, photosystem I based sensors have received less attention and could be used to identify novel substances displaying phytotoxic effects, including those obtained from natural product extracts. We have developed a robust procedure to functionalize gold electrodes with photo- and redox-active photosystem I complexes based on transparent gold and a thiolate self-assembled monolayer, and we have obtained reproducible electrochemical photoresponses. Chronoamperometric recordings have allowed us to measure photocurrents in the presence of the viologen derivative paraquat at concentrations below 100 nM under lock-in operation and a sensor dynamic range spanning six orders of magnitude up to 100 mM. We have modeled their time course to identify the main electrochemical processes and limiting steps in the electron transport chain. Our results allow us to isolate the contributions from photosystem I and the redox mediator, and evaluate photocurrent features (spectral and power dependence, fast transient kinetics) that could be used as a sensing signal to detect other inhibitors and modulators of photosystem I activity.

Evergreen conifers in boreal forests can survive extremely cold (freezing) temperatures during long dark winter and fully recover during summer. A phenomenon called “sustained quenching” putatively provides photoprotection and enables their survival, but its precise molecular and physiological mechanisms are not understood. To unveil them, here we have analyzed seasonal adjustment of the photosynthetic machinery of Scots pine (Pinus sylvestris) trees by monitoring multi-year changes in weather, chlorophyll fluorescence, chloroplast ultrastructure, and changes in pigment-protein composition. Analysis of Photosystem II and Photosystem I performance parameters indicate that highly dynamic structural and functional seasonal rearrangements of the photosynthetic apparatus occur. Although several mechanisms might contribute to ‘sustained quenching’ of winter/early spring pine needles, time-resolved fluorescence analysis shows that extreme down-regulation of photosystem II activity along with direct energy transfer from photosystem II to photosystem I play a major role. This mechanism is enabled by extensive thylakoid destacking allowing for the mixing of PSII with PSI complexes. These two linked phenomena play crucial roles in winter acclimation and protection., Evergreen conifers rely on ‘sustained quenching’ to protect their photosynthetic machinery during long, cold winters. Here, Bag et al. show that direct energy transfer (spillover) from photosystem II to photosystem I triggered by loss of grana stacking in chloroplast is the major component of sustained quenching in Scots pine.

Background Lytic polysaccharide monooxygenases (LPMOs) are indispensable redox enzymes used in industry for the saccharification of plant biomass. LPMO-driven cellulose oxidation can be enhanced considerably through photobiocatalysis using chlorophyll derivatives and light. Water soluble chlorophyll binding proteins (WSCPs) make it is possible to stabilize and solubilize chlorophyll in aqueous solution, allowing for in vitro studies on photostability and ROS production. Here we aim to apply WSCP–Chl a as a photosensitizing complex for photobiocatalysis with the LPMO, TtAA9. Results We have in this study demonstrated how WSCP reconstituted with chlorophyll a (WSCP–Chl a) can create a stable photosensitizing complex which produces controlled amounts of H2O2 in the presence of ascorbic acid and light. WSCP–Chl a is highly reactive and allows for tightly controlled formation of H2O2 by regulating light intensity. TtAA9 together with WSCP–Chl a shows increased cellulose oxidation under low light conditions, and the WSCP–Chl a complex remains stable after 24 h of light exposure. Additionally, the WSCP–Chl a complex demonstrates stability over a range of temperatures and pH conditions relevant for enzyme activity in industrial settings. Conclusion With WSCP–Chl a as the photosensitizer, the need to replenish Chl is greatly reduced, enhancing the catalytic lifetime of light-driven LPMOs and increasing the efficiency of cellulose depolymerization. WSCP–Chl a allows for stable photobiocatalysis providing a sustainable solution for biomass processing.

Carotenoids are essential in oxygenic photosynthesis: they stabilize the pigment–protein complexes, are active in harvesting sunlight and in photoprotection. In plants, they are present as carotenes and their oxygenated derivatives, xanthophylls. While mutant plants lacking xanthophylls are capable of photoautotrophic growth, no plants without carotenes in their photosystems have been reported so far, which has led to the common opinion that carotenes are essential for photosynthesis. Here, we report the first plant that grows photoautotrophically in the absence of carotenes: a tobacco plant containing only the xanthophyll astaxanthin. Surprisingly, both photosystems are fully functional despite their carotenoid-binding sites being occupied by astaxanthin instead of β-carotene or remaining empty (i.e. are not occupied by carotenoids). These plants display non-photochemical quenching, despite the absence of both zeaxanthin and lutein and show that tobacco can regulate the ratio between the two photosystems in a very large dynamic range to optimize electron transport., eLife digest Most life on Earth depends on photosynthesis, the process used by plants and many other organisms to store energy from sunlight and produce oxygen. The first steps of photosynthesis, the capture and conversion of sunlight into chemical energy, happen in large assemblies of proteins containing many pigment molecules called photosystems. In plants, the pigments involved in photosynthesis are green chlorophylls and carotenoids. In addition to harvesting light, carotenoids have an important role in preventing damage caused by overexposure to sunlight There are over one thousand different carotenoids in living beings, but only one, β-carotene, is present in every organism that performs the type of photosynthesis in which oxygen is released, and is thought to be essential for the process. However, this could never be proved because it is impossible to remove β-carotene from cells using typical genetic approaches without affecting all other carotenoids. Xu et al. used genetic engineering to create tobacco plants that produced a pigment called astaxanthin in place of β-carotene. Astaxanthin is a carotenoid from salmon and shrimp, not normally found in plants. These plants are the first living things known to perform photosynthesis without β-carotene and demonstrate that this pigment is not essential for photosynthesis as long as other carotenoids are present. Xu et al. also show that the photosystems can adapt to using different carotenoids, and can even operate with a reduced number of them. Xu et al’s findings show the high flexibility of photosynthesis in plants, which are able to incorporate non-native elements to the process. These results are also important in the context of increasing the photosynthetic efficiency, and thus the productivity of crops, since they show that a radical redesign of the photosynthetic machinery is feasible.

