Carotenoids are essential constituents of plant light-harvesting complexes (LHCs), being involved in protein stability, light harvesting, and photoprotection. Unlike chlorophylls, whose binding to LHCs is known to require coordination of the central magnesium, carotenoid binding relies on weaker intermolecular interactions (such as hydrogen bonds and van der Waals forces), whose character is far more elusive. Here we addressed the key interactions responsible for carotenoid binding to LHCs by combining molecular dynamics simulations and polarizable quantum mechanics/molecular mechanics calculations on the major LHC, LHCII. We found that carotenoid binding is mainly stabilized by van der Waals interactions with the surrounding chlorophyll macrocycles rather than by hydrogen bonds to the protein, the latter being more labile than predicted from structural data. Furthermore, the interaction network in the binding pockets is relatively insensitive to the chemical structure of the embedded carotenoid. Our results are consistent with a number of experimental data and challenge the role played by specific interactions in the assembly of pigment-protein complexes.

Photosynthetic organisms utilize dynamic and complex networks of pigments bound within light-harvesting complexes to transfer solar energy from antenna complexes to reaction centers. Understanding the principles underlying the efficiency of these energy transfer processes, and how they may be incorporated into artificial light-harvesting systems, is facilitated by the construction of easily tunable model systems. We describe a protein-based model to mimic directional energy transfer between light-harvesting complexes using a circular permutant of the tobacco mosaic virus coat protein (cpTMV), which self-assembles into a 34-monomer hollow disk. Two populations of cpTMV assemblies, one labeled with donor chromophores and another labeled with acceptor chromophores, were coupled using a direct protein-protein bioconjugation method. Using potassium ferricyanide as an oxidant, assemblies containing o-aminotyrosine were activated toward the addition of assemblies containing p-aminophenylalanine. Both of these noncanonical amino acids were introduced into the cpTMV monomers through amber codon suppression. This coupling strategy has the advantages of directly, irreversibly, and site-selectively coupling donor with acceptor protein assemblies and avoids cross-reactivity with native amino acids and undesired donor-donor or acceptor-acceptor combinations. The coupled donor-acceptor model was shown to transfer energy from an antenna disk containing donor chromophores to a downstream disk containing acceptor chromophores. This model ultimately provides a controllable and modifiable platform for understanding photosynthetic interassembly energy transfer and may lead to the design of more efficient functional light-harvesting materials.

Plants are designed to utilize visible light for photosynthesis. Expanding this light absorption toward the far-red could boost growth in low-light conditions and potentially increase crop productivity in dense canopies. A promising strategy is broadening the absorption of antenna complexes to the far-red. In this study, we investigated the capacity of the photosystem I antenna protein Lhca4 to incorporate far-red absorbing chlorophylls d and f and optimize their spectra. We demonstrate that these pigments can successfully bind to Lhca4, with the protein environment further red-shifting the chlorophyll d absorption, markedly extending the absorption range of this complex above 750 nm. Notably, chlorophyll d substitutes the canonical chlorophyll a red-forms, resulting in the most red-shifted emission observed in a plant light-harvesting complex. Using ultrafast spectroscopy, we show that the introduction of these novel chlorophylls does not interfere with the excited state decay or the energy equilibration processes within the complex. The results demonstrate the feasibility of engineering plant antennae to absorb deeper into the far-red region while preserving their functional and structural integrity, paving the way for innovative strategies to enhance photosynthesis.

