Molecular functions of PsbP and PsbQ proteins in the photosystem II supercomplex

The PsbP and PsbQ proteins are extrinsic subunits of the photosystem II (PSII) supercomplex, which are found in green plants including higher plants and green algae. These proteins are thought to have evolved from their cyanobacterial homologs; cyanoP and cyanoQ respectively. It has been suggested that the functions of PsbP and PsbQ have largely changed from those of cyanoP and cyanoQ. In addition, multiple isoforms and homologs of PsbP and PsbQ were found in green plants, indicating that the acquisition of PsbP and PsbQ in PSII is not a direct path but a result of intensive functional divergence during evolution from cyanobacterial endosymbiont to chloroplast. In this review, we highlight newly introduced topics related to the functions and structures of both PsbP and PsbQ proteins. The present data suggest that PsbP together with PsbQ have specific and important roles in coordinating the activity of the donor and acceptor sides of PSII and stabilizing the active form of the PSII-light-harvesting complex II (LHCII) supercomplex.     

Auxiliary functions of the PsbO, PsbP and PsbQ proteins of higher plant Photosystem II: a critical analysis

Numerous studies over the last 25 years have established that the extrinsic PsbO, PsbP and PsbQ proteins of Photosystem II play critically important roles in maintaining optimal manganese, calcium and chloride concentrations at the active site of Photosystem II. Chemical or genetic removal of these components induces multiple and profound defects in Photosystem II function and oxygen-evolving complex stability. Recently, a number of studies have indicated possible additional roles for these proteins within the photosystem. These include putative enzymatic activities, regulation of reaction center protein turnover, modulation of thylakoid membrane architecture, the mediation of PS II assembly/stability, and effects on the reducing side of the photosystem. In this review we will critically examine the findings which support these auxiliary functions and suggest additional lines of investigations which could clarify the nature of the functional interactions of these proteins with the photosystem.     

Function and association of CyanoP in photosystem II of Synechocystis sp. PCC 6803

The CyanoP protein is a cyanobacterial homolog of the PsbP protein, which is an extrinsic subunit of photosystem II (PSII) in green plant species. The molecular function of CyanoP has been investigated in mutant strains of Synechocystis but inconsistent results have been reported by different laboratories. In this study, we generated and characterized a Synechocystis mutant in which entire region of the CyanoP gene was eliminated. After repeated subculture in CaCl₂-depleted medium, growth retardation was clearly observed for a CyanoP knockout mutant of Synechocystis sp. PCC 6803 (?P). The PSII-mediated oxygen-evolving activity of the ?P cells was more susceptible to depletion of CaCl₂ than that of wild-type cells. The 77 K fluorescence emission spectra indicated that energy coupling between phycobilisome and PSII was perturbed in both wild-type and ?P cells under CaCl₂-depleted conditions, and was more evident for the ?P mutant. To examine the association of CyanoP with PSII complexes, we tested several detergents for solubilization of thylakoid membranes and showed that CyanoP was partly included in fractions containing large protein complexes in gel-filtration analysis. These results indicate that CyanoP constitutively stabilizes PSII functionality in vivo.     

Dual selective nitration in Arabidopsis: Almost exclusive nitration of PsbO and PsbP,and highly susceptible nitration of four non‐PSII proteins,including peroxiredoxin II E

Protein tyrosine nitration is a selective process, as revealed in studies of animals. However, evidence for selective protein nitration in plants is scarce. In this study, Arabidopsis plants were exposed to air with or without nitrogen dioxide at 40 ppm for 8 h in light. Proteins extracted from whole leaves or isolated chloroplasts were subjected to 2D PAGE followed by SYPRO Ruby staining and immunoblotting using an anti‐3‐nitrotyrosine antibody. We determined the relative intensity of a spot on an immunoblot (designated RISI), and relative intensity of the corresponding spot on SYPRO Ruby gel (designated RISS). Proteins that exhibited a high RISI value and/or a high RISI/RISS ratio were considered selectively nitrated. In whole leaf proteins from exposed plants, all immunopositive spots were identified as PsbO1, PsbO2 or PsbP1 by PMF. Thus, nitration was exclusive to PsbO and PsbP, extrinsic proteins of photosystem II (PSII). Their RISI/RISS ratio was ≤1.5. Non‐exposed plants showed very faint nitration. In purified chloroplast proteins, PsbO and PsbP accounted for >80% of the total RISI values, while four non‐PSII proteins, including peroxiredoxin II E, exhibited high RISI/RISS ratios (2.5～6.6). Tyr⁹ of PsbO1 was identified as a nitration site. Thus, nitration is selective for two PSII and four non‐PSII proteins in Arabidopsis.     