Photobiocatalysis holds great promise toward the development of sustainable and environmentally friendly processes, harnessing light to drive biocatalytic reactions. However, photobiocatalysis at the interface of insoluble substrates, such as cellulose, has not been studied in much detail. In this context, the catalytic enhancement of lytic polysaccharide monooxygenases (LPMOs) by light is of great interest to the biorefinery field due to their capacity to oxidatively cleave such recalcitrant polysaccharides which can facilitate the degradation of lignocellulose. It has previously been reported that light-driven LPMO reactions have a huge catalytic potential, but effective continuous illumination in reactors may be challenging. Therefore, we investigated the impact of intermittent illumination. We show that illumination intervals as short as 1 s/min enable LPMO catalysis on phosphoric acid-swollen cellulose (PASC) to the same level as continuous illumination. Additionally, time-resolved measurements indicate that reductant depletion, and not enzyme inactivation, limits light-driven LPMO reactions. This study shows that a 60-fold reduction in illumination time enhances LPMO catalysis while protecting reaction elements, e.g. the reductant. Most importantly, the significant enhancement of LPMO catalysis with minimal and intermittent illumination is promising toward an application of photobiocatalytic depolymerization of lignocellulose where shading and light scattering minimize light availability and continuity., SCOPUS: ar.j, info:eu-repo/semantics/published

The colonial green alga Botryococcus braunii (BB) is a potential source of biofuel due to its natural high hydrocarbon content. Unfortunately, its slow growth limits its biotechnological potential. Understanding its photosynthetic machinery could help to identify possible growth limitations. Here, we present the first study on BB light-harvesting complexes (LHCs). We purified two LHC fractions containing the complexes in monomeric and trimeric form. Both fractions contained at least two proteins with molecular weight (MW) around 25 kDa. The chlorophyll composition is similar to that of the LHCII of plants; in contrast, the main xanthophyll is loroxanthin, which substitutes lutein in most binding sites. Circular dichroism and 77 K absorption spectra lack typical differences between monomeric and trimeric complexes, suggesting that intermonomer interactions do not play a role in BB LHCs. This is in agreement with the low stability of the BB LHCII trimers as compared to the complexes of plants, which could be related to loroxanthin binding in the central (L1 and L2) binding sites. The properties of BB LHCII are similar to those of plant LHCII, indicating a similar pigment organization. Differences are a higher content of red chlorophyll a, similar to plant Lhcb3. These differences and the different Xan composition had no effect on excitation energy transfer or fluorescence lifetimes, which were similar to plant LHCII. Electronic supplementary material The online version of this article (doi:10.1007/s11120-017-0405-8) contains supplementary material, which is available to authorized users.

Photoprotective nonphotochemical quenching (NPQ) of absorbed solar energy is vital for survival of photosynthetic organisms, and NPQ modifications significantly improve plant productivity. However, the exact NPQ quenching mechanism is obscured by discrepancies between reported mechanisms, involving xanthophyll-chlorophyll (Xan-Chl) and Chl-Chl interactions. We present evidence of an experimental artifact that may explain the discrepancies: strong laser pulses lead to the formation of a novel electronic species in the major plant light-harvesting complex (LHCII). This species evolves from a high excited state of Chl a and is absent with weak laser pulses. It resembles an excitonically coupled heterodimer of Chl a and lutein (or other Xans at site L1) and acts as a de-excitation channel. Laser powers, and consequently amounts of artifact, vary strongly between NPQ studies, thereby explaining contradicting spectral signatures attributed to NPQ. Our results offer pathways toward unveiling NPQ mechanisms and highlight the necessity of careful attention to laser-induced artifacts.

In this work, we studied the changes in high-light tolerance and photosynthetic activity in leaves of the Arabidopsis (Arabidopsis thaliana) rosette throughout the vegetative stage of growth. We implemented an image-analysis work flow to analyze the capacity of both the whole plant and individual leaves to cope with excess excitation energy by following the changes in absorbed light energy partitioning. The data show that leaf and plant age are both important factors influencing the fate of excitation energy. During the dark-to-light transition, the age of the plant affects mostly steady-state levels of photochemical and nonphotochemical quenching, leading to an increased photosynthetic performance of its leaves. The age of the leaf affects the induction kinetics of nonphotochemical quenching. These observations were confirmed using model selection procedures. We further investigated how different leaves on a rosette acclimate to high light and show that younger leaves are less prone to photoinhibition than older leaves. Our results stress that both plant and leaf age should be taken into consideration during the quantification of photosynthetic and photoprotective traits to produce repeatable and reliable results.

In contrast to single cellular species, detailed information is lacking on the processes of photosynthetic acclimation for colonial algae, although these algae are important for biofuel production, ecosystem biodiversity, and wastewater treatment. To investigate differences between single cellular and colonial species, we studied the regulation of photosynthesis and photoprotection during photoacclimation for the colonial green alga Botryococcus braunii and made a comparison with the properties of the single cellular species Chlamydomonas reinhardtii. We show that B. braunii shares some high-light (HL) photoacclimation strategies with C. reinhardtii and other frequently studied green algae: decreased chlorophyll content, increased free carotenoid content, and increased nonphotochemical quenching (NPQ). Additionally, B. braunii has unique HL photoacclimation strategies, related to its colonial form: strong internal shading by an increase of the colony size and the accumulation of extracellular echinenone (a ketocarotenoid). HL colonies are larger and more spatially heterogenous than low-light colonies. Compared with surface cells, cells deeper inside the colony have increased pigmentation and larger photosystem II antenna size. The core of the largest of the HL colonies does not contain living cells. In contrast with C. reinhardtii, but similar to other biofilm-forming algae, NPQ capacity is substantial in low light. In HL, NPQ amplitude increases, but kinetics are unchanged. We discuss possible causes of the different acclimation responses of C. reinhardtii and B. braunii. Knowledge of the specific photoacclimation processes for this colonial green alga further extends the view of the diversity of photoacclimation strategies in photosynthetic organisms.

