PsbO Protein Research Articles

Protein crystallization and initial neutron diffraction studies of the photosystem II subunit PsbO

The PsbO protein of photosystem II stabilizes the active-site manganese cluster and is thought to act as a proton antenna. To enable neutron diffraction studies, crystals of the β-barrel core of PsbO were grown in capillaries. The crystals were optimized by screening additives in a counter-diffusion setup in which the protein and reservoir solutions were separated by a 1% agarose plug. Crystals were cross-linked with glutaraldehyde. Initial neutron diffraction data were collected from a 0.25 mm3 crystal at room temperature using the MaNDi single-crystal diffractometer at the Spallation Neutron Source, Oak Ridge National Laboratory.

Charged groups at binding interfaces of the PsbO subunit of photosystem II: A combined bioinformatics and simulation study

PsbO is an extrinsic subunit of photosystem II engaged in complex binding interactions within photosystem II. At the interface between PsbO, D1 and D2 subunits of photosystem II, a cluster of charged and polar groups of PsbO is part of an extended hydrogen-bond network thought to participate in proton transfer. The precise role of specific amino acid residues at this complex binding interface remains a key open question. Here, we address this question by carrying out extensive bioinformatics analyses and molecular dynamics simulations of PsbO proteins with mutations at the binding interface. We find that PsbO proteins from cyanobacteria vs. plants have specific preferences for the number and composition of charged amino acid residues that may ensure that PsbO proteins avoid aggregation and expose long unstructured loops for binding to photosystem II. A cluster of conserved charged groups with dynamic hydrogen bonds provides PsbO with structural plasticity at the binding interface with photosystem II.

N-Terminal Lipid Modification Is Required for the Stable Accumulation of CyanoQ in Synechocystis sp. PCC 6803.

The CyanoQ protein has been demonstrated to be a component of cyanobacterial Photosystem II (PS II), but there exist a number of outstanding questions concerning its physical association with the complex. CyanoQ is a lipoprotein; upon cleavage of its transit peptide by Signal Peptidase II, which targets delivery of the mature protein to the thylakoid lumenal space, the N-terminal cysteinyl residue is lipid-modified. This modification appears to tether this otherwise soluble component to the thylakoid membrane. To probe the functional significance of the lipid anchor, mutants of the CyanoQ protein have been generated in Synechocystis sp. PCC 6803 to eliminate the N-terminal cysteinyl residue, preventing lipid modification. Substitution of the N-terminal cysteinyl residue with serine (Q-C22S) resulted in a decrease in the amount of detectable CyanoQ protein to 17% that of the wild-type protein. Moreover, the physical properties of the accumulated Q-C22S protein were consistent with altered processing of the CyanoQ precursor. The Q-C22S protein was shifted to a higher apparent molecular mass and partitioned in the hydrophobic phase in TX-114 phase-partitioning experiments. These results suggest that the hydrophobic N-terminal 22 amino acids were not properly cleaved by a signal peptidase. Substitution of the entire CyanoQ transit peptide with the transit peptide of the soluble lumenal protein PsbO yielded the Q-SS mutant and resulted in no detectable accumulation of the modified CyanoQ protein. Finally, the CyanoQ protein was present at normal amounts in the PS II mutant strains ΔpsbB and ΔpsbO, indicating that an association with PS II was not a prerequisite for stable CyanoQ accumulation. Together these results indicate that CyanoQ accumulation in Synechocystis sp. PCC 6803 depends on the presence of the N-terminal lipid anchor, but not on the association of CyanoQ with the PS II complex.

Crystallographic and Computational Analysis of the Barrel Part of the PsbO Protein of Photosystem II: Carboxylate-Water Clusters as Putative Proton Transfer Relays and Structural Switches.

