Light-harvesting Pigment-protein Complexes Research Articles

Unravelling the fluorescence kinetics of light-harvesting proteins with simulated measurements

The plant light-harvesting pigment-protein complex LHCII is the major antenna sub-unit of PSII and is generally (though not universally) accepted to play a role in photoprotective energy dissipation under high light conditions, a process known Non-Photochemical Quenching (NPQ). The underlying mechanisms of energy trapping and dissipation within LHCII are still debated. Various models have been proposed for the underlying molecular detail of NPQ, but they are often based on different interpretations of very similar transient absorption measurements of isolated complexes. Here we present a simulated measurement of the fluorescence decay kinetics of quenched LHCII aggregates to determine whether this relatively simple measurement can discriminate between different potential NPQ mechanisms. We simulate not just the underlying physics (excitation, energy migration, quenching and singlet-singlet annihilation) but also the signal detection and typical experimental data analysis. Comparing this to a selection of published fluorescence decay kinetics we find that: (1) Different proposed quenching mechanisms produce noticeably different fluorescence kinetics even at low (annihilation free) excitation density, though the degree of difference is dependent on pulse width. (2) Measured decay kinetics are consistent with most LHCII trimers becoming relatively slow excitation quenchers. A small sub-population of very fast quenchers produces kinetics which do not resemble any observed measurement. (3) It is necessary to consider at least two distinct quenching mechanisms in order to accurately reproduce experimental kinetics, supporting the idea that NPQ is not a simple binary switch.

Responses of the photosynthetic characteristics of summer maize to shading stress

AbstractThe vast majority of the energy and substance required for maize growth and development and related metabolic processes come from the photosynthesis of leaves. Over the past 60 years, the amount of solar radiation reaching the earth's surface has decreased significantly, reducing the photosynthetic rate of crops and leading to a significant reduction in yields. However, (1) the effects of shading stress on stomatal characteristics, electron transport efficiency of summer maize leaves and (2) the adaptability of summer maize to low light are still unclear. In order to investigate this problems, maize hybrid Denghai605 (DH605) was used as the experimental materials, and two treatments of shading (shading degree is 60%) and CK (natural light as control) were set in the field. The results showed that shading stress resulted in chloroplast dysplasia, and the overall performance of the two photosystems was reduced by 27.85%. The number of stomata per unit area decreased by 15.6% and the proportion of stomatal opening decreased by 73.1% after shading, which resulted in a significant 27.7% reduction in intercellular CO2 concentration. Shading significantly reduced photosynthetic rate by affecting stomatal characteristics and electron transport, and the former was the main influencing factor. The decrease in carbon assimilation capacity resulted in insufficient assimilate supply and significantly reduced grain allocation ratio, resulting in a significant yield reduction of 57.5%. However, in the low‐light environment, the expression of chlorophyll‐binding protein in the leaf of summer maize was upregulated, the content of Lhcb1 protein of the light harvesting pigment protein complex II (LHCII) was increased, and the content of photosynthetic pigment, especially chlorophyll b, was increased, which increased the absorption and capture of blue light, improved the efficiency of light energy utilization, and was a stress manifestation of summer maize adapting to shading stress.

Enhancing the spectral range of plant and bacterial light-harvesting pigment-protein complexes with various synthetic chromophores incorporated into lipid vesicles.

The Light-Harvesting (LH) pigment-protein complexes found in photosynthetic organisms have the role of absorbing solar energy with high efficiency and transferring it to reaction centre complexes. LH complexes contain a suite of pigments that each absorb light at specific wavelengths, however, the natural combinations of pigments within any one protein complex do not cover the full range of solar radiation. Here, we provide an in-depth comparison of the relative effectiveness of five different organic "dye" molecules (Texas Red, ATTO, Cy7, DiI, DiR) for enhancing the absorption range of two different LH membrane protein complexes (the major LHCII from plants and LH2 from purple phototrophic bacteria). Proteoliposomes were self-assembled from defined mixtures of lipids, proteins and dye molecules and their optical properties were quantified by absorption and fluorescence spectroscopy. Both lipid-linked dyes and alternative lipophilic dyes were found to be effective excitation energy donors to LH protein complexes, without the need for direct chemical or generic modification of the proteins. The Förster theory parameters (e.g., spectral overlap) were compared between each donor-acceptor combination and found to be good predictors of an effective dye-protein combination. At the highest dye-to-protein ratios tested (over 20:1), the effective absorption strength integrated over the full spectral range was increased to ∼180% of its natural level for both LH complexes. Lipophilic dyes could be inserted into pre-formed membranes although their effectiveness was found to depend upon favourable physicochemical interactions. Finally, we demonstrated that these dyes can also be effective at increasing the spectral range of surface-supported models of photosynthetic membranes, using fluorescence microscopy. The results of this work provide insight into the utility of self-assembled lipid membranes and the great flexibility of LH complexes for interacting with different dyes.