In nature, plants experience large fluctuations in light intensity and they need to balance the absorption and utilization of this energy appropriately. Non-photochemical quenching (NPQ) is a rapidly switchable mechanism that protects plants from photodamage caused by high light exposure by dissipating the excess absorbed energy as heat. It is triggered by the pH gradient across the thylakoid membrane and requires the protein PsbS and the xanthophyll zeaxanthin. However, the site and mechanism of the quencher(s) remain unknown. Here, we constructed a mutant of Arabidopsis thaliana that lacks light-harvesting complex II (LHCII), the main antenna complex of plants, to verify its contribution to NPQ. The mutant plant has normally stacked thylakoid membranes, displays no upregulation of other LHCs but shows a relative decrease in Photosystem I (PSI), which compensates for the decrease of the PSII antenna. The mutant plant exhibits a reduction in NPQ of about 60% and the remaining NPQ resembles that of mutant plants lacking chlorophyll (Chl) b, which lack all PSII peripheral antenna complexes. We thus report that PsbS-dependent NPQ occurs mainly in LHCII, but there is an additional quenching site in the PSII core.

LHCSR3 is a member of the Light-Harvesting Complexes (LHC) family, which is mainly composed of pigment-protein complexes responsible for collecting photons during the first steps of photosynthesis. Unlike related LHCs, LHCSR3 is expressed in stress conditions and has been shown to be essential for the fast component of photoprotection, non-photochemical quenching (NPQ), in the green alga Chlamydomonas reinhardtii. In plants, which do not possess LHCSR homologs, NPQ is triggered by the PSBS protein. Both PSBS and LHCSR3 possess the ability to sense pH changes but, unlike PSBS, LHCSR3 binds multiple pigments. In this work we have analyzed the properties of the pigments bound to LHCSR3 and their excited state dynamics. The data show efficient excitation energy transfer between pigments with rates similar to those observed for the other LHCs. Application of an exciton model based on a template of LHCII, the most abundant LHC, satisfactorily explains the collected steady state and time-resolved spectroscopic data, indicating that LHCSR3 has a LHC-like molecular architecture, although it probably binds less pigments. The model suggests that most of the chlorophylls have similar energy and interactions as in LHCII. The most striking difference is the localization of the lowest energy state, which is not on the Chlorophyll a (Chl a) 610-611-612 triplet as in all the LHCB antennas, but on Chl a613, which is located close to the lumen and to the pH-sensing region of the protein.

We aimed to ascertain if cholesterol levels within the reference standards for healthy non-pregnant women are a risk factor for depressive and anxiety symptoms in the immediate postpartum period. During the first week after delivery, total cholesterol levels of 120 new mothers were measured and their mood state was assessed with the Profile of Mood States (POMS). Two weeks before delivery, mothers' personal and family history of mood disturbances was assessed with the Maternal History of Mood Disturbances (MHMD) scale. Only 26 (22%) of the new mothers had normal cholesterol levels (≤200 mg/dL). Mothers with normal levels did not differ on psychometric measures from those with high levels. However, in the subgroup of mothers with normal cholesterol, those with lower levels experienced more symptoms of anxiety, depression and fatigue and scored higher on the MHMD scale. In the larger group of mothers with high cholesterol levels, history of mood disturbances and postpartum depressive and anxiety symptoms were not correlated with total cholesterol. Measuring cholesterol levels in the peripartum can be useful to identify a subgroup of women with naturally low cholesterol levels and an increased risk for postpartum depressive and anxiety symptoms.

LHCII is the most abundant membrane protein on earth. It participates in the first steps of photosynthesis by harvesting sunlight and transferring excitation energy to the core complex. Here we have analyzed the LHCII complex of the green alga Chlamydomonas reinhardtii and its association with the core of Photosystem II (PSII) to form multiprotein complexes. Several PSII supercomplexes with different antenna sizes have been purified, the largest of which contains three LHCII trimers (named S, M and N) per monomeric core. A projection map at a 13 angstrom resolution was obtained allowing the reconstruction of the 3D structure of the supercomplex. The position and orientation of the S trimer are the same as in plants; trimer M is rotated by 45 degrees and the additional trimer (named here as LHCII-N), which is taking the position occupied in plants by CP24, is directly associated with the core. The analysis of supercomplexes with different antenna sizes suggests that LhcbM1, LhcbM2/7 and LhcbM3 are the major components of the trimers in the PSII supercomplex, while LhcbM5 is part of the "extra" LHCII pool not directly associated with the supercomplex. It is also shown that Chlamydomonas LHCII has a slightly lower Chlorophyll a/b ratio than the complex from plants and a blue shifted absorption spectrum. Finally the data indicate that there are at least six LHCII trimers per dimeric core in the thylakoid membranes, meaning that the antenna size of PSII of C reinhardtii is larger than that of plants. (C) 2013 The Authors. Published by Elsevier B.V. All rights reserved.

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.

Light energy harvested by the pigments in Photosystem I (PSI) is used for charge separation in the reaction center (RC), after which the positive charge resides on a special chlorophyll dimer called P700. In studies on the PSI trapping kinetics, P700(+) is usually chemically reduced to re-open the RCs. So far, the information available about the reduction rate and possible chlorophyll fluorescence quenching effects of these reducing agents is limited. This information is indispensible to estimate the fraction of open RCs under known experimental conditions. Moreover, it would be important to understand if these reagents have a chlorophyll fluorescence quenching effects to avoid the introduction of exogenous singlet excitation quenching in the measurements. In this study, we investigated the effect of the commonly used reducing agent phenazine methosulfate (PMS) on the RC and fluorescence emission of higher plant PSI-LHCI. We measured the P700(+) reduction rate for different PMS concentrations, and show that we can give a reliable estimation on the fraction of closed RCs based on these rates. The data show that PMS is quenching chlorophyll fluorescence emission. Finally, we determined that the fluorescence quantum yield of PSI with closed RCs is 4% higher than if the RCs are open.