Crystal structure of the oxygen-evolving complex of photosystem II

The oxygen in our atmosphere is derived from and maintained by the water-splitting process of photosynthesis. The enzyme that facilitates this reaction and therefore underpins virtually all life on our planet is known as photosystem II (PSII). It is a multisubunit enzyme embedded in the lipid environment of the thylakoid membranes of plants, algae, and cyanobacteria. Powered by light, PSII catalyzes the chemically and thermodynamically demanding reaction of water splitting. In so doing, it releases molecular oxygen into the atmosphere and provides the reducing equivalents required for the conversion of carbon dioxide into the organic molecules of life. Recently, a fully refined structure of an isolated 700 kDa cyanobacterial dimeric PSII complex was elucidated by X-ray crystallography, which gave organizational and structural details of the 19 subunits (16 intrinsic and 3 extrinsic) that make up each monomer and provided information about the position and protein environments of the many different cofactors it binds. The water-splitting site was revealed as a cluster of four Mn ions and a Ca ion surrounded by amino acid side chains, of which six or seven form direct ligands to the metals. The metal cluster was originally modeled as a cubane-like structure composed of three Mn ions and the Ca (2+) linked by oxo bonds and the fourth Mn attached to the cubane via one of its O atoms. New data from X-ray diffraction and X-ray spectroscopy suggest some alternative arrangements. Nevertheless, all of the models are sufficiently similar to provide a basis for discussing the chemistry by which PSII splits water and makes oxygen.     

Psb30 is a photosystem II reaction center subunit and is required for optimal growth in high light in Chlamydomonas reinhardtii

The psb30 (ycf12) gene is conserved in a wide variety of oxygenic-photosynthetic organisms except angiosperms and some marine cyanobacteria. Psb30 protein is found in cyanobacterial photosystem II (PSII) core complexes and is dispensable for PSII structure and function. The most recent three-dimensional structure of cyanobacterial PSII core complex has revealed that Psb30 is located in proximity of PsbJ, PsbK, and PsbZ. However Psb30 has not yet been detected in PSII complexes from eukaryotic photosynthetic organisms. Here we found the expression of the chloroplast psb30 gene in the green alga Chlamydomonas reinhardtii by immunoblotting and Psb30 is exclusively co-purifies with PSII core complex and is significantly reduced in PSII-deficient mutants. Partial disintegration of PSII core complex and subsequent fractionation of the resulting subcomplexes revealed that Psb30 is exclusively associated with PSII reaction center. We have generated chloroplast transformants in which the psb30 gene is disrupted and the resulting ΔPsb30 cells showed decreased oxygen evolution activity by 15%, grew photosynthetically under moderate light, and displayed increased sensitivity to high light relative to wild type. We conclude that Psb30 is a PSII reaction center subunit and is required for optimal PSII function under high light environments.     

Proteomic study of the peripheral proteins from thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803

Thylakoid membranes of cyanobacteria and plants contain enzymes that function in diverse metabolic reactions. Many of these enzymes and regulatory proteins are associated with the membranes as peripheral proteins. To identify these proteins, we separated and identified the peripheral proteins of thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803. Trichloroacetic acid (TCA)-acetone extraction was used to enrich samples with peripheral proteins and to remove integral membrane proteins. The proteins were separated by two-dimensional electrophoresis (2-DE) and identified by peptide mass fingerprinting. More than 200 proteins were detected on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel that was stained with colloidal Coomassie blue. We analyzed 116 spots by peptide mass fingerprinting and identified 78 spots that were derived from 51 genes. Some proteins were found in multiple spots, indicating differential modifications resulting in charge differences. Therefore, a significant fraction of the peripheral proteins in thylakoid membranes is modified post-translationally. In our analysis, products of 17 hypothetical genes could be identified in the peripheral protein fraction. Therefore, proteomic analysis is a powerful tool to identify location of the products of hypothetical genes and to characterize complexity in gene expression due to post-translational modifications.     