Arthrospira platensis Mutagenesis for Protein and C-Phycocyanin Improvement and Proteomics Approaches.

Arthrospira (Spirulina) platensis is known for its use as a food supplement, with reported therapeutic properties including antiviral, anti-inflammatory and antioxidant activity. Arthrospira is also an excellent source of proteins and C-phycocyanin. The latter is a light-harvesting pigment-protein complex in cyanobacteria, located on the outer surface of the thylakoid membrane and comprising 40 to 60% of the total soluble protein in cells. Random mutagenesis is a useful tool as a non-genetically modified mutation method that has been widely used to generate mutants of different microorganisms. Exposure of microalgae or cyanobacteria to chemical stimuli affects their growth and many biological processes. Chemicals influence several proteins, including those involved in carbohydrate and energy metabolisms, photosynthesis and stress-related proteins (oxidative stress-reactive oxygen species (ROS) scavenging enzymes). Signal transduction pathways and ion transportation mechanisms are also impacted by chemical treatment, with changes causing the production of numerous biomolecules and stimulation of defence responses. This study compared the protein contents of A. platensis control and after mutagenesis using diethyl sulphate (DES) under various treatment concentrations for effective mutation of A. platensis. Results identified 1152 peptides using proteomics approaches. The proteins were classified into 23 functional categories. Random mutagenesis of A. platensis by DES was found to be highly effective for C-phycocyanin and protein production.

Engineering purple bacterial carotenoid biosynthesis to study the roles of carotenoids in light-harvesting complexes.

Carotenoids are important photosynthetic pigments that play key roles in light harvesting and energy transfer, photoprotection, and in the folding, assembly, and stabilization of light-harvesting pigment-protein complexes. The genetically tractable purple phototrophic bacteria have been useful for investigating the biosynthesis and function of photosynthetic pigments and cofactors, including carotenoids. Here, we give an overview of the roles of carotenoids in photosynthesis and of their biosynthesis, focusing on the extensively studied purple bacterium Rhodobacter sphaeroides as a model organism. We provide detailed procedures for manipulating carotenoid biosynthesis, and for the preparation and analysis of the light-harvesting and photosynthetic reaction center complexes that bind them. Using appropriate examples from the literature, we discuss how such approaches have enhanced our understanding of the biosynthesis of carotenoids and the photosynthesis-related functions of these fascinating molecules.

Excited States of Xanthophylls Revisited: Toward the Simulation of Biologically Relevant Systems

Xanthophylls are a class of oxygen-containing carotenoids, which play a fundamental role in light-harvesting pigment–protein complexes and in many photoresponsive proteins. The complexity of the manifold of the electronic states and the large sensitivity to the environment still prevent a clear and coherent interpretation of their photophysics and photochemistry. In this Letter, we compare cutting-edge ab initio methods (CC3 and DMRG/NEVPT2) with time-dependent DFT and semiempirical CI (SECI) on model keto-carotenoids and show that SECI represents the right compromise between accuracy and computational cost to be applied to real xanthophylls in their biological environment. As an example, we investigate canthaxanthin in the orange carotenoid protein and show that the conical intersections between excited states and excited–ground states are mostly determined by the effective bond length alternation coordinate, which is significantly tuned by the protein through geometrical constraints and electrostatic effects.

Exciton Origin of Color-Tuning in Ca2+-Binding Photosynthetic Bacteria.