The light-harvesting complexes of photosystem I and II (Lhcas and Lhcbs) of plants display a high structural homology and similar pigment content and organization. Yet, the spectroscopic properties of these complexes, and accordingly their functionality, differ substantially. This difference is primarily due to the charge-transfer (CT) character of a chlorophyll dimer in all Lhcas, which mixes with the excitonic states of these complexes, whereas this CT character is generally absent in Lhcbs. By means of single-molecule spectroscopy near room temperature, we demonstrate that the presence or absence of such a CT state in Lhcas and Lhcbs can occasionally be reversed; i.e., these complexes are able to interconvert conformationally to quasi-stable spectral states that resemble the Lhcs of the other photosystem. The high structural similarity of all the Lhca and Lhcb proteins suggests that the stable conformational states that give rise to the mixed CT-excitonic state are similar for all these proteins, and similarly for the conformations that involve no CT state. This indicates that the specific functions related to Lhca and Lhcb complexes are realized by different stable conformations of a single generic protein structure. We propose that this functionality is modulated and controlled by the protein environment.

In this work we have investigated the origin of the multi-exponential fluorescence decay and of the short excited-state lifetime of Lhca4. Lhca4 is the antenna complex of Photosystem I which accommodates the red-most chlorophyll forms and it has been proposed that these chlorophylls can play a role in fluorescence quenching. Here we have compared the fluorescence decay of Lhca4 with that of several Lhca4 mutants that are affected in their red form content. The results show that neither the multi-exponentiality of the decay nor the fluorescence quenching is due to the red forms. The data indicate that Lhca4 exists in multiple conformations. The presence of the red forms, which are very sensitive to changes in the environment, allows to spectrally resolve the different conformations: a "blue" conformation with a short lifetime and a "red" one with a long lifetime. This finding strongly Supports the idea that the members of the Lhc family are able to adopt different conformations associated with their light-harvesting and photoprotective roles. The ratio between the conformations is modified by the substitution of lutein by violaxanthin. Finally, it is demonstrated that the red forms cannot be present in the quenched conformation. (C) 2010 Elsevier B.V. All rights reserved.

CP24 is a minor antenna complex of Photosystem II, which is specific for land plants. It has been proposed that this complex is involved in the process of excess energy dissipation, which protects plants from photodamage in high light conditions. Here, we have investigated the functional architecture of the complex, integrating mutation analysis with time-resolved spectroscopy. A comprehensive picture is obtained about the nature, the spectroscopic properties, and the role in the quenching in solution of the pigments in the individual binding sites. The lowest energy absorption band in the chlorophyll a region corresponds to chlorophylls 611/612, and it is not the site of quenching in CP24. Chlorophylls 613 and 614, which are present in the major light-harvesting complex of Photosystem appear to be absent in CP24. In contrast to all other light-harvesting complexes, CP24 is stable when the L1 carotenoid binding site is empty and upon mutations in the third helix, whereas mutations in the first helix strongly affect the folding/stability of the pigment-protein complex. The absence of lutein in L1 site does not have any effect on the quenching, whereas substitution of violaxanthin in the L2 site with lutein or zeaxanthin results in a complex with enhanced quenched fluorescence. Triplet-minus-singlet measurements indicate that zeaxanthin and lutein in site L2 are located closer to chlorophylls than violaxanthin, thus suggesting that they can act as direct quenchers via a strong interaction with a neighboring chlorophyll. The results provide the molecular basis for the zeaxanthin-dependent quenching in isolated CP24.

In this work the photoprotective role of all xanthophylls in LHCII, Lhcb4, and Lhcb5 is investigated by laser-induced Triplet-minus-Singlet (TmS) spectroscopy. The comparison of native LHCII trimeric complexes with different carotenoid composition shows that the xanthophylls in sites V1 and N1 do not directly contribute to the chlorophyll triplet quenching. The largest part of the triplets is quenched by the lutein bound in site L1, which is located in close proximity to the chlorophylls responsible for the low energy state of the complex. The lutein in the L2 site is also active in triplet quenching, and it shows a longer triplet lifetime than the lutein in the L1 site. This lifetime difference depends on the occupancy of the N1 binding site, where neoxanthin acts as an oxygen barrier, limiting the access of O2 to the inner domain of the Lhc complex, thereby strongly contributing to the photostability. The carotenoid triplet decay of monomeric Lhcb1, Lhcb4, and Lhcb5 is mono-exponential, with shorter lifetimes than observed for trimeric LHCII, suggesting that their inner domains are more accessible for O2. As for trimeric LHCII, only the xanthophylls in sites L1 and L2 are active in triplet quenching. Although the chlorophyll to carotenoid triplet transfer is efficient (95%) in all complexes, it is not perfect, leaving 5% of the chlorophyll triplets unquenched. This effect appears to be intrinsically related to the molecular organization of the Lhcb proteins.

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

In this work the spectroscopic properties of the special low-energy absorption bands of the outer antenna complexes of higher plant Photosystem I have been investigated by means of low-temperature absorption, fluorescence, and fluorescence line- narrowing experiments. It was found that the red- most absorption bands of Lhca3, Lhca4, and Lhca1-4 peak, respectively, at 704, 708, and 709 nm and are responsible for 725-, 733-, and 732- nm fluorescence emission bands. These bands are more red shifted compared to ``normal'' chlorophyll a (Chl a) bands present in light-harvesting complexes. The low-energy forms are characterized by a very large bandwidth (400-450 cm(-1)), which is the result of both large homogeneous and inhomogeneous broadening. The observed optical reorganization energy is untypical for Chl a and resembles more that of BChl a antenna systems. The large broadening and the changes in optical reorganization energy are explained by a mixing of an Lhca excitonic state with a charge transfer state. Such a charge transfer state can be stabilized by the polar residues around Chl 1025. It is shown that the optical reorganization energy is changing through the inhomogeneous distribution of the red- most absorption band, with the pigments contributing to the red part of the distribution showing higher values. A second red emission form in Lhca4 was detected at 705 nm and originates from a broad absorption band peaking at 690 nm. This fluorescence emission is present also in the Lhca4-N-47H mutant, which lacks the 733-nm emission band.