Light-harvesting and structural organization of Photosystem II: from individual complexes to thylakoid membrane

Photosystem II (PSII) is responsible for the water oxidation in photosynthesis and it consists of many proteins and pigment-protein complexes in a variable composition, depending on environmental conditions. Sunlight-induced charge separation lies at the basis of the photochemical reactions and it occurs in the reaction center (RC). The RC is located in the PSII core which also contains light-harvesting complexes CP43 and CP47. The PSII core of plants is surrounded by external light-harvesting complexes (lhcs) forming supercomplexes, which together with additional external lhcs, are located in the thylakoid membrane where they perform their functions. In this paper we provide an overview of the available information on the structure and organization of pigment-protein complexes in PSII and relate this to experimental and theoretical results on excitation energy transfer (EET) and charge separation (CS). This is done for different subcomplexes, supercomplexes, PSII membranes and thylakoid membranes. Differences in experimental and theoretical results are discussed and the question is addressed how results and models for individual complexes relate to the results on larger systems. It is shown that it is still very difficult to combine all available results into one comprehensive picture.     

The Lack of Lutein Accelerates the Extent of Light‐induced Bleaching of Photosynthetic Pigments in Thylakoid Membranes of Arabidopsis thaliana

The high light‐induced bleaching of photosynthetic pigments and the degradation of proteins of light‐harvesting complexes of PSI and PSII were investigated in isolated thylakoid membranes of Arabidopsis thaliana, wt and lutein‐deficient mutant lut2, with the aim of unraveling the role of lutein for the degree of bleaching and degradation. By the means of absorption spectroscopy and western blot analysis, we show that the lack of lutein leads to a higher extent of pigment photobleaching and protein degradation in mutant thylakoid membranes in comparison with wt. The highest extent of bleaching is suffered by chlorophyll a and carotenoids, while chlorophyll b is bleached in lut2 thylakoids during long periods at high illumination. The high light‐induced degradation of Lhca1, Lhcb2 proteins and PsbS was followed and it is shown that Lhca1 is more damaged than Lhcb2. The degradation of analyzed proteins is more pronounced in lut2 mutant thylakoid membranes. The lack of lutein influences the high light‐induced alterations in organization of pigment–protein complexes as revealed by 77 K fluorescence.     

Proteomic Analyses of Changes in Synechococcus sp. PCC7942 Following UV‐C Stress

UV‐C's effects on the physiological and biochemical processes of cyanobacteria have been well characterized. However, the molecular mechanisms of cyanobacteria's tolerance to UV‐C still need further investigation. This research attempts to decode the variation in protein abundances in cyanobacteria after UV‐C stress. Different expression levels of proteins in the cytoplasm of Synechococcus sp. PCC7942 under UV‐C stress were investigated using a comparative proteomic approach. In total, 47 UV‐C‐regulated proteins were identified by MALDI‐TOF analysis and classified by Gene Ontology (GO). After studying their pathways, the proteins were mainly enriched in the groups of protein folding, inorganic ion transport and energy production. By focusing on these areas, this study reveals the correlation between UV‐C stress‐responsive proteins and the physiological changes of Synechococcus sp. PCC7942 under UV‐C radiation. These findings may open up new areas for further exploration in the homeostatic mechanisms associated with cyanobacteria responses to UV‐C radiation.     