In all organisms that employ oxygenic photosynthesis, the membrane-extrinsic PsbO protein is a functionally important component of photosystem II. To study the previously proposed proton antenna function of carboxylate clusters at the protein-water interface, we combined crystallography and simulations of a truncated cyanobacterial (Thermosynechococcus elongatus) PsbO without peripheral loops. We expressed the PsbO β-barrel heterologously and determined crystal structures at resolutions of 1.15-1.5 Å at 100 K at various pH values and at 297 K and pH 6. (1) Approximately half of the 177 surface waters identified at 100 K are resolved at 297 K, suggesting significant occupancy of specific water sites at room temperature, and loss of resolvable occupancy for other sites. (2) Within a loop region specific to cyanobacterial PsbO, three residues and four waters coordinating a calcium ion are well ordered even at 297 K; the ligation differs for manganese. (3) The crystal structures show water-carboxylate clusters that could facilitate fast Grotthus-type proton transfer along the protein surface and/or store protons. (4) Two carboxylate side chains, which are part of a structural motif interrupting two β-strands and connecting PsbO to photosystem II, are within hydrogen bonding distance at pH 6 (100 K). Simulations indicate coupling between protein structure and carboxylate protonation. The crystal structure determined at 100 K and pH 10 indicates broken hydrogen bonding between the carboxylates and local structural change. At pH 6 and 297 K, both conformations were present in the crystal, suggesting conformational dynamics in the functionally relevant pH regime. Taken together, crystallography and molecular dynamics underline a possible mechanism for pH-dependent structural switching.

The extrinsic PsbO protein modulates the oxidation/reduction rate of the exogenous Mn cation at the high-affinity Mn-binding site of Mn-depleted PSII membranes.

The oxidation of exogenous Mn(II) cations at the high-affinity (HA) Mn-binding site in Mn-depleted photosystem II (PSII) membranes with or without the presence of the extrinsic PsbO polypeptide was studied by EPR. The six-lines EPR spectrum of Mn(II) cation disappears in the absence of the PsbO protein in membranes under illumination, but there was no effect when PSII preparations bound the PsbO protein. Our study demonstrates that such effect is determined by significant influence of the PsbO protein on the ratio between the rates of Mn oxidation and reduction at the HA site when the membranes are illuminated.

Parallel subfunctionalisation of PsbO protein isoforms in angiosperms revealed by phylogenetic analysis and mapping of sequence variability onto protein structure.

BackgroundPsbO, the manganese-stabilising protein, is an indispensable extrinsic subunit of photosystem II. It plays a crucial role in the stabilisation of the water-splitting Mn4CaO5 cluster, which catalyses the oxidation of water to molecular oxygen by using light energy. PsbO was also demonstrated to have a weak GTPase activity that could be involved in regulation of D1 protein turnover. Our analysis of psbO sequences showed that many angiosperm species express two psbO paralogs, but the pairs of isoforms in one species were not orthologous to pairs of isoforms in distant species.ResultsPhylogenetic analysis of 91 psbO sequences from 49 land plant species revealed that psbO duplication occurred many times independently, generally at the roots of modern angiosperm families. In spite of this, the level of isoform divergence was similar in different species. Moreover, mapping of the differences on the protein tertiary structure showed that the isoforms in individual species differ from each other on similar positions, mostly on the luminally exposed end of the β-barrel structure. Comparison of these differences with the location of differences between PsbOs from diverse angiosperm families indicated various selection pressures in PsbO evolution and potential interaction surfaces on the PsbO structure.ConclusionsThe analyses suggest that similar subfunctionalisation of PsbO isoforms occurred parallelly in various lineages. We speculate that the presence of two PsbO isoforms helps the plants to finely adjust the photosynthetic apparatus in response to variable conditions. This might be mediated by diverse GTPase activity, since the isoform differences predominate near the predicted GTP-binding site.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0523-4) contains supplementary material, which is available to authorized users.

The green alga Chlamydomonas reinhardtii as a tool for in vivo study of site-directed mutations in PsbO protein of photosystem II.

The photosynthetic water oxidation in photosystem II (PS II) takes place in a special water-oxidizing complex (WOC) that consists of a catalytic center, Mn4CaO5 cluster, and also includes a group of extrinsic proteins needed for its stability. The most important of these is PsbO, which binds to the donor side of PS II near the Mn cluster and is directly involved in the regulation of its stability and activity. However, the molecular mechanism of PsbO involvement in photosynthetic water oxidation remains unclear. One of the main approaches to solving this problem is site-directed mutagenesis. Until recently, the effect of mutations in PsbO in vivo has been studied only in cyanobacteria (prokaryotes). In eukaryotic organisms, such studies (site-directed mutagenesis of PsbO) have not been carried out, though it is known that the role of PsbO protein in plants and cyanobacteria may be different. In this review, we consider the possibility of using for this purpose the unicellular green alga Chlamydomonas reinhardtii, a eukaryotic organism with a set of extrinsic proteins of the WOC similar to that of the higher plants. However, in contrast to higher plants, the ΔpsbO mutant of C. reinhardtii is viable. Another reason to use this alga is that the ΔpsbO strain of C. reinhardtii grown in the dark (heterotrophically) is able to build the minimal photochemically active complex of PS II, allowing investigation of the role of individual amino acid substitutions in PsbO in vivo without damaging PS II due to photoinactivation.