The mature architecture of the photosynthetic membrane of the purple phototroph Rhodobacter sphaeroides has been characterised to a level where an atomic-level membrane model is available, but the roles of the putative assembly proteins LhaA and PucC in establishing this architecture are unknown. Here we investigate the assembly of light-harvesting LH2 and reaction centre-light-harvesting1-PufX (RC-LH1-PufX) photosystem complexes using spectroscopy, pull-downs, native gel electrophoresis, quantitative mass spectrometry and fluorescence lifetime microscopy to characterise a series of lhaA and pucC mutants. LhaA and PucC are important for specific assembly of LH1 or LH2 complexes, respectively, but they are not essential; the few LH1 subunits found in ΔlhaA mutants assemble to form normal RC-LH1-PufX core complexes showing that, once initiated, LH1 assembly round the RC is cooperative and proceeds to completion. LhaA and PucC form oligomers at sites of initiation of membrane invagination; LhaA associates with RCs, bacteriochlorophyll synthase (BchG), the protein translocase subunit YajC and the YidC membrane protein insertase. These associations within membrane nanodomains likely maximise interactions between pigments newly arriving from BchG and nascent proteins within the SecYEG-SecDF-YajC-YidC assembly machinery, thereby co-ordinating pigment delivery, the co-translational insertion of LH polypeptides and their folding and assembly to form photosynthetic complexes.

The light-harvesting protein complexes (Lhc) play key roles in the processes of light absorption and protection in diatoms. However, different Lhc protein carries out distinct function in photosynthesis. For now, roles of many Lhc proteins in light acclimation are largely unknown. Here, function of Lhcx3 in marine diatom Phaeodactylum tricornutum was examined by using reverse genetic technologies. The overexpression of Lhcx3 led to increased diadinoxanthin + diatoxanthin content and elevated non-photochemical fluorescence quenching (NPQ) while knockdown of Lhcx3 reduced NPQ level. In addition, the expression of Lhcx3 could be induced by blue light but not by red light. After addition of the photosynthetic inhibitor, upregulation of Lhcx3 transcript in high light could be inhibited by NH4Cl, but not by DCMU (3-(3,4-dichlorophenyl)-l,l-dim ethylurea). In contrast, DCMU addition increased expression of Lhcx3 in high light. In combination with changes of NPQ after addition of inhibitor, we concluded that the Lhcx3 played key roles in high light acclimation of diatoms. This finding will provide new clues for genetic improvement of P. tricornutum with an aim to cultivate new strains with high growth rate.

Diverse algae of the red lineage possess chlorophyll a-binding proteins termed LHCR, comprising the PSI light-harvesting system, which represent an ancient antenna form that evolved in red algae and was acquired through secondary endosymbiosis. However, the function and regulation of LHCR complexes remain obscure. Here we describe isolation of a Nannochloropsis oceanica LHCR mutant, named hlr1, which exhibits a greater tolerance to high-light (HL) stress compared to the wild type. We show that increased tolerance to HL of the mutant can be attributed to alterations in PSI, making it less prone to ROS production, thereby limiting oxidative damage and favoring growth in HL. HLR1 deficiency attenuates PSI light-harvesting capacity and growth of the mutant under light-limiting conditions. We conclude that HLR1, a member of a conserved and broadly distributed clade of LHCR proteins, plays a pivotal role in a dynamic balancing act between photoprotection and efficient light harvesting for photosynthesis.

The mildly thermophilic purple phototrophic bacterium Allochromatium tepidum provides a unique model for investigating various intermediate phenotypes observed between those of thermophilic and mesophilic counterparts. The core light-harvesting (LH1) complex from A. tepidum exhibits an absorption maximum at 890 nm and mildly enhanced thermostability, both of which are Ca2+-dependent. However, it is unknown what structural determinants might contribute to these properties. Here, we present a cryo-EM structure of the reaction center-associated LH1 complex at 2.81 Å resolution, in which we identify multiple pigment-binding α- and β-polypeptides within an LH1 ring. Of the 16 α-polypeptides, we show that six (α1) bind Ca2+ along with β1- or β3-polypeptides to form the Ca2+-binding sites. This structure differs from that of fully Ca2+-bound LH1 from Thermochromatium tepidum, enabling determination of the minimum structural requirements for Ca2+-binding. We also identified three amino acids (Trp44, Asp47, and Ile49) in the C-terminal region of the A. tepidum α1-polypeptide that ligate each Ca ion, forming a Ca2+-binding WxxDxI motif that is conserved in all Ca2+-bound LH1 α-polypeptides from other species with reported structures. The partial Ca2+-bound structure further explains the unusual phenotypic properties observed for this bacterium in terms of its Ca2+-requirements for thermostability, spectroscopy, and phototrophic growth, and supports the hypothesis that A. tepidum may represent a "transitional" species between mesophilic and thermophilic purple sulfur bacteria. The characteristic arrangement of multiple αβ-polypeptides also suggests a mechanism of molecular recognition in the expression and/or assembly of the LH1 complex that could be regulated through interactions with reaction center subunits.