Photosynthetic organisms protect themselves from high-light stress by dissipating excess absorbed energy as heat in a process called non-photochemical quenching (NPQ). Zeaxanthin is essential for the full development of NPQ, but its role remains debated. The main discussion revolves around two points: where does zeaxanthin bind and does it quench? To answer these questions we have followed the zeaxanthin-dependent quenching from leaves to individual complexes, including supercomplexes. We show that small amounts of zeaxanthin are associated with the complexes, but in contrast to what is generally believed, zeaxanthin binding per se does not cause conformational changes in the complexes and does not induce quenching, not even at low pH. We show that in NPQ conditions zeaxanthin does not exchange for violaxanthin in the internal binding sites of the antennas but is located at the periphery of the complexes. These results together with the observation that the zeaxanthin-dependent quenching is active in isolated membranes, but not in functional supercomplexes, suggests that zeaxanthin is acting in between the complexes, helping to create/participating in a variety of quenching sites. This can explain why none of the antennas appears to be essential for NPQ and the multiple quenching mechanisms that have been observed in plants.

It is known that aggregation of isolated light-harvesting complex II (LHCII) in solution results in high fluorescence quenching, reduced chlorophyll fluorescence lifetime, and increased electronic coupling of carotenoid (Car) S-1 and chlorophyll (Chl) Q(y) states, as determined by two-photon studies. It has been suggested that this behavior of aggregated LHCII mimics aspects of non-photochemical quenching processes of higher plants and algae. However, several studies proposed that the minor photosystem II proteins CP24 and CP29 also play a significant role in regulation of photosynthesis. Therefore, we use a simple protocol that allows gradual aggregation also of CP24 and CP29. Similarly, as observed for LHCII, aggregation of CP24 and CP29 also leads to increasing fluorescence quenching and increasing electronic Car S-1-Chl Q(y) coupling. Furthermore, a direct comparison of the three proteins revealed a significant higher electronic coupling in the two minor proteins already in the absence of any aggregation. These differences become even more prominent upon aggregation. A red-shift of the Q(y) absorption band known from LHCII aggregation was also observed for CP29 but not for CP24. We discuss possible implications of these results for the role of CP24 and CP29 as potential valves for excess excitation energy in the regulation of photosynthetic light harvesting.

The light-harvesting complex II (LHCII) is the main energy absorber for photosynthesis in green plants, and its translocation between photosystems I and II is the primary means of energy redistribution between them. Using single-particle tracking, we performed the first measurement of the mobility of LHCII in the photosynthetic membranes in both the nonphosphorylated and the phosphorylated (P-LHCII) conformations. These are part of an important, reversible, energy re-equilibration process called the state transition. We found that the population of P-LHCII in unappressed membranes is more mobile than the population of non-P-LHCII from the same regions.

The red-most fluorescence emission of photosystem I (733 nm at 4 K) is associated with the Lhca4 subunit of the antenna complex. It has been proposed that this unique spectral feature originates from the low energy absorption band of an excitonic interaction involving chlorophyll A5 and a second chlorophyll a molecule, probably B5 (Morosinotto, T., Breton, J., Bassi, R., and Croce, R. (2003) J. Biol. Chem. 278, 49223-49229). Because of the short distances between chromophores in Lhc proteins, the possibility that other pigments are involved in the red-shifted spectral forms could not be ruled out. In this study, we have analyzed the pigment-pigment interactions between nearest neighboring chromophores in Lhca4. This was done by deleting individual chlorophyll binding sites by mutagenesis, and analyzing the changes in the spectroscopic properties of recombinant proteins refolded in vitro. The red-shifted (733 nm) fluorescence peak, the major target of this analysis, was lost upon mutations affecting sites A4, A5, and B5 and was modified by mutating site B6. In agreement with the shorter distance between chlorophylls A5 and B5 (7.9 Å) versus A4 and A5 (12.2 Å) in Lhca4 (Ben-Shem, A., Frolow, F., and Nelson, N. (2003) Nature 426, 630-635), we conclude that the low energy spectral form originates from an interaction involving pigments in sites A5 and B5. Mutation at site B6, although inducing a 15-nm blue-shift of the emission peak, maintains the red-shifted emission. This implies that chromophores responsible for the interaction are conserved and suggests a modification in the pigment organization. Besides the A5-B5 pair, evidence for additional pigment-pigment interactions between chlorophylls in sites B3-A3 and B6-A6 was obtained. However, these features do not affect the red-most spectral form responsible for the 733-nm fluorescence emission band.

The binding sites for xanthophylls in the CP29 antenna protein of higher plant Photosystem II have been investigated using recombinant proteins refolded in vitro. Despite the presence of three xanthophyll species CP29 binds two carotenoids per polypeptide. The localization of neoxanthin was studied producing a chimeric protein constructed by swapping the C-helix domain from CP29 to LHCII. The resulting holoprotein did not bind neoxanthin, confirming that the N1 site is not present in CP29. Neoxanthin in CP29 was, instead, bound to the L2 site, which is thus shown to have a wider specificity with respect to the homologous site L2 in LHCII. Lutein was found in the L1 site of CP29. For each site the selectivity for individual xanthophyll species was studied as well as its role in protein stabilization, energy transfer, and photoprotection. Putative xanthophyll binding sequences, identified by primary structure analysis as a stretch of hydrophobic residues including an acidic term, were analyzed by site-directed mutagenesis or, in one case, by deleting the entire sequence. The mutant proteins were unaffected in their xanthophyll composition, thus suggesting that the target motifs had little influence in determining xanthophyll binding, whereas hydrophobic sequences in the membrane-spanning helices are important.