Flexible color adaptation to available ecological niches is vital for the photosynthetic organisms to thrive. Hence, most purple bacteria living in the shade of green plants and algae apply bacteriochlorophyll a pigments to harvest near infra-red light around 850–875 nm. Exceptions are some Ca2+-containing species fit to utilize much redder quanta. The physical basis of such anomalous absorbance shift equivalent to ~5.5 kT at ambient temperature remains unsettled so far. Here, by applying several sophisticated spectroscopic techniques, we show that the Ca2+ ions bound to the structure of LH1 core light-harvesting pigment–protein complex significantly increase the couplings between the bacteriochlorophyll pigments. We thus establish the Ca-facilitated enhancement of exciton couplings as the main mechanism of the record spectral red-shift. The changes in specific interactions such as pigment–protein hydrogen bonding, although present, turned out to be secondary in this regard. Apart from solving the two-decade-old conundrum, these results complement the list of physical principles applicable for efficient spectral tuning of photo-sensitive molecular nano-systems, native or synthetic.

Orange light spectra filtered through transparent colored polyvinyl chloride sheet enhanced pigment content and growth of Arthrospira cells

Microalgae are significantly affected by the spectra composition with various wavelengths. The development of light harvesting pigments can be controlled with specific wavelength of filtered light received by microalgae. Coverage of open raceway pond using transparent colored polyvinyl chloride sheets (PVCS) to filter light spectra, was assessed for the capacity to enhance biomass growth rate. Results showed that orange PVCS filtered light spectra at wavelengths from 480 to 665 nm, increased biomass dry weight (3.3 g/L) by 61% compared with control condition (white PVCS = 350–750 nm). Light spectra filtered through orange PVCS were more easily absorbed by the light harvesting pigment protein complex (phycobilisome) of Arthrospira platensis cells and subsequently transferred to intracellular photosynthesis reaction centers. Therefore, A. platensis cells cultivated with light spectra filtered through orange PVCS contained 62.7 mg/L chlorophyll-a and 23.5 mg/L carotenoid, which were 40% and 29% higher than control condition (with white PVCS).

Preparation of high-purity R-phycoerythrin and R-phycocyanin from Pyropia yezoensis in membrane chromatography

R-phycoerythrin and R-phycocyanin are the main light-harvesting pigment-protein complexes in Pyropia yezoensis. They have bright colors and a high degree of fluorescence, and are now widely used in biomedicine, food, and cosmetic industries. Therefore, membrane chromatography was used to isolate and purify phycobiliprotein from P. yezoensis. Algal cells were disrupted by repeated freeze-thaw cycles. Then ammonium sulfate was added into the crude phycobiliprotein extract, so that the saturation of ammonium sulfate in the solution reached 20%, 30%, and 40% in sequence. At 30% and 40% ammonium sulfate saturation, precipitates of R-phycoerythrin and R-phycocyanin mixture in different proportions were obtained. The precipitates were ultra-filtrated to remove the salt, and then, the retentate was taken for membrane chromatographic treatment. In the membrane chromatographic treatment, salt-sensitive PVDF (polyvinyl difluoride) membranes were used for the follow-up experiments. By adjusting the concentration of ammonium sulfate, the membrane system changed the binding strength of various proteins, and then separated different types of phycobiliproteins. The final products of R-phycoerythrin (Aλmax/A280 purity 4.25, yield 45%) and R-phycocyanin (two purity grades: purity 5.8, yield 14.70% and purity 4.18, yield 39.89%) were obtained and then characterized by spectrophotometric and fluorometric measurements and SDS-PAGE. However, the large-scale production of R-phycocyanin and R-phycoerythrin remains a bottleneck as the column chromatography is difficult to be applied for industrial production. This method avoids the problem of easy clogging of column chromatography and can be useful for mass-production of R-phycoerythrin and R-phycocyanin in the future based on the laboratory results.

Photoprotective mechanisms in the core LH1 antenna pigment-protein complex from the purple photosynthetic bacterium, Rhodospirillum rubrum

Time-resolved absorption spectra from the femtoscecond to sub-millisecond time regimes were recorded in order to eluciate the photoprotective mechanisms of carotenoid (spirilloxanthin) in core LH1 light-harvesting pigment-protein complexes from a purple photosynthetic bacterium, Rhodospirillum (Rsp.) rubrum. Carotenoids can prevent the production of singlet oxygen by rapidly quenching the Bchl a triplet state. Through this triplet-triplet energy-transfer reaction the carotenoid is believed to protect the biological system from exposure to excess light. We set out to investigate this paricular function in the purple bacterium, Rsp. rubrum strain S1. We also investigated Rsp. rubrum strain G9+, which is the carotenoidless mutant of Rsp. rubrum, to make sure that the carotenoid is truly playing this important role. As a result, we could demonstrate that strain S1 realizes nearly 100% excitation energy transfer from triplet Bchl a to carotenoid. In addition, we found another major excited state deactivation channel of carotenoids, which takes away excess energy directly from the singlet excited state of Bchl a and so prevents the production of triplet excited Bchl a.