Biotechnological applications of protein complexes require detailed information about their structure and composition, which can be challenging to obtain for proteins from natural sources. Prominent examples are the ring-shaped phycoerythrin (PE) and phycocyanin (PC) complexes isolated from the light-harvesting antennae of red algae and cyanobacteria. Despite their widespread use as fluorescent probes in biotechnology and medicine, the structures and interactions of their noncrystallizable central subunits are largely unknown. Here, we employ ion mobility mass spectrometry to reveal varying stabilities of the PC and PE complexes and identify their closest architectural homologues among all protein assemblies in the Protein Data Bank (PDB). Our results suggest that the central subunits of PC and PE complexes, although absent from the crystal structures, may be crucial for their stability, and thus of unexpected importance for their biotechnological applications.

Fucoxanthin chlorophyll (Chl) a/c-binding proteins (FCPs) function as light harvesters in diatoms. The structure of a diatom photosystem II-FCPII (PSII-FCPII) supercomplex have been solved by cryo-electron microscopy (cryo-EM) previously; however, the FCPII subunits that constitute the FCPII tetramers and monomers are not identified individually due to their low resolutions. Here, we report a 2.5 Å resolution structure of the PSII-FCPII supercomplex using cryo-EM. Two types of tetrameric FCPs, S-tetramer, and M-tetramer, are identified as different types of hetero-tetrameric complexes. In addition, three FCP monomers, m1, m2, and m3, are assigned to different gene products of FCP. The present structure also identifies the positions of most Chls c and diadinoxanthins, which form a complicated pigment network. Excitation-energy transfer from FCPII to PSII is revealed by time-resolved fluorescence spectroscopy. These structural and spectroscopic findings provide insights into an assembly model of FCPII and its excitation-energy transfer and quenching processes.

The light-harvesting complex II (LHCII) is the main component of the antenna system of plants and green algae and plays a major role in the capture of sun light for photosynthesis. The LHCII complexes have also been proposed to play a key role in the optimization of photosynthetic efficiency through the process of state 1-state 2 transitions and are involved in down-regulation of photosynthesis under excess light by energy dissipation through non-photochemical quenching (NPQ). We present here the first solid-state magic-angle spinning (MAS) NMR data of the major light-harvesting complex (LHCII) of Chlamydomonas reinhardtii, a eukaryotic green alga. We are able to identify nuclear spin clusters of the protein and of its associated chlorophyll pigments in ¹³C-¹³C dipolar homonuclear correlation spectra on a uniformly ¹³C-labeled sample. In particular, we were able to resolve several chlorophyll 13¹ carbon resonances that are sensitive to hydrogen bonding to the 13¹-keto carbonyl group. The data show that ¹³C NMR signals of the pigments and protein sites are well resolved, thus paving the way to study possible structural reorganization processes involved in light-harvesting regulation through MAS solid-state NMR.

A simple and robust nanolithographic method that allows sub-100 nm chemical patterning on a range of oxide surfaces was developed in order to fabricate nanoarrays of plant light-harvesting LHCII complexes. The site-specific immobilization and the preserved functionality of the LHCII complexes were confirmed by fluorescence emission spectroscopy. Nanopatterned LHCII trimers could be reversibly switched between fluorescent and quenched states by controlling the detergent concentration in the imaging buffer. A 3-fold quenching of the average fluorescence intensity was accompanied by a decrease in the average (amplitude-weighted) fluorescence lifetime from approximately 2.24 ns to approximately 0.4 ns, attributed to the intrinsic ability of LHCII to switch between fluorescent and quenched states upon changes in its conformational state. The nanopatterning methodology was extended by immobilizing a second protein, the enhanced green fluorescent protein (EGFP), onto LHCII-free areas of the chemically patterned surfaces. This very simple surface chemistry, which allows simultaneous selective immobilization and therefore sorting of the two types of protein molecules on the surface, is a key underpinning step toward the integration of LHCII into switchable biohybrid antenna constructs.