Sulfite-stress induced functional and structural changes in the complexes of photosystems I and II in a cyanobacterium, Synechococcus elongatus PCC 7942.

Excess sulfite is well known to have toxic effects on photosynthetic activities and growth in plants, however, so far, the behavior of the photosynthetic apparatus during sulfite-stress has not been characterized as to the responsible proteins or genes. Here, the effects of sulfite on photosystem complexes were investigated in a cyanobacterium, Synechococcus elongatus PCC 7942, a possible model organism of chloroplasts. Culturing of the cells for 24 h in the presence of 10 mM sulfite retarded cell growth of the wild type, concomitantly with synthesis of Chl and phycobilisome repressed. The excess sulfite simultaneously repressed photosynthesis by more than 90%, owing largely to structural destabilization and resultant inactivation of the PSII complex, which seemed to consequently retard the cell growth. Notably, the PsbO protein, one of the subunits that construct the water-splitting system of PSII, was retained at a considerable level, and disruption of the psbO gene led to higher sensitivity of photosynthesis and growth to sulfite. Meanwhile, the PSI complex showed monomerization of its trimeric configuration with little effect on the activity. The structural alterations of these PS complexes depended on light. Our data provide evidence for quantitative decreases in the photosystem complex(es) including their antenna(e), structural alterations of the PSI and PSII complexes that would modulate their functions, and a crucial role of psbO in PSII protection, in Synechococcus cells during sulfite-stress. We suggest that the reconstruction of the photosystem complexes is beneficial to cell survival.

Localization of the CyanoP binding site on photosystem II by surface plasmon resonance spectroscopy.

Photosystem II (PSII), a large multi subunit membrane protein complex localized in the thylakoid membrane of cyanobacteria and chloroplasts, is the only known enzyme that catalyzes the light-driven oxidation of water. In addition to the membrane intrinsic part of PSII, efficient oxygen evolution requires soluble protein subunits at its luminal interface. In contrast to the detailed crystal structure of the active cyanobacterial complex the characterization of intermediate PSII species related to its assembly and repair is hampered by their instability or low abundance. As most structural variations of the corresponding PSII species are based on a different set of protein factors bound to the luminal interface of the complex we developed a system for interaction analysis between PSII and its soluble interaction partners based on surface plasmon resonance (SPR) spectroscopy. The assay was validated by the correct localization of the extrinsic PSII proteins PsbO, PsbV, and PsbU on the luminal PSII surface and used to determine the unknown binding position of CyanoP, the cyanobacterial homolog of higher plant PsbP. The CyanoP binding site was clearly localized in the center of PSII at a position, which is occupied by the PsbO subunit in mature PSII complexes. Consistently, we demonstrate selective binding of CyanoP to an inactive PSII assembly intermediate that lacks the extrinsic subunits PsbO, PsbV, and PsbU. These findings suggest, that CyanoP functions in the dynamic lifecycle of PSII, possibly in the association of CP47 and CP43 or in photoactivation of the oxygen-evolving complex.

Cellular capacities for high-light acclimation and changing lipid profiles across life cycle stages of the green alga Haematococcus pluvialis.

