The oxygen-evolving complex of chloroplast membranes

The oxygen-evolving complex (OEC) of plants is the main energy-transforming structure of chloroplast membranes, in which light energy is used for photosynthetic oxidation of intracellular water and oxygen formation. The conducted research has resulted in isolation of functionally active OEC of higher plants and elucidation of its molecular composition, photochemical properties and structural organization. The OEC has been revealed to represent the dimer of the pigment-lipoprotein complexes of photosystem 2 (PLPC PS-2) associated in a chloroplast membrane according to the mirror symmetry rule into an integrate structure based on hydrophobic bonds. The model has been developed for the structure of the dimeric complex of PS-2 that has the function of oxygen formation. This model was confirmed by the X-ray analysis of crystals of the dimeric complex of PS-2. The concept about the fact that the “hydrophobic boiler” determining the formation of the water-oxidizing center of the OEC is formed in the area of association of the reaction centers of monomeric PLPCs PS-2 was advanced based on the regularities of change in the functional activity of the OEC under the action of stress-factors. The new scheme has been advanced for the two-anode organization of the water-oxidizing center as the main condition for realizing the process of molecular oxygen formation. The mechanism of the process of photosynthetic water oxidation and molecular oxygen formation has been developed based on the experimental data about the structural organization of the OEC and its water-oxidizing center. The quantum-chemical modeling of the process showed that its course corresponds to the mechanism suggested.     

3D map of the plant photosystem II supercomplex obtained by cryoelectron microscopy and single particle analysis

Here we describe the first 3D structure of the photosystem II (PSII) supercomplex of higher plants, constructed by single particle analysis of images obtained by cryoelectron microscopy. This large multisubunit membrane protein complex functions to absorb light energy and catalyze the oxidation of water and reduction of plastoquinone. The resolution of the 3D structure is 24 A and emphasizes the dimeric nature of the supercomplex. The extrinsic proteins of the oxygen-evolving complex (OEC) are readily observed as a tetrameric cluster bound to the lumenal surface. By considering higher resolution data, obtained from electron crystallography, it has been possible to relate the binding sites of the OEC proteins with the underlying intrinsic membrane subunits of the photochemical reaction center core. The model suggests that the 33 kDa OEC protein is located towards the CP47/D2 side of the reaction center but is also positioned over the C-terminal helices of the D1 protein including its CD lumenal loop. In contrast, the model predicts that the 23/17 kDa OEC proteins are positioned at the N-terminus of the D1 protein incorporating the AB lumenal loop of this protein and two other unidentified transmembrane helices. Overall the 3D model represents a significant step forward in revealing the structure of the photosynthetic OEC whose activity is required to sustain the aerobic atmosphere on our planet.     

Carbonic anhydrase activity of the photosystem II OEC33 protein from pea

The purpose of this study was to identify the location of one of the two sources of carbonic anhydrase (CA) activity associated with the PSII complex in chloroplast membranes. We tested the hypothesis that the extrinsic 33 kDa protein, OEC33, associated with the oxygen-evolving complex (OEC), is one source of CA activity. We found that precursor OEC33 expressed in Escherichia coli exhibits CA activity, but the expressed precursors of OEC24 or OEC17 do not. The CA activity of OEC33 remained after treatment at 90 degrees C for 15 min. Additional biochemical evidence supports the hypothesis. Only those wash treatments that remove the OEC33 from PSII also remove CA activity. Both immunoblot and CA activity show that the CA tracks the OEC33, in parallel, when PSII undergoes washing at different CaCl2 concentrations. The OEC33 protein purified by HiTrap Q anion exchange chromatography has CA activity that is inhibited by an antibody against OEC33. PSII membranes washed with 1 M CaCl2 to remove OEC33 can be reconstituted either with extracted, purified, OEC33 or with the E. coli-expressed precursor OEC33. Reconstitution partially restores both oxygen evolution and CA activity. For maximal CA activity, OEC33 requires manganese as a cofactor.     