Light quality significantly influences plant metabolism, growth and development. Recently, we have demonstrated that leaves of barley and other plant species grown under monochromatic green light (500-590 nm) accumulated a large pool of chlorophyll a (Chl a) intermediates with incomplete hydrogenation of their phytyl chains. In this work, we studied accumulation of these geranylgeranylated Chls a and b in pigment-protein complexes (PPCs) of Arabidopsis plants acclimated to green light and their structural-functional consequences on the photosynthetic apparatus. We found that geranylgeranylated Chls are present in all major PPCs, although their presence was more pronounced in light-harvesting complex II (LHCII) and less prominent in supercomplexes of photosystem II (PSII). Accumulation of geranylgeranylated Chls hampered the formation of PSII and PSI super- and megacomplexes in the thylakoid membranes as well as their assembly into chiral macrodomains; it also lowered the temperature stability of the PPCs, especially that of LHCII trimers, which led to their monomerization and an anomaly in the photoprotective mechanism of non-photochemical quenching. Role of geranylgeranylated Chls in adverse effects on photosynthetic apparatus of plants acclimated to green light is discussed.

The genomes of some purple photosynthetic bacteria contain a multigene puc family encoding a series of α- and β-polypeptides that together form a heterogeneous antenna of light-harvesting 2 (LH2) complexes. To unravel this complexity, we generated four sets of puc deletion mutants in Rhodopseudomonas palustris, each encoding a single type of pucBA gene pair and enabling the purification of complexes designated as PucA-LH2, PucB-LH2, PucD-LH2, and PucE-LH2. The structures of all four purified LH2 complexes were determined by cryogenic electron microscopy (cryo-EM) at resolutions ranging from 2.7 to 3.6 Å. Uniquely, each of these complexes contains a hitherto unknown polypeptide, γ, that forms an extended undulating ribbon that lies in the plane of the membrane and that encloses six of the nine LH2 αβ-subunits. The γ-subunit, which is located near to the cytoplasmic side of the complex, breaks the C9 symmetry of the LH2 complex and binds six extra bacteriochlorophylls (BChls) that enhance the 800-nm absorption of each complex. The structures show that all four complexes have two complete rings of BChls, conferring absorption bands centered at 800 and 850 nm on the PucA-LH2, PucB-LH2, and PucE-LH2 complexes, but, unusually, the PucD-LH2 antenna has only a single strong near-infared (NIR) absorption peak at 803 nm. Comparison of the cryo-EM structures of these LH2 complexes reveals altered patterns of hydrogen bonds between LH2 αβ-side chains and the bacteriochlorin rings, further emphasizing the major role that H bonds play in spectral tuning of bacterial antenna complexes.

The main function of the transmembrane light-harvesting complexes in photosynthetic organisms is the absorption of a light quantum and its subsequent rapid transfer to a reaction center where a charge separation occurs. A combination of freeze-thaw and dialysis methods were used to reconstitute the detergent-solubilized Light Harvesting 2 complex (LH2) of the purple bacterium Rhodopseudomonas acidophila strain 10050 into preformed egg phosphatidylcholine liposomes, without the need for extra chemical agents. The LH2-containing liposomes opened up to a flat bilayer, which were imaged with tapping and contact mode atomic force microscopy under ambient and physiological conditions, respectively. The LH2 complexes were packed in quasicrystalline domains. The endoplasmic and periplasmic sides of the LH2 complexes could be distinguished by the difference in height of the protrusions from the lipid bilayer. The results indicate that the complexes entered in intact liposomes. In addition, it was observed that the most hydrophilic side, the periplasmic, enters first in the membrane. In contact mode the molecular structure of the periplasmic side of the transmembrane pigment-protein complex was observed. Using Föster's theory for describing the distance dependent energy transfer, we estimate the dipole strength for energy transfer between two neighboring LH2s, based on the architecture of the imaged unit cell.