The unicellular microalga Haematococcus pluvialis has emerged as a promising biomass feedstock for the ketocarotenoid astaxanthin and neutral lipid triacylglycerol. Motile flagellates, resting palmella cells, and cysts are the major life cycle stages of H. pluvialis. Fast-growing motile cells are usually used to induce astaxanthin and triacylglycerol biosynthesis under stress conditions (high light or nutrient starvation); however, productivity of biomass and bioproducts are compromised due to the susceptibility of motile cells to stress. This study revealed that the Photosystem II (PSII) reaction center D1 protein, the manganese-stabilizing protein PsbO, and several major membrane glycerolipids (particularly for chloroplast membrane lipids monogalactosyldiacylglycerol and phosphatidylglycerol), decreased dramatically in motile cells under high light (HL). In contrast, palmella cells, which are transformed from motile cells after an extended period of time under favorable growth conditions, have developed multiple protective mechanisms—including reduction in chloroplast membrane lipids content, downplay of linear photosynthetic electron transport, and activating nonphotochemical quenching mechanisms—while accumulating triacylglycerol. Consequently, the membrane lipids and PSII proteins (D1 and PsbO) remained relatively stable in palmella cells subjected to HL. Introducing palmella instead of motile cells to stress conditions may greatly increase astaxanthin and lipid production in H. pluvialis culture.

Isolation and characterization of oxygen-evolving photosystem II particles and photosystem II core complex from the filamentous cyanobacterium Spirulina platensis

Photosystem (PS) II particles retaining a high rate of O2 evolution were isolated from the mesophilic filamentous cyanobacterium, Spirulina platensis. To achieve high production of PSII complexes in the cells, irradiance from halogen incandescent lamps was used. Disruption of cells by vibration of glass beads proved to be the most suitable procedure for isolation of thylakoid membranes. The selectivity of detergents for PSII particle preparation rose in the order of Triton X-100 < decyl-β-D-glucopyranoside < dodecyldimethyl-aminooxide < n-heptyl-β-D-thioglucoside < N-dodecyl-N,N-dimethylammonio-3-propane sulphonate < n-octyl-β-thioglycoside < octylglucoside < n-dodecyl-β-D-maltoside. The last four detergents yielded extracts, from which pure PSII particles not contaminated by PSI complexes could be obtained by sucrose-gradient centrifugation (20–45%) at the 43% sucrose level. We assumed both the acceptor and donor sides of the isolated n-dodecyl-β-D-maltoside (DM) particles to be intact due to high oxygen production by DM particles [1,500 meq(e−) mol−1 (Chl) s−1] achieved in the presence of all artificial acceptors tested. The PSII particle fraction from the sucrose gradient was used with immobilized metal (Cu2+) affinity chromatography (IMAC) for the preparation of the PSII core complex. By washing the column with a MES buffer containing MgCl2 and CaCl2, the phycobiliproteins were stripped off. The PSII core complex was eluted in a buffer containing 1% DM, mannitol, MgCl2, NaCl, CaCl2, and ɛ-aminocaproic acid. SDS-PAGE of the core complex provided pure bands of D1 and D2 proteins and PsbO protein from thylakoid membrane, which were used to raise polyclonal antibodies in rabbits. These antibodies recognized D1 and D2 not only as monomers of 31 and 32 kDa proteins, but also as heterodimers of D1, D2 corresponding to the band of 66 kDa on SDS-PAGE. This was in contrast to antibodies of synthetic determinants, which reacted only with the monomers of D1 and D2 proteins. These negative reactions against heterodimers of D1, D2 supported the hypothesis that dimeric forms of PSII reaction centre proteins have a C-terminal sequence sterically protected against a reaction with specific antibodies.

Knockdown of PsbO leads to induction of HydA and production of photobiological H2 in the green alga Chlorella sp. DT

Green algae are able to convert solar energy to H2 via the photosynthetic electron transport pathway under certain conditions. Algal hydrogenase (HydA, encoded by HYDA) is in charge of catalyzing the reaction: 2H(+)+2e(-)↔H2 but usually inhibited by O2, a byproduct of photosynthesis. The aim of this study was to knockdown PsbO (encoded by psbO), a subunit concerned with O2 evolution, so that it would lead to HydA induction. The alga, Chlorella sp. DT, was then transformed with short interference RNA antisense-psbO (siRNA-psbO) fragments. The algal mutants were selected by checking for the existence of siRNA-psbO fragments in their genomes and the low amount of PsbO proteins. The HYDA transcription and the HydA expression were observed in the PsbO-knockdown mutants. Under semi-aerobic condition, PsbO-knockdown mutants could photobiologically produce H2 which increased by as much as 10-fold in comparison to the wild type.