A combinatorial approach to studying protein complex composition by employing size-exclusion chromatography and proteome analysis

The genome sequences of numerous organisms are available now, but gene sequences alone do not provide sufficient information to accurately deduce protein functions. Protein function is largely dependent on the association of multiple polypeptide chains into large structures with interacting subunits that regulate and support each other. Therefore, the mapping of protein interaction networks in a physiological context is conducive to deciphering protein functions, including those of hypothetical proteins. Although several high-throughput methods to globally identify protein interactions have been reported in recent years, these approaches often have a high rate of nonspecific or artificial interactions detected. For instance, the fraction of false positives of the protein interactions identified by yeast two-hybrid assay has been predicted to be of the order of 50%. We have developed a strategy to globally map Bacillus subtilis protein-protein interactions in a physiological context by fractionating the cell lysates using size-exclusion chromatography (SEC), followed by proteome analysis. Components of both known and unknown protein complexes, multisubunits and multiproteins, have been identified using this strategy. In one case, the partners of the B. subtilis protein complex have been coexpressed in Escherichia coli, and the formation of the overexpressed protein complex has been further confirmed by a pull-down assay.     

Function of ROC4 in the Efficient Repair of Photodamaged Photosystem II in Arabidopsis†

ROC4 is the only cyclophilin in the chloroplast stroma. Here, we used the T‐DNA knockout mutant of roc4 to study the physiological role of ROC4 in vivo in Arabidopsis thaliana. Our results showed that ROC4 is not required for the biogenesis and functional operation of photosystem II (PSII). However, growth in greenhouse and PSII activity, as detected by photoinhibition measurements showed increased sensitivity to high light irradiance in the mutant. In the presence of chloroplast protein synthesis inhibitor lincomycin, which blocks de novo protein synthesis and thus the repair of PSII, wild‐type and mutant plants showed a similar extent of inactivation of PSII activity. The recovery of PSII activity in roc4 leaves from photoinhibition is also impaired compared with that of wild‐type plants. Immunoblot analysis showed that the degradation of PSII reaction center proteins occurred at a similar rate in the presence of lincomycin in wild‐type and mutant plants. Thus, these results suggest that ROC4 functions in the repair of photodamaged PSII.     

Phylogenetic distribution and structural analyses of cyanobacterial glutaredoxins (Grxs)

Glutaredoxins (Grxs), the oxidoreductase proteins, are involved in several cellular processes, including maintenance of cellular redox potential and iron-sulfur homeostasis. The analysis of 503 amino acid sequences from 167 cyanobacterial species led to the identification of four classes of cyanobacterial Grxs, i.e., class I, II, V, and VI Grxs. Class III and IV Grxs were absent in cyanobacteria. Class I and II Grxs are single module oxidoreductase while class V and VI Grxs are multimodular proteins having additional modules at their C-terminal and N-terminal end, respectively. Furthermore, class VI Grxs were exclusively present in marine cyanobacteria. We also report the identification of class VI Grxs with two novel active site motif compositions. Detailed phylogenetic analysis of all four classes of Grxs revealed the presence of several subgroups within each class of Grx having variable dithiol and/or monothiol catalytic active site motif and putative glutathione binding sites. However, class II Grxs possess CGFS-type highly conserved monothiol catalytic active site motif. Sequence analysis confirmed the highly diverse nature of Grx proteins in terms of their amino acid composition; though, sequence diversity does not affect the overall 3D structure of cyanobacterial Grxs. The active site residues and putative GSH binding residues are uncharged amino acids which are present on the surface of the protein. Additionally, the presence of hydrophilic residues at the surface of Grxs confirms their solubility. Protein-ligand interaction analysis identified novel glutathione binding sites on Grxs. Regulation of Grxs encoding genes expression by light quality and quantity as well as salinity suggests their role in determining the fitness of organisms under abiotic factors.     

Mass spectrometric characterization of photooxidative protein modifications in Arabidopsis thaliana thylakoid membranes

Oxidative and nitrosative stress leaves footprints in the plant chloroplast in the form of oxidatively modified proteins. Using a mass spectrometric approach, we identified 126 tyrosine and 12 tryptophan nitration sites in 164 nitrated proteolytic peptides, mainly from photosystem I (PSI), photosystem II (PSII), cytochrome b(6) /f and ATP-synthase complexes and 140 oxidation products of tyrosine, tryptophan, proline, phenylalanine and histidine residues. While a high number of nitration sites were found in proteins from four photosynthetic complexes indicating that the nitration belongs to one of the prominent posttranslational protein modifications in photosynthetic apparatus, amino acid oxidation products were determined mostly in PSII and to a lower extent in PSI. Exposure of plants to light stress resulted in an increased level of tyrosine and tryptophan nitration and tryptophan oxidation in proteins of PSII reaction center and the oxygen-evolving complex, as compared to low light conditions. In contrast, the level of nitration and oxidation of these amino acid residues strongly decreased for all light-harvesting proteins of PSII under the same conditions. Based on these data, we propose that oxidative modifications of proteins by reactive oxygen and nitrogen species might represent an important regulatory mechanism of protein turnover under light stress conditions, especially for PSII and its antenna proteins.     