A nucleus-encoded factor, CRR2, is essential for the expression of chloroplast ndhB in Arabidopsis

The chloroplast NDH complex, NAD(P)H dehydrogenase, reduces the plastoquinone pool non-photochemically and is involved in cyclic electron flow around photosystem I (PSI). A transient increase in chlorophyll fluorescence after turning off actinic light is a result of NDH activity. We focused on this subtle change in chlorophyll fluorescence to isolate nuclear mutants affected in chloroplast NDH activity in Arabidopsis by using chlorophyll fluorescence imaging. crr2-1 and crr2-2 (chlororespiratory reduction) are recessive mutant alleles in which accumulation of the NDH complex is impaired. Except for the defect in NDH activity, photosynthetic electron transport was unaffected. CRR2 encodes a member of the plant combinatorial and modular protein (PCMP) family consisting of more than 200 genes in Arabidopsis. CRR2 functions in the intergenic processing of chloroplast RNA between rps7 and ndhB, which is possibly essential for ndhB translation. We have determined the function of a PCMP family member, indicating that the family is closely related to pentatrico-peptide PPR proteins involved in the maturation steps of organellar RNA.     

Recovery of net CO2 assimilation after heat stress is correlated with recovery of oxygen-evolving-complex proteins in Zea mays L.

Photosystem 2 (PS2) in general, and the oxygen-evolving complex (OEC) in particular, is one of the most thermolabile components of photosynthesis. We examined the effects of heat stress on net photosynthetic rate (PN) and content of several stromal and thylakoid-membrane proteins (including OEC proteins) in maize (Zea mays L.) in order to determine if decreases in PN during, and especially after, heat stress were correlated with decreases in the content of OEC proteins. The PN decreased with heat stress in maize, and post-heat stress recovery of PN required 4 d following the second of two heat-shocks. The decrease in PN was not the result of stomatal closure. Cellular levels of the 33, 23, and 16 kDa OEC proteins decreased with heat stress, and the decreases were greatest and most closely correlated with decreases in PN for OEC16. Following the second heat stress, full recovery of OEC levels (especially OEC16 and 33) coincided with full recovery of PN, more so than with other photosynthetic proteins examined. For example, decreases in levels of the 32-kDa QB-binding protein of the PS2 reaction center (D1), ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit, and phosphoenolpyruvate carboxylase were generally smaller than for the OEC proteins and full recovery of these proteins occurred at least 2 d prior to full recovery of photosynthesis. These results are consistent with previous fluorescence and in vitro studies by others in suggesting that heat-relaed effects on PS2 and the OEC are an important limitation to Pn during heat stress. Additionally, these results suggest that heat-related decreases in the content of OEC proteins may limit post-heat stress recovery of carbon fixation.     

The extrinsic proteins of Photosystem II

Years of genetic, biochemical, and structural work have provided a number of insights into the oxygen evolving complex (OEC) of Photosystem II (PSII) for a variety of photosynthetic organisms. However, questions still remain about the functions and interactions among the various subunits that make up the OEC. After a brief introduction to the individual subunits Psb27, PsbP, PsbQ, PsbR, PsbU, and PsbV, a current picture of the OEC as a whole in cyanobacteria, red algae, green algae, and higher plants will be presented. Additionally, the role that these proteins play in the dynamic life cycle of PSII will be discussed.     

Recovery of net CO2 assimilation after heat stress is correlated with recovery of oxygen-evolving-complex proteins in Zea mays L.

Photosystem 2 (PS2) in general, and the oxygen-evolving complex (OEC) in particular, is one of the most thermolabile components of photosynthesis. We examined the effects of heat stress on net photosynthetic rate (PN) and content of several stromal and thylakoid-membrane proteins (including OEC proteins) in maize (Zea mays L.) in order to determine if decreases in PN during, and especially after, heat stress were correlated with decreases in the content of OEC proteins. The PN decreased with heat stress in maize, and post-heat stress recovery of PN required 4 d following the second of two heat-shocks. The decrease in PN was not the result of stomatal closure. Cellular levels of the 33, 23, and 16 kDa OEC proteins decreased with heat stress, and the decreases were greatest and most closely correlated with decreases in PN for OEC16. Following the second heat stress, full recovery of OEC levels (especially OEC16 and 33) coincided with full recovery of PN, more so than with other photosynthetic proteins examined. For example, decreases in levels of the 32-kDa QB-binding protein of the PS2 reaction center (D1), ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit, and phosphoenolpyruvate carboxylase were generally smaller than for the OEC proteins and full recovery of these proteins occurred at least 2 d prior to full recovery of photosynthesis. These results are consistent with previous fluorescence and in vitro studies by others in suggesting that heat-relaed effects on PS2 and the OEC are an important limitation to Pn during heat stress. Additionally, these results suggest that heat-related decreases in the content of OEC proteins may limit post-heat stress recovery of carbon fixation. This revised version was published online in June 2006 with corrections to the Cover Date.     