The PsbO homolog from Symbiodinium kawagutii (Dinophyceae) characterized using biochemical and molecular methods

A photosystem II component, the PsbO protein is essential for maximum rates of oxygen production during photosynthesis, and has been extensively characterized in plants and cyanobacteria but not in symbiotic dinoflagellates. Its close interaction with D1 protein has important environmental implications since D1 has been identified as the primary site of damage in endosymbiotic dinoflagellates after thermal stress. We identified and biochemically characterized the PsbO homolog from Symbiodinium kawagutii as a 28-kDa protein, and immunolocalized it to chloroplast membranes. Chloroplast association was further confirmed by western blot on photosynthetic membrane preparations. TX-114 phase partitioning, chromatography, and SDS-PAGE for single band separation and partial peptide sequencing yielded peptides identical or with high identity to PsbO from dinoflagellates. Analysis of a cDNA library revealed three genes differing by only one aminoacid residue in the in silico-translated ORFs despite greater differences at nucleotide level in the untranslated, putative regulatory sequences. The consensus full amino acid sequence displayed all the characteristic domains and features of PsbO from other sources, but changes in functionally critical, highly conserved motifs were detected. Our biochemical, molecular, and immunolocalization data led to the conclusion that the 28-kDa protein from S. kawagutii is the PsbO homolog, thereby named SkPsbO. We discuss the implications of critical amino acid substitutions for a putative regulatory role of this protein.

Oxidative Folding in Chloroplasts

Disulfide-bonded proteins in chloroplasts from green plants exist in the envelope and the thylakoid membrane, and in the stroma and the lumen. The formation of disulfide bonds in proteins is referred to as oxidative folding and is linked to the import and folding of chloroplast proteins as well as the assembly and repair of thylakoid complexes. It is also important in the redox regulation of enzymes and signal transfer. Green-plant chloroplasts contain enzymes that can form and isomerize disulfide bonds in proteins. In Arabidopsis thaliana, four proteins are identified that are relevant for the catalysis of disulfide bond formation in chloroplast proteins. The proteins' low quantum yield of Photosystem II 1 (LQY1, At1g75690) and snowy cotyledon 2 (SCO2, At3g19220) exhibits protein disulfide isomerase activity and is suggested to function in the assembly and repair of Photosystem II (PSII), and the biogenesis of thylakoids in cotyledons, respectively. The thylakoid-located Lumen thiol oxidoreductase 1 (LTO1, At4g35760) can catalyze the formation of the disulfide bond of the extrinsic PsbO protein of PSII. In addition, the stroma-located protein disulfide isomerase PDIL1-3 (At3g54960) may have a role in oxidative folding. Research on oxidative folding in chloroplasts plants is in an early stage and little is known about the mechanisms of disulfide bond formation in chloroplast proteins. The close link between the import and folding of chloroplast proteins suggests that Hsp93, a component of the inner envelope's import apparatus, may have co-chaperones that can catalyze disulfide bond formation in newly imported proteins.

Insights into Alternanthera mosaic virus TGB3 Functions: Interactions with Nicotiana benthamiana PsbO Correlate with Chloroplast Vesiculation and Veinal Necrosis Caused by TGB3 Over-Expression

Alternanthera mosaic virus (AltMV) triple gene block 3 (TGB3) protein is involved in viral movement. AltMV TGB3 subcellular localization was previously shown to be distinct from that of Potato virus X (PVX) TGB3, and a chloroplast binding domain identified; veinal necrosis and chloroplast vesiculation were observed in Nicotiana benthamiana when AltMV TGB3 was over-expressed from PVX. Plants with over-expressed TGB3 showed more lethal damage under dark conditions than under light. Yeast-two-hybrid analysis and bimolecular fluorescence complementation (BiFC) reveal that Arabidopsis thaliana PsbO1 has strong interactions with TGB3; N. benthamiana PsbO (NbPsbO) also showed obvious interaction signals with TGB3 through BiFC. These results demonstrate an important role for TGB3 in virus cell-to-cell movement and virus-host plant interactions. The Photosystem II oxygen-evolving complex protein PsbO interaction with TGB3 is presumed to have a crucial role in symptom development and lethal damage under dark conditions. In order to further examine interactions between AtPsbO1, NbPsbO, and TGB3, and to identify the binding domain(s) in TGB3 protein, BiFC assays were performed between AtPsbO1 or NbPsbO and various mutants of TGB3. Interactions with C-terminally deleted TGB3 were significantly weaker than those with wild-type TGB3, and both N-terminally deleted TGB3 and a TGB3 mutant previously shown to lose chloroplast interactions failed to interact detectably with PsbO in BiFC. To gain additional information about TGB3 interactions in AltMV-susceptible plants, we cloned 12 natural AltMV TGB3 sequence variants into a PVX expression vector to examine differences in symptom development in N. benthamiana. Symptom differences were observed on PVX over-expression, with all AltMV TGB3 variants showing more severe symptoms than the WT PVX control, but without obvious correlation to sequence differences.