Adaptation of light-harvesting and energy-transfer processes of a diatom Phaeodactylum tricornutum to different light qualities.

Fucoxanthin-chlorophyll (Chl) a/c-binding proteins (FCPs) are light-harvesting pigment-protein complexes found in diatoms and brown algae. Due to the characteristic pigments, such as fucoxanthin and Chl c, FCPs can capture light energy in blue-to green regions. A pennate diatom Phaeodactylum tricornutum synthesizes a red-shifted form of FCP under weak or red light, extending a light-absorption ability to longer wavelengths. In the present study, we examined changes in light-harvesting and energy-transfer processes of P. tricornutum cells grown under white- and single-colored light-emitting diodes (LEDs). The red-shifted FCP appears in the cells grown under the green, yellow, and red LEDs, and exhibited a fluorescence peak around 714nm. Additional energy-transfer pathways are established in the red-shifted FCP; two forms (F713 and F718) of low-energy Chl a work as energy traps at 77K. Averaged fluorescence lifetimes are prolonged in the cells grown under the yellow and red LEDs, whereas they are shortened in the blue-LED-grown cells. Based on these results, we discussed the light-adaptation machinery of P. tricornutum cells involved in the red-shifted FCP.

The Singlet–Triplet Fission of Carotenoid Excitation in Light-Harvesting Complexes from Thermochromatium tepidum

Abstract—Illumination of purple phototrophic bacteria in the carotenoid absorption band of light harvesting complexes often leads to low energy efficiency of absorbed light. This is due to singlet–triplet fission of carotenoid excitation. In the present study, the nature of this process was explored with a phototrophic bacterium Thermochromatium tepidum as an example. It was demonstrated by time-resolved EPR spectroscopy and magnetic field modulation of the fluorescence yield that the concept of intramolecular excitation fission that was developed in several publications was not confirmed experimentally. Evidence of the intermolecular character of excitation fission that involves two carotenoid molecules of the light-harvesting pigment–protein complexes, LH1-RC and LH2, has been obtained. The advantages of intermolecular excitation fission for application in photovoltaic solar energy converters are discussed.

Quantum Chemical Modeling of the Photoinduced Activity of Multichromophoric Biosystems.

Multichromophoric biosystems represent a broad family with very diverse members, ranging from light-harvesting pigment–protein complexes to nucleic acids. The former are designed to capture, harvest, efficiently transport, and transform energy from sunlight for photosynthesis, while the latter should dissipate the absorbed radiation as quickly as possible to prevent photodamages and corruption of the carried genetic information. Because of the unique electronic and structural characteristics, the modeling of their photoinduced activity is a real challenge. Numerous approaches have been devised building on the theoretical development achieved for single chromophores and on model Hamiltonians that capture the essential features of the system. Still, a question remains: is a general strategy for the accurate modeling of multichromophoric systems possible? By using a quantum chemical point of view, here we review the advancements developed so far highlighting differences and similarities with the single chromophore treatment. Finally, we outline the important limitations and challenges that still need to be tackled to reach a complete and accurate picture of their photoinduced properties and dynamics.

Far-red light acclimation in diverse oxygenic photosynthetic organisms.

Oxygenic photosynthesis has historically been considered limited to be driven by the wavelengths of visible light. However, in the last few decades, various adaptations have been discovered that allow algae, cyanobacteria, and even plants to utilize longer wavelength light in the far-red spectral range. These adaptations provide distinct advantages to the species possessing them, allowing the effective utilization of shade light under highly filtered light environments. In prokaryotes, these adaptations include the production of far-red-absorbing chlorophylls d and f and the remodeling of phycobilisome antennas and reaction centers. Eukaryotes express specialized light-harvesting pigment-protein complexes that use interactions between pigments and their protein environment to spectrally tune the absorption of chlorophyll a. If these adaptations could be applied to crop plants, a potentially significant increase in photon utilization in lower shaded leaves could be realized, improving crop yields.