STRUCTURAL RELATIONSHIPS OF THE PHOTOSYSTEM I AND PHOTOSYSTEM II CHLOROPHYLL a/b AND a/c LIGHT-HARVESTING APOPROTEINS OF PLANTS AND ALGAE

Polyclonal antibodies against four different apoproteins of either the chlorophyll (Chl) a/b light-harvesting antenna of photosystem I or II, or a chlorophyll-protein complex homologous to CP26 from Chlamydomonas reinhardtii, crossreact with11–13 thylakoid proteins of Chlamydomonas, Euglena gracilis and higher plants. The number of antigenically-related proteins correlates with the quantity of light-harvesting chlorophyll-protein complex (LHC) gene types that have been sequenced in higher plants. The antibodies also react specifically with Chi a/c-binding proteins of three diatoms and Coccolithophora sp. as determined by immunoblot and Ouchterlony assays. Four to six crossreacting proteins are observed in each chromophyte species and a functional role for some can be deduced by antibody reactivity. It appears that despite major differences in the structures of their pigment ligands, at least some domains of Chl-binding LHC apoproteins have been conserved during their evolution, possibly functioning in protein: protein, as opposed to pigment: protein, interactions in photosynthetic membranes.     

2-DE proteomic analysis of the model cyanobacterium Anabaena variabilis

Cyanobacteria are photosynthetic bacteria capable of producing hydrogen and secondary metabolites with potential pharmaceutical applications. A limited number of cyanobacterial 2-DE proteomic studies have been published, most of which are based on Synechocystis sp. PCC 6803. Here, we report the use of 2-DE, ESI-MS/MS and protein bioinformatics tools to characterize the proteome of Anabaena variabilis ATCC 29413, a heterocystous nitrogen-fixing cyanobacterium that is a model organism for the study of nitrogen fixation. Using a 2-DE workflow that included the use of a detergent-based extraction buffer and 3-10 nonlinear IPG strips resulted in the identification of 254 unique proteins, with significantly better coverage of basic and low-abundance proteins that has been reported in 2-DE analyses of Synechocystis sp. A set of protein bioinformatics tools was employed to provide estimates of protein localization, hydrophobicity, abundance and other properties. The characteristics of the A. variabilis proteins identified in this study were compared against the theoretical proteome for this organism, and more generally within the cyanobacteria, to identify opportunities for further development of 2-DE-based cyanobacterial proteomics.     

Cyano2Dbase updated: linkage of 234 protein spots to corresponding genes through N-terminal microsequencing.

The cyanobacterium Synechocystis sp. strain PCC6803 is an interesting model organism for preoteome study because it is a photosynthetic procaryote and its genomic sequence has already been determined at our institute. We thus initiated characterization of this organism from a proteomic viewpoint by exploiting two-dimensional (2-D) gel electrophoresis coupled with N-terminal protein sequencing. In a previous study, we linked 130 protein spots on two dimensional gels with the genes that encoded them. As an extension of the previous study, the number of protein spots linked to their corresponding genes was increased to 227 in this study by separately analyzing cyanobacterial proteins in four different fractions (soluble, insoluble, thylakoid membrane, and secretory protein fractions). The resultant updated 2-D protein-gene linkage database, named Cyano2Dbase, will serve as an indispensable tool in future cyanobacterial proteomic studies. From the data compiled in the Cyano2Dbase, we can extract many items of information concerning translation, posttranslational processing including characteristics of cyanobacterial signal sequences and modification of cyanobacterial proteins. The Cyano2Dbase is available to the public through the World Wide Web (http://www.kazusa.or.jp/tech/sazuka/cyano/pr oteome.html).     