The multiple roles of light-harvesting chlorophyll a/b-protein complexes in the structure and function of Arabidopsis chloroplasts were investigated using two chlorophyll b-less mutants grown under metal halide lamps with a significant far-red component. In ch1-3, all six light-harvesting proteins of photosystem (PS) II were greatly decreased; in ch1-3lhcb5, Lhcb5 was completely absent while the other five proteins were further decreased. The thylakoids of ch1-3 were less negatively-charged than the wild type, and those of ch1-3lhcb5 were even less so. Despite the expected weaker electrostatic repulsion, however, thylakoids in leaves of the mutants were not well stacked, an effect we attribute to lower van der Waals attraction, lower electrostatic attraction between opposite charges, and the absence or instability of PSII supercomplexes and peripheral light-harvesting trimers. The quantum yield of oxygen evolution in leaves decreased from 0.109 (wild type) to 0.087 (ch1-3) and 0.081 (ch1-3lhcb5) O(2) (photon absorbed)(-1); we attribute this decrease to an excessive spillover from PSII to PSI, a limited PSII antenna, and increased light-independent thermal dissipation in PSII in the mutants. Destabilization of the donor side of PSII, indicated by slower electron donation to the redox-active tyrosine Y(Z)(*) in ch1-3, probably enhanced PSII susceptibility to photoinactivation, increased the non-functional PSII complexes in vivo, and further inactivated PSII complexes in vitro. The evolution of chlorophyll b-containing chloroplasts seems to fine-tune oxygenic photosynthesis.

Photosynthetic light-harvesting proteins are the mechanism by which energy enters the marine ecosystem. The dominant prokaryotic photoautotrophs are the cyanobacterial genera Prochlorococcus and Synechococcus that are defined by two distinct light-harvesting systems, chlorophyll-bound protein complexes or phycobilin-bound protein complexes, respectively. Here, we use the Global Ocean Sampling (GOS) Project as a unique and powerful tool to analyze the environmental diversity of photosynthetic light-harvesting genes in relation to available metadata including geographical location and physical and chemical environmental parameters.

Photosynthetic organisms adapt to changing light conditions by manipulating their light harvesting complexes. Biophysical, biochemical, physiological and genetic aspects of these processes are studied extensively. The structural basis for these studies is lacking. In this study we address this gap in knowledge by focusing on phycobilisomes (PBS), which are large structures found in cyanobacteria and red algae. In this study we focus on the phycobilisomes (PBS), which are large structures found in cyanobacteria and red algae. Specifically, we examine red algae (Porphyridium purpureum) grown under a low light intensity (LL) and a medium light intensity (ML). Using cryo-electron microscopy, we resolve the structure of ML-PBS and compare it to the LL-PBS structure. The ML-PBS is 13.6 MDa, while the LL-PBS is larger (14.7 MDa). The LL-PBS structure have a higher number of closely coupled chromophore pairs, potentially the source of the red shifted fluorescence emission from LL-PBS. Interestingly, these differences do not significantly affect fluorescence kinetics parameters. This indicates that PBS systems can maintain similar fluorescence quantum yields despite an increase in LL-PBS chromophore numbers. These findings provide a structural basis to the processes by which photosynthetic organisms adapt to changing light conditions.