The role of far-red spectral states in the energy regulation of phycobilisomes

The main light-harvesting pigment-protein complex of cyanobacteria and certain algae is the phycobilisome, which harvests sunlight and regulates the flow of absorbed energy to provide the photochemical reaction centres with a constant energy throughput. At least two light-driven mechanisms of excited energy quenching in phycobilisomes have been identified: the dominant mechanism in many strains of cyanobacteria depends on the orange carotenoid protein (OCP), while the second mechanism is intrinsically available to a phycobilisome and is possibly activated faster than the former. Recent single molecule spectroscopy studies have shown that far-red (FR) emission states are related to the OCP-dependent mechanism and it was proposed that the second mechanism may involve similar states. In this study, we examined the dynamics of simultaneously measured emission spectra and intensities from a large set of individual phycobilisome complexes from Synechocystis PCC 6803. Our results suggest a direct relationship between FR spectral states and thermal energy dissipating states and can be explained by a single phycobilin pigment in the phycobilisome core acting as the site of both quenching and FR emission likely due to the presence of a charge-transfer state. Our experimental method provides a means to accurately resolve the fluorescence lifetimes and spectra of the FR states, which enabled us to quantify a kinetic model that reproduces most of the experimentally determined properties of the FR states.

Characterization of the coherent dynamics of bacteriochlorophyll a in solution

Disclosing the physical origin of quantum beatings in the early dynamics of biological light-harvesting pigment-protein complexes is one of the major challenges in the ultrafast spectroscopy community. 2D electronic spectroscopy (2DES) is a powerful tool for this purpose, but the complexity of the beating behavior in multichromophoric systems complicates the interpretation. For this reason, the availability of control datasets providing a full characterization of the response of isolated chromophores is highly desirable to untangle the features of intermolecular interactions from the properties of individual pigments. Here, a thorough 2DES characterization of the frequencies and dephasing times of intramolecular vibrational coherences of bacteriochlorophyll a in solution is provided. Several beating modes in the ground and excited state have been found and their dephasing times have been determined. The obtained results represent an essential interpretation key of the beating dynamics of pigment-protein complexes binding bacteriochlorophyll a.

Progress in biosynthesis of phycobiliprotein

Photosynthesis has existed on the earth for about 4 billion years, and the photosynthetic organisms have emerged with the production of photosynthesis. In order to effectively capture light energy and adapt their living environment, algae and higher plants have evolved different light-harvesting antenna systems during the long evolution, one is the chlorophyll based light harvesting systems present mainly in terrestrial organisms, such as higher plants; and the other is the phycobiliprotein (PBP) based phycobilisome system mainly in aquatic organisms, such as cyanobacteria, red algae and cryptophytes. As a special light-harvesting pigment-protein complex, PBP can not only capture light energy, also shows many unique characteristics, such as good fluorescent properties and lots of pharmaceutical activities. Therefore, PBPs have been extensively studied and commercially used in foods, cosmetics, biotechnology detection, pharmacological and medicine fields. Due to its broad excitation spectrum, nontoxic, stable fluorescence and high quantum yield, PBPs have also employed as valuable fluorescent probes for immunological diagnosis. At present, there are mainly two ways to obtain fluorescent PBPs, one is to purify PBP directly from natural algae, and the other is to express recombinant PBP using genetic engineered host cells. Purification of PBPs from natural algae has many disadvantages, such as higher cost, complicated processes, and many by-products. Using genetically engineered cell biotechnology, large-scale and low-cost production of recombinant PBPs can be achieved, which can help solve problems such as source, quality control and so on. Since natural PBPs are mostly in the form of polymers such as trimers or hexamers, it is difficult to study the assembly process. Through genetic engineering techniques, monomers or subunits can be obtained, which will help to study the assembly process of PBP. In addition, novel recombinant PBPs can be produced by molecular design to improve their fluorescence and biological activity. The study of the biosynthesis will help to elucidate the relationship between the structure and spectral properties of PBPs and provide a basis for the construction of solar energy utilization systems. This review mainly focuses on the progress of combinational biosynthesis and in vivo reorganization of PBP in heterologous genetically engineered host cells, with emphasis on different genes coding for apo-PBPs and genes coding for different enzymes which catalyze the production of phycobilins from endogenous heme, along with the recent progress of three types of lyases which catalyze the covalently binding of phycobilins with apo-PBPs. The possible applications of these valuable recombinant PBPs were proposed as well.