Leaf plasticity to light intensity in Italian cypress (Cupressus sempervirens L.): Adaptability of a Mediterranean conifer cultivated in the Alps

Italian cypress (Cupressus sempervirens L.) is native to the eastern Mediterranean, an area characterised by hot, dry summers and mild winters. Over the centuries, however, the species has been introduced into more northerly regions, a long way from its native range. The current, generally warmer climatic conditions brought about by global warming have favoured its cultivation in even more northerly areas in the Alps and other European alpine regions. Given that not only temperature, but also light availability are limiting factors for the spread of cypress in these environments, it is important to ascertain how this species copes with low light conditions. The photosynthetic characteristics of cypress leaves collected from different portions of the crown with contrasting light availability were evaluated by several methods. Chlorophyll a (Chl a), chlorophyll b (Chl b) and carotenoid (Car) content was found to be higher in shade leaves than in sun leaves when measured on a fresh mass basis, although enzymatic activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) and nitrate reductase were lower in shade leaves. When the efficiency of PSII was measured by chlorophyll fluorescence, a marked reduction in Fm was found in shade leaves, while Fo remained unchanged. The use of exogenous electron donors diphenyl carbazide (DPC) and NH2OH actually improved the photosynthetic efficiency of shade leaves, and the same effect was found when PSII electron transport activity was measured as O2 evolution. Altogether, these results seem to indicate lesser photosynthetic efficiency in shade leaves, probably an impairment on the donor side of the PSII. At the same time, analysis by SDS–PAGE revealed differences in the polypeptide composition of the thylakoid membranes of sun and shade leaves: the bands corresponding to 23kDa, 28–25kDa and 33kDa polypeptides were less intense in the thylakoid membranes extracted from shade leaves. These results were further confirmed by an immunological study showing that the content of the 33kDa protein, corresponding to the extrinsic PSII protein PsbO, was significantly diminished in shade leaves. The high plasticity of cypress leaves appears to be an advantageous trait in the plant’s response to variations in environmental conditions, including global change. Implications for the management of this Mediterranean species at the northern edge of its distribution are discussed.

Antisense reductions in the PsbO protein of photosystem II leads to decreased quantum yield but similar maximal photosynthetic rates.

Photosystem (PS) II is the multisubunit complex which uses light energy to split water, providing the reducing equivalents needed for photosynthesis. The complex is susceptible to damage from environmental stresses such as excess excitation energy and high temperature. This research investigated the in vivo photosynthetic consequences of impairments to PSII in Arabidopsis thaliana (ecotype Columbia) expressing an antisense construct to the PsbO proteins of PSII. Transgenic lines were obtained with between 25 and 60% of wild-type (WT) total PsbO protein content, with the PsbO1 isoform being more strongly reduced than PsbO2. These changes coincided with a decrease in functional PSII content. Low PsbO (less than 50% WT) plants grew more slowly and had lower chlorophyll content per leaf area. There was no change in content per unit area of cytochrome b6f, ATP synthase, or Rubisco, whereas PSI decreased in proportion to the reduction in chlorophyll content. The irradiance response of photosynthetic oxygen evolution showed that low PsbO plants had a reduced quantum yield, but matched the oxygen evolution rates of WT plants at saturating irradiance. It is suggested that these plants had a smaller pool of PSII centres, which are inefficiently connected to antenna pigments resulting in reduced photochemical efficiency.