In this study, two gene products (Lhca2 and Lhca3), encoding higher plants (Arabidopsis thaliana) Photosystem I antenna complexes, were overexpressed in bacteria and reconstituted in vitro with purified chloroplast pigments. The chlorophyll-xanthophyll proteins thus obtained were characterized by biochemical and spectroscopic methods. Both complexes were shown to bind 10 chlorophyll (a and b) molecules per polypeptide, Lhca2 having higher chlorophyll b content as compared to Lhca3. The two proteins differed for the number of carotenoid binding sites: two and three for Lhca2 and Lhca3, respectively. β-carotene was specifically bound to Lhca3 in addition to the xanthophylls violaxanthin and lutein, indicating a peculiar structure of carotenoid binding sites in this protein since it is the only one so far identified with the ability of binding β-carotene. Analysis of the spectroscopic properties of the two pigment proteins showed the presence of low energy absorption forms (red forms) in both complexes, albeit with different energies and amplitudes. The fluorescence emission maximum at 77 K of Lhca2 was found at 701 nm, while in Lhca3 the major emission was at 725 nm. Reconstitution of Lhca3 without Chl b reveals that Chl b is not involved in originating the low energy absorption forms of this complex. The present data are discussed in comparison to the properties of the recombinant Lhca1 and Lhca4 complexes and of the native LHCI preparation, previously analyzed, thus showing a comprehensive description of the gene products composing the Photosystem I light harvesting system of A. thaliana.

The Lhca antenna complexes of photosystem I (PSI) have been characterized by comparison of native and recombinant preparations. Eight Lhca polypeptides have been found to be all organized as dimers in the PSI–LHCI complex. The red emission fluorescence is associated not only with Lhca1–4 heterodimer, but also with dimers containing Lhca2 and/or Lhca3 complexes. Reconstitution of Lhca1 and Lhca4 monomers as well as of the Lhca1–4 dimer in vitro was obtained. The biochemical and spectroscopic features of these three complexes are reported. The monomers Lhca1 and Lhca4 bind 10 Chls each, while the Chl a/b ratio is lower in Lhca4 as compared to Lhca1. Three carotenoid binding sites have been found in Lhca1, while only two are present in Lhca4. Both complexes contain lutein and violaxanthin while β-carotene is selectively bound to the Lhca1–4 dimer in substoichiometric amounts upon dimerization. Spectral analysis revealed the presence of low energy absorption forms in Lhca1 previously thought to be exclusively associated with Lhca4. It is shown that the process of dimerization changes the spectroscopic properties of some chromophores and increases the amplitude of the red absorption tail of the complexes. The origin of these spectroscopic features is discussed.

Mutant proteins from light-harvesting complexes of higher plants may be obtained by expressing modified apoproteins in Escherichia coli, and reconstituting them in the presence of chlorophyll and carotenoid cofactors. This method has allowed, in particular, the engineering of mutant LHCs in which each of the residues coordinating the central Mg atoms of the chlorophylls was replaced by noncoordinating amino acids [Bassi, R., Croce, R., Cugini, D., and Sandonà, D. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 10056-10061]. The availability of these mutants is of particular importance for determining the precise position of absorption bands for the different chlorophyll molecules, as well as the sequence of energy transfer events that occur within LHC complexes, provided that the structural impact of each mutation is precisely evaluated. Using resonance Raman spectroscopy, we have characterized the pigment-protein interactions in the minor photosystem II antenna protein, Lhcb4 (CP29), in which each of three of the four central chlorophyll a molecules has been removed by such mutations. By comparing the spectra of these mutants with those of the wild-type protein, the state of interaction of the carbonyl group, the coordination state of the central magnesium ion, and the dielectric constant (polarity) of the immediate environment in the binding pocket of the chlorophyll a molecule were defined for each cofactor binding site. In addition, the structural impact of the absence of one chlorophyll a molecule and the quality of protein folding were evaluated for each of these mutated polypeptides.

In the last ten years, a large series of studies have targeted antenna complexes of plants (Lhc) with the aim of understanding the mechanisms of light harvesting and photoprotection. Combining spectroscopy, modeling and mutation analyses, the role of individual pigments in these processes has been highlighted in vitro. In plants, however, these proteins are associated with multiple complexes of the photosystems and function within this framework. In this work, we have envisaged a way to bridge the gap between in vitro and in vivo studies by knocking out in vivo pigments that have been proposed to play an important role in excitation energy transfer between the complexes or in photoprotection. We have complemented a CP24 knock-out mutant of Arabidopsis thaliana with the CP24 (Lhcb6) gene carrying a His-tag and with a mutated version lacking the ligand for chlorophyll 612, a specific pigment that in vitro experiments have indicated as the lowest energy site of the complex. Both complexes efficiently integrated into the thylakoid membrane and assembled into the PSII supercomplexes, indicating that the His-tag does not impair the organization in vivo. The presence of the His-tag allowed the purification of CP24-WT and of CP24-612 mutant in their native states. It is shown that CP24-WT coordinates 10 chlorophylls and 2 carotenoid molecules and has properties identical to those of the reconstituted complex, demonstrating that the complex self-assembled in vitro assumes the same folding as in the plant. The absence of the ligand for chlorophyll 612 leads to the loss of one Chl a and of lutein, again as in vitro, indicating the feasibility of the method. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy. © 2014 Elsevier B.V.

The energy transfer kinetics from carotenoids to chlorophylls and among chlorophylls has been measured by femtosecond transient absorption kinetics in a monomeric unit of the major light-harvesting complex (LHCII) from higher plants. The samples were reconstituted complexes with different carotenoid contents. The kinetics was measured both in the carotenoid absorption region and in the chlorophyll Qy region using two different excitation wavelengths suitable for selective excitation of the carotenoids. Analysis of the data shows that the overwhelming part of the energy transfer from the carotenoids occurs directly from the initially excited S2 state of the carotenoids. Only a small part (1 pathway. All the S2 energy transfer from carotenoids to chlorophylls occurs with time constants x state of Chl b and to the Qx state, while the transfer to Chl a occurs only via the Qx state. We find no compelling evidence in favor of a substantial S1 transfer path of the carotenoids, although some transfer via the S1 state of neoxanthin can not be entirely excluded. The S1 lifetimes of the two luteins were determined to be 15 ps and 3.9 ps. A detailed quantitative analysis and kinetic model of the processes described here will be presented in a separate paper.