From isolated light-harvesting complexes to the thylakoid membrane: a single-molecule perspective

Abstract The conversion of solar radiation to chemical energy in plants and green algae takes place in the thylakoid membrane. This amphiphilic environment hosts a complex arrangement of light-harvesting pigment-protein complexes that absorb light and transfer the excitation energy to photochemically active reaction centers. This efficient light-harvesting capacity is moreover tightly regulated by a photoprotective mechanism called non-photochemical quenching to avoid the stress-induced destruction of the catalytic reaction center. In this review we provide an overview of single-molecule fluorescence measurements on plant light-harvesting complexes (LHCs) of varying sizes with the aim of bridging the gap between the smallest isolated complexes, which have been well-characterized, and the native photosystem. The smallest complexes contain only a small number (10–20) of interacting chlorophylls, while the native photosystem contains dozens of protein subunits and many hundreds of connected pigments. We discuss the functional significance of conformational dynamics, the lipid environment, and the structural arrangement of this fascinating nano-machinery. The described experimental results can be utilized to build mathematical-physical models in a bottom-up approach, which can then be tested on larger in vivo systems. The results also clearly showcase the general property of biological systems to utilize the same system properties for different purposes. In this case it is the regulated conformational flexibility that allows LHCs to switch between efficient light-harvesting and a photoprotective function.

A Key Role of Xanthophylls That Are Not Embedded in Proteins in Regulation of the Photosynthetic Antenna Function in Plants, Revealed by Monomolecular Layer Studies.

The main physiological function of LHCII (light-harvesting pigment-protein complex of photosystem II), the largest photosynthetic antenna complex of plants, is absorption of light quanta and transfer of excitation energy toward the reaction centers, to drive photosynthesis. However, under strong illumination, the photosynthetic apparatus faces the danger of photodegradation and therefore excitations in LHCII have to be down-regulated, e.g., via thermal energy dissipation. One of the elements of the regulatory system, operating in the photosynthetic apparatus under light stress conditions, is a conversion of violaxanthin, the xanthophyll present under low light, to zeaxanthin, accumulated under strong light. In the present study, an effect of violaxanthin and zeaxanthin on the molecular organization and the photophysical properties of LHCII was studied in a monomolecular layer system with application of molecular imaging (atomic force microscopy, fluorescence lifetime imaging microscopy) and spectroscopy (UV-Vis absorption, FTIR, fluorescence spectroscopy) techniques. The results of the experiments show that violaxanthin promotes the formation of supramolecular LHCII structures preventing dissipative excitation quenching while zeaxanthin is involved in the formation of excitonic energy states able to quench chlorophyll excitations in both the higher (B states) and lower (Q states) energy levels. The results point to a strategic role of xanthophylls that are not embedded in a protein environment, in regulation of the photosynthetic light harvesting activity in plants.

Carotenoids and Photosynthesis.

Carotenoids are ubiquitous and essential pigments in photosynthesis. They absorb in the blue-green region of the solar spectrum and transfer the absorbed energy to (bacterio-)chlorophylls, and so expand the wavelength range of light that is able to drive photosynthesis. This is an example of singlet-singlet energy transfer, and so carotenoids serve to enhance the overall efficiency of photosynthetic light reactions. Carotenoids also act to protect photosynthetic organisms from the harmful effects of excess exposure to light. Triplet-triplet energy transfer from chlorophylls to carotenoids plays a key role in this photoprotective reaction. In the light-harvesting pigment-protein complexes from purple photosynthetic bacteria and chlorophytes, carotenoids have an additional role of structural stabilization of those complexes. In this article we review what is currently known about how carotenoids discharge these functions. The molecular architecture of photosynthetic systems will be outlined first to provide a basis from which to describe carotenoid photochemistry, which underlies most of their important functions in photosynthesis.