Probing the N-terminal sequence of spinach PsbO: evidence that essential threonine residues bind to different functional sites in eukaryotic photosystem II

The N-terminal ¹E-⁶L domain of the manganese-stabilizing protein (PsbO) from spinach prevents non-specific binding of the subunit to photosystem II (PSII) and deletions of the ¹E-⁷T or ¹E-¹⁵T sequences from the PsbO N-terminus reduce or impair, respectively, functional binding of PsbO to PSII (Popelkova et al., Biochemistry 42:6193-6200, 2003). The work presented here provides deeper insights into the interaction of PsbO with PSII. The data show that a single mutation, ¹⁵T → A in mature PsbO from spinach reduces the stoichiometry of its functional binding from two to one subunit per PSII and decreases reconstitution of activity to about 45 % of the wild-type control. Replacement of the ¹E-⁶L domain with ⁶M in the T15A PsbO mutant has no additional negative effect on recovery of O₂ evolution activity, but it significantly weakens both functional and nonspecific binding of the truncated mutant to PSII. These results suggest that the ¹⁵T side-chain by itself is essential for binding of one of two PsbO subunits to eukaryotic PSII and that specific PSII-binding sites for PsbO are distinguishable; one PSII-binding site does not require PsbO-¹⁵T and probably interacts with the other N-terminal domain of PsbO. Identity of the latter domain is revealed by a requirement for the presence of the ¹E-⁶L sequence that is shown here to be necessary for high-affinity binding of PsbO to PSII. When combined with previous results, the data presented here lead to a more detailed model for PsbO binding in eukaryotic PSII.

Expression pattern of the psbO gene and its involvement in acclimation of the photosynthetic apparatus during abiotic stresses in Festuca arundinacea and F. pratensis

PsbO, the manganese-stabilizing protein, plays a crucial role in oxygen-evolving complex functioning and stabilization, by maintaining optimal manganese, calcium and chloride concentrations at the active state of PSII. In this paper we present a study focused on recognizing the relationship between psbO gene activity and acclimation of the photosynthetic apparatus under abiotic stresses in the grasses Festuca arundinacea and F. pratensis. PsbO expression was compared between two distinct genotypes within each species which differed in their levels of stress tolerance (drought and frost, respectively) during drought treatment (F. arundinacea) and cold acclimation (F. pratensis). The research involved: (1) the analyses of psbO gene expression profiles using real-time PCR, and (2) the analyses of PsbO protein accumulation profiles using protein gel blot hybridization. The results indicate that PsbO plays a protective function with respect to the photosynthetic apparatus during abiotic stresses. In cold-treated F. pratensis plants the accumulation of PsbO seems to be responsible for differences in the PSII photochemical efficiency. Higher stability of PSII during drought, observed in the high-drought tolerant F. arundinacea genotype, is not associated with PsbO accumulation, although the degradation of this protein affects destabilization of the oxygen-evolving complex in drought.

Effect of K223E and K226E amino acid substitutions in PsbO protein of photosystem 2 on stability and functional activity of the water-oxidizing complex in Chlamydomonas reinhardtii

Site-directed mutations were introduced into PsbO protein of photosystem 2 to study the role of two lysine residues, 223 and 226 (LGAKPPK), in the green alga Chlamydomonas reinhardtii. Lysines 223 and 226 homologous to His228 and His231 from cyanobacteria are located on the protein side facing the lumen and can participate in formation of a channel connecting the Mn cluster with the intrathylakoid space. The K223E and K226E mutants were generated on the basis of the ΔpsbO strain of C. reinhardtii with the substitution of glutamic acid for the lysine residues. The K226E mutation leads to a decrease in stability of the protein and development of the ΔpsbO phenotype (the absence of both photosynthetic activity of photosystem 2 and photoautotrophic growth), with substantially decreased PsbO content in the cells. In the case of K223E, the mutant strain accumulated the normal level of PsbO protein and was able to grow photoautotrophically and to evolve oxygen. However, the rate of oxygen evolution and the F(v)/F(m) ratio were reduced by 15-20% compared to the control. Also, the time of the dark decay of F(v) in the presence of DCMU in the cells of the K223E mutant was increased, indicating impairment in the water-oxidizing complex. In general, our study shows the importance of amino acids K223 and K226 located at the lumenal surface of PsbO protein for the activity of the water-oxidizing complex.