A photosystem I preparation from maize, containing its full antenna complement (PSI-200) and in which detergent effects on chlorophyll coupling are almost completely absent, has been studied by time-resolved fluorescence techniques with approximately 5 ps resolution at 280 and 170 K in the wavelength interval of 690-780 nm. The data have been analyzed in terms of both the decay-associated spectra (DAS) and the time-resolved emission spectra (TRES). As in a previous room temperature study [Turconi, S., Weber, N., Schweitzer, D., Strotmann, H., and Holzwarth, A. R. (1994) Biochim. Biophys. Acta 1187, 324-334], the 280 K decay is well described by three DAS components in the 11-130 ps time range, the fastest of which displays both positive and negative amplitudes characteristic of excitation transfer from the bulk to the red antenna forms. Both the 57 and 130 ps components have all positive amplitudes and describe complex decay and equilibration processes involving the red forms. At 170 K, four major components in the 10-715 ps time range are required to describe the decay. The fastest represents bulk to red form transfer processes, while the 55, 216, and 715 ps decays, with all positive amplitudes, have maxima near 720, 730, and 740 nm, respectively, in accord with previous steady-state fluorescence measurements. The width and asymmetry of these DAS indicate that they are spectrally complex and represent decay and equilibration processes involving the red forms. Spectral evolution during the fluorescence decay process was analyzed in terms of the TRES. The red shifting of the TRES was analyzed in terms of the first central spectral moment (mean spectral energy) which is biexponential at both temperatures. The slower component, which describes equilibration between the red forms, leads to spectral red shifting during the entire fluorescence decay process, and the mean lifetimes of the spectral moments at 280 and 170 K (86 and 291 ps, respectively) are similar to the mean lifetimes of the fluorescence decays (119 and 384 ps, respectively). Thus, both spectral evolution and the trapping-associated fluorescence decay occur on a similar time scale, and both processes display a very similar temperature sensitivity. On the basis of these data, it is concluded that trapping in PSI-200 is to a large extent rate-limited by excitation diffusion in the antenna and in particular by the slow "uphill" transfer from the low-energy forms to the bulk and/or inner core chlorophyll molecules.

The spectral forms of the two chlorophyll species in higher plant Photosystem II antenna proteins have been experimentally determined within their protein environment. Recombinant CP29 and LHC II antenna proteins missing individual chromophores were obtained by over-expression in bacteria without any changing of the primary protein sequence and in vitro reconstitution. Difference absorption spectroscopy with respect to the corresponding proteins binding the complete pigment complement yielded the spectral shape and extinction of single chlorophyll a and b. A functional relation of their absorption was given by Gaussian subband decomposition covering the entire Qx and Qy optical region together with the absolute value of the molar extinction coefficient. With respect to analogous determinations reported in the literature for organic solvents, this information is valuable for further understanding the in-protein chlorophyll excited states and excited state dynamics: in particular, for the calculation of Forster transfer rates by means of chlorophyll–chlorophyll overlap integral employing the Stepanov relation for emission and single chromophore transition energies according to the results of mutational analysis of chlorophyll binding sites [Bassi et al. (1999) Proc Natl Acad Sci USA 96: 10056–10061; Remelli et al. (1999) J Biol Chem 274: 33510–33521].

The chromophore binding properties of the higher plant light-harvesting complex II have been studied by site-directed mutagenesis of pigment-binding residues. Mutant apoproteins were overexpressed in Escherichia coli and then refolded in vitro with purified chromophores to yield holoproteins selectively affected in chlorophyll-binding sites. Biochemical and spectroscopic characterization showed a specific loss of pigments and absorption spectral forms for each mutant, thus allowing identification of the chromophores bound to most of the binding sites. On these bases a map for the occupancy of individual sites by chlorophyll a and chlorophyll b is proposed. In some cases a single mutation led to the loss of more than one chromophore indicating that four chlorophylls and one xanthophyll could be bound by pigment-pigment interactions. Differential absorption spectroscopy allowed identification of the Q(y) transition energy level for each chlorophyll within the complex. It is shown that not only site selectivity is largely conserved between light-harvesting complex II and CP29 but also the distribution of absorption forms among different protein domains, suggesting conservation of energy transfer pathways within the protein and outward to neighbor subunits of the photosystem.

Recombinant light-harvesting complex II (LHCII) proteins with modified carotenoid composition have been obtained by in vitro reconstitution of the Lhcb1 protein overexpressed in bacteria. The monomeric protein possesses three xanthophyll-binding sites. The L1 and L2 sites, localized by electron crystallography in the helix A/helix B cross, have the highest affinity for lutein, but also bind violaxanthin and zeaxanthin with lower affinity. The latter xanthophyll causes disruption of excitation energy transfer. The occupancy of at least one of these sites, probably L1, is essential for protein folding. Neoxanthin is bound to a distinct site (N1) that is highly selective for this species and whose occupancy is not essential for protein folding. Whereas xanthophylls in the L1 and L2 sites interact mainly with chlorophyll a, neoxanthin shows strong interaction with chlorophyll b, inducing the hyperchromic effect of the 652 nm absorption band. This observation explains the recent results of energy transfer from carotenoids to chlorophyll b obtained by femtosecond absorption spectroscopy. Whereas xanthophylls in the L1 and L2 sites are active in photoprotection through chlorophyll-triplet quenching, neoxanthin seems to act mainly in 1O2/* scavenging.