The X-ray crystal structure of the Rhodopseudomonas (Rps.) palustris reaction center-light harvesting 1 (RC-LH1) core complex revealed the presence of a sixth protein component, variably referred to in the literature as helix W, subunit W or protein W. The position of this protein prevents closure of the LH1 ring, possibly to allow diffusion of ubiquinone/ubiquinol between the RC and the cytochrome bc1 complex in analogous fashion to the well-studied PufX protein from Rhodobacter sphaeroides. The identity and function of helix W have remained unknown for over 13years; here we use a combination of biochemistry, mass spectrometry, molecular genetics and electron microscopy to identify this protein as RPA4402 in Rps. palustris CGA009. Protein W shares key conserved sequence features with PufX homologs, and although a deletion mutant was able to grow under photosynthetic conditions with no discernible phenotype, we show that a tagged version of protein W pulls down the RC-LH1 complex. Protein W is not encoded in the photosynthesis gene cluster and our data indicate that only approximately 10% of wild-type Rps. palustris core complexes contain this non-essential subunit; functional and evolutionary consequences of this observation are discussed. The ability to purify uniform RC-LH1 and RC-LH1-protein W preparations will also be beneficial for future structural studies of these bacterial core complexes.

The purple nonsulfur bacterium Rhodopseudomonas palustris is a model for understanding how a phototrophic organism adapts to changes in light intensity because it produces different light-harvesting (LH) complexes under high light (LH2) and low light intensities (LH3 and LH4). Outside of this change in the composition of the photosystem, little is understood about how R. palustris senses and responds to low light intensity. On the basis of the results of transcription analysis of 17 R. palustris strains grown in low light, we found that R. palustris strains downregulate many genes involved in iron transport and homeostasis. The only operon upregulated in the majority of R. palustris exposed to low light intensity was pucBAd, which encodes LH4. In previous work, pucBAd expression was shown to be modulated in response to light quality by bacteriophytochromes that are part of a low-light signal transduction system. Here we found that this signal transduction system also includes a redox-sensitive protein, LhfE, and that its redox sensitivity is required for LH4 synthesis in response to low light. Our results suggest that R. palustris upregulates its LH4 system when the cellular redox state is relatively oxidized. Consistent with this, we found that LH4 synthesis was upregulated under high light intensity when R. palustris was grown semiaerobically or under nitrogen-fixing conditions. Thus, changes in the LH4 system in R. palustris are not dependent on light intensity per se but rather on cellular redox changes that occur as a consequence of changes in light intensity.IMPORTANCE An essential aspect of the physiology of phototrophic bacteria is their ability to adjust the amount and composition of their light-harvesting apparatus in response to changing environmental conditions. The phototrophic purple bacterium R. palustris adapts its photosystem to a range of light intensities by altering the amount and composition of its peripheral LH complexes. Here we found that R. palustris regulates its LH4 complex in response to the cellular redox state rather than in response to light intensity per se Relatively oxidizing conditions, including low light, semiaerobic growth, and growth under nitrogen-fixing conditions, all stimulated a signal transduction system to activate LH4 expression. By understanding how LH composition is regulated in R. palustris, we will gain insight into how and why a photosynthetic organism senses and adapts its photosystem to multiple environmental cues.

Photosynthetic light-harvesting complexes (LHCs) of higher plants, moss, and green algae can undergo dynamic conformational transitions, which have been correlated to their ability to adapt to fluctuations in the light environment. Herein, we demonstrate the application of solid-state NMR spectroscopy on native, heterogeneous thylakoid membranes of Chlamydomonas reinhardtii (Cr) and on Cr light-harvesting complex II (LHCII) in thylakoid lipid bilayers to detect LHCII conformational dynamics in its native membrane environment. We show that membrane-reconstituted LHCII contains selective sites that undergo fast, large-amplitude motions, including the phytol tails of two chlorophylls. Protein plasticity is also observed in the N-terminal stromal loop and in protein fragments facing the lumen, involving sites that stabilize the xanthophyll-cycle carotenoid violaxanthin and the two luteins. The results report on the intrinsic flexibility of LHCII pigment-protein complexes in a membrane environment, revealing putative sites for conformational switching. In thylakoid membranes, fast dynamics of protein and pigment sites is significantly reduced, which suggests that in their native organelle membranes, LHCII complexes are locked in specific conformational states.