Alternatives to Styrene- and Diisobutylene-Based Copolymers for Membrane Protein Solubilization via Nanodisc Formation

Styrene-maleic acid copolymers (SMAs), and related amphiphilic copolymers, are promising tools for isolating and studying integral membrane proteins in a native-like state. However, they do not exhibit this ability universally, as several reports have found that SMAs and related amphiphilic copolymers show little to no efficiency when extracting specific membrane proteins. Recently, it was discovered that esterified SMAs could enhance the selective extraction of trimeric Photosystem I from the thylakoid membranes of thermophilic cyanobacteria; however, these polymers are susceptible to saponification that can result from harsh preparation or storage conditions. To address this concern, we herein describe the development of α-olefin-maleic acid copolymers (αMAs) that can extract trimeric PSI from cyanobacterial membranes with the highest extraction efficiencies observed when using any amphiphilic copolymers, including diisobutylene-co-maleic acid (DIBMA) and functionalized SMA samples. Furthermore, we will show that αMAs facilitate the formation of photosystem I-containing nanodiscs that retain an annulus of native lipids and a native-like activity. We also highlight how αMAs provide an agile, tailorable synthetic platform that enables fine-tuning hydrophobicity, controllable molar mass, and consistent monomer incorporation while overcoming shortcomings of prior amphiphilic copolymers.     

Effects of (60)Co gamma radiation on thylakoid membrane functions in Anacystis nidulans

In photosynthetic organisms oxidative stress is known to result in photoinactivation of photosynthetic machinery. We investigated effects of ⁶⁰Co γ radiation, which generates oxidative stress, on thylakoid structure and function in cyanobacteria. Cells of unicellular, non-nitrogen fixing cyanobacterium Anacystis nidulans (Synechococcus sp.) showed D₁₀ value of 257 Gy of ⁶⁰Co γ radiation. When measured immediately after exposure, cells irradiated with 1500 Gy (lethal dose) of ⁶⁰Co γ radiation did not show any differences in photosynthetic functions such as CO₂ fixation, O₂ evolution and partial reactions of photosynthetic electron transport in comparison to unirradiated cells. Incubation of irradiated cells for 24 h in light or dark resulted in decline in photosynthesis. The decline in photosynthesis was higher in the cells incubated in light as compared to the cells incubated in dark. Among the partial reactions of electron transport, only PSII activity declined drastically after incubation of irradiated samples. This was also supported by the analysis of membrane functions using thermoluminescence. Exposure of cyanobacteria to high doses of ⁶⁰Co γ radiation did not affect the thylakoid membrane ultrastructure immediately after exposure as shown by electron microscopy. The level of reactive oxygen species (ROS) in irradiated cells was 20 times higher as compared to control. In irradiated cells de novo protein synthesis was reduced considerably immediately after irradiation. Treatment of cells with tetracycline also affected photosynthesis as in irradiated cells. The results showed that photoinhibition of photosynthetic apparatus after incubation of irradiated cells was probably augmented due to reduced protein synthesis. Active photosynthesis is known to require uninterrupted replenishment of some of the proteins involved in electron transport chain. The defective thylakoid membrane biogenesis may be leading to photosynthetic decline post-irradiation.     

Typology of secondary cyanobacterial metabolites from minimum spanning tree analysis

Recently, two main events have spurred a rapid increase in cyanobacteria chemical, toxicological, and ecological research. The first deals with the interest in isolating compounds from these organisms as source of active products with potential therapeutic applications. The second pertains the crucial problem of harmful cyanobacterial blooms in the aquatic environments. In this context, 594 secondary metabolites belonging to more than 30 genera of cyanobacteria were retrieved from literature. In order to perform their typology, they were first associated with 87 different molecular archetypes and two orphan classes. These 89 groups of molecular structures were then confronted to minimum spanning tree analysis. Attempts were made to graphically derive chemotaxonomical relationships. The interest of QSAR models for estimating the potential pharmacological interest of the cyanobacterial secondary metabolites was also discussed.