The Q(y) transition dipole moment vectors of all eight chlorophylls in the higher-plant antenna protein CP29 were calculated by an original method on the basis of linear dichroism and absorption spectroscopy. The contribution of individual chromophores was determined from difference spectra between wild type and mutant proteins in which a single chlorophyll has been removed by mutating pigment-binding residues. Recombinant proteins were constructed by overexpressing the apoprotein in bacteria and refolding of the pigment-protein complex in vitro [Bassi, R., Croce, R., Cugini, D., and Sandona, D. (1999) Proc. Natl. Acad. Sci. U.S.A. (in press)]. The spectroscopic data are interpreted on the basis of a protein structural model obtained via the homology with the major antenna complex LHCII [Kuhlbrandt, W., Wang, D. N., and Fujiyoshi, Y. (1994) Nature 367, 614-621]. The results allow us to determine the orientation of six chromophores within the protein structure. The orientations of the two remaining chromophores are inferred by considering the symmetry properties of CP29 and fitting steady state absorption and linear dichroism spectra by independent chlorophyll spectral forms. As a consequence, four 'mixed' sites with different chlorophyll a and b binding affinities are identified in CP29. Geometrical data and the Forster mechanism for energy transfer suggest that excitation energy equilibrates rapidly among chlorophyll 'pure' sites while energy preferentially flows outward from chlorophyll 'mixed' sites. The orientation of the dipole moments of two chlorophyll molecules symmetrically located at the center of the protein and parallel to the carotenoid transition vectors suggests a role in energy transfer from xanthophyll to chlorophyll.

The chromophore-binding properties of the higher plant light-harvesting protein CP29 have been studied by using site-directed mutagenesis of pigment-binding residues. Overexpression of the apoproteins in bacteria was followed by reconstitution in vitro with purified pigments, thus obtaining a family of mutant CP29 proteins lacking individual chromophore-binding sites. Biochemical characterization allowed identification of the eight porphyrins and two xanthophyll-binding sites. It is shown that the four porphyrin-binding sites (A1, A2, A4, and A5) situated in the central, twofold-symmetrical domain of the protein are selective for Chl-a, whereas the four peripheral sites (A3, B3, B5, and B6) have mixed Chl-a–Chl-b specificity. Within a site, porphyrin coordination by glutamine increases affinity for Chl-b as compared with glutamate. Xanthophyll site L1 is occupied by lutein, whereas site L2 can bind violaxanthin or neoxanthin. The protein is relatively stable when site L2 site is empty, suggesting that xanthophylls can be exchanged during operation of xanthophyll cycle-dependent photoprotection mechanism. Differential absorption spectroscopy allowed determination of transition energy levels for individual chromophores, thus opening the way to calculation of energy-transfer rates between Chl in higher plant antenna proteins.

The distribution of xanthophyll cycle pigments (violaxanthin plus antheraxanthin plus zeaxanthin [VAZ]) among photosynthetic pigment-protein complexes was examined in Vinca majorbefore, during, and subsequent to a photoinhibitory treatment at low temperature. Four pigment-protein complexes were isolated: the core of photosystem (PS) II, the major light-harvesting complex (LHC) protein of PSII (LHCII), the minor light-harvesting proteins (CPs) of PSII (CP29, CP26, and CP24), and PSI with its LHC proteins (PSI-LHCI). In isolated thylakoids 80% of VAZ was bound to protein independently of the de-epoxidation state and was found in all complexes. Plants grown outside in natural sunlight had higher levels of VAZ (expressed per chlorophyll), compared with plants grown in low light in the laboratory, and the additional VAZ was mainly bound to the major LHCII complex, apparently in an acid-labile site. The extent of de-epoxidation of VAZ in high light and the rate of reconversion of Z plus A to V following 2.5 h of recovery were greatest in the free-pigment fraction and varied among the pigment-protein complexes. Photoinhibition caused increases in VAZ, particularly in low-light-acclimated leaves. The data suggest that the photoinhibitory treatment caused an enrichment in VAZ bound to the minor CPs caused by de novo synthesis of the pigments and/or a redistribution of VAZ from the major LHCII complex.

Singlet energy transfer between the carotenoids (Cars) and chlorophylls (Chls) in the light-harvesting complex II (LHC II) from higher plants has been studied using ultrafast transient absorption spectroscopy by exciting the Cars directly in the 475-515 nm wavelength range. LHC II trimers from Arabidopsis thaliana with well-defined Car compositions have been used. From HPLC, the wild type (WT) monomer contains two luteins (Ls), one neoxanthin (N), and a trace of violaxanthin (V) per 12 Chls. The ABA-3 mutant contains 1.4 Ls and 0.6 zeaxanthin (Z) per monomer. Though exploitation of the difference in Car constitution and exciting the WT at 475 and 490 nm, and the ABA-3 mutant at 490 and 515 nm, the different Car contributions to energy transfer have been probed. Evidence for energy transfer mainly from the Car to Chl b is observed in the WT. In the mutant, additional transfer from Car to Chl a correlates with the presence of Z. The results imply predominant energy transfer from the central Ls to Chl b which requires a modification of the currently accepted arrangement of Chl pigments in LHC II.

The minor light-harvesting chlorophyll-a/b-binding protein CP29 (Lhcb4), overexpressed in Escherichia coil, has been reconstituted in vitro with pigments. The recombinant pigment-protein complexes show biochemical and spectral properties identical to the native CP29 purified from maize thylakoids. The xanthophyll lutein is the only carotenoid necessary for reconstitution, a finding consistent with the structural role of two lutein molecules/polypeptide suggested by the crystallographic data for: the homologous protein light-harvesting chlorophyll-a/b-binding protein of photosystem II (LHCII). The CP29 protein scaffold can accommodate different chromophores. This conclusion was deduced by the observation that the pigment composition of the reconstituted protein depends on the pigments present in the reconstitution mixture. Thus, in addition to a recombinant CP29 identical to the native One, two additional forms of the complex could be obtained by increasing chlorophyll b content. This finding is typical of CP29 because the major LHCII complex shows an absolute selectivity for chromophore binding [Plumley, E G. and Schmidt, G. W. (1987) Proc. Natl Acad. Sci. USA 84, 146-1501 Paulsen, H., Rumler, U. and Rudiger, W (1990) Planta (Neidelb.) 181, 204-211], and it is consistent with the higher stability of CP29 during greening and in chlorophyll b mutants compared with LHCII.