NITROGEN LIMITATION IN ISOCHRYSIS GALBANA (HAPTOPHYCEAE). II. RELATIVE ABUNDANCE OF CHLOROPLAST PROTEINS1

Using spectroscopic, biophysical and immunological techniques, we assayed the relative abundance often chloroplast proteins and protein complexes in the marine haptophyte, Isochrysis galbana Green, grown at nine steady-state dilution rates in nitrogen-limited chemostats. The proteins included Photosystem I reaction center (RCI) chlorophyll protein, CP1; Photosystem II reaction center (RC II) protein, D1; two chlorophyll a-binding apoproteins, CP 43 and CP 47; 33 KDa oxygen evolving protein, OEC 33; α subunit of coupling factor, CF1α; large (LSU) and small subunits (SSU) of ribulose 1,5-bisphosphate carboxylase, RuBisCO; the chlorophyll a/c/fucoxanthin protein complex, LHCP; and cytochrome b₆/f. Seven of the ten protein complexes are encoded in the chloroplast, two are encoded in the nucleus and one shares chloroplast and nuclear genomes. Over the range of dilution rates (0.96-0.18 d^?1) cell N decreased 42% and cellular chlorophyll a decreased 50%; however, the stoichiometric proportion of RC II: cytochrome b₆/f: RC I remained constant, averaging 1:3.3:0.8. In contrast, RuBisCO / PS II decreased by 58%. The light harvesting chlorophyll a/c/fucoxanthin protein complex increased relative to RC II; however, as cells became more nitrogen limited the fraction of total cell nitrogen contained in RuBisCO decreased from 21.3 to 6.7%, whereas that of the light harvesting complex remained relatively constant, averaging 6.8%. Our results generally support the hypothesis that in nitrogen limited cells, proteins encoded in the nuclear genome are synthesized preferentially over those encoded in the chloroplast.     

Protein assembly of photosystem II and accumulation of subcomplexes in the absence of low molecular mass subunits PsbL and PsbJ.

The protein assembly and stability of photosystem II (PSII) (sub)complexes were studied in mature leaves of four plastid mutants of tobacco (Nicotiana tabacum L), each having one of the psbEFLJ operon genes inactivated. In the absence of psbL, no PSII core dimers or PSII-light harvesting complex (LHCII) supercomplexes were formed, and the assembly of CP43 into PSII core monomers was extremely labile. The assembly of CP43 into PSII core monomers was found to be necessary for the assembly of PsbO on the lumenal side of PSII. The two other oxygen-evolving complex (OEC) proteins, PsbP and PsbQ, were completely lacking in Delta psbL. In the absence of psbJ, both intact PSII core monomers and PSII core dimers harboring the PsbO protein were formed, whereas the LHCII antenna remained detached from the PSII dimers, as demonstrated by 77 K fluorescence measurements and by the lack of PSII-LHCII supercomplexes. The Delta psbJ mutant was characterized by a deficiency of PsbQ and a complete lack of PsbP. Thus, both the PsbL and PsbJ subunits of PSII are essential for proper assembly of the OEC. The absence of psbE and psbF resulted in a complete absence of all central PSII core and OEC proteins. In contrast, very young, vigorously expanding leaves of all psbEFLJ operon mutants accumulated at least traces of D2, CP43 and the OEC proteins PsbO and PsbQ, implying developmental control of the expression of the PSII core and OEC proteins. Despite severe problems in PSII assembly, the thylakoid membrane complexes other than PSII were present and correctly assembled in all psbEFLJ operon mutants.     

Expression, assembly and auxiliary functions of photosystem II oxygen-evolving proteins in higher plants

The oxygen-evolving complex (OEC) of higher plant photosystem II (PSII) consists of an inorganic Mn₄Ca cluster and three nuclear-encoded proteins, PsbO, PsbP and PsbQ. In this review, we focus on the assembly of these OEC proteins, and especially on the role of the small intrinsic PSII proteins and recently found “novel” PSII proteins in the assembly process. The numerous auxiliary functions suggested during the past few years for the OEC proteins will likewise be discussed. For example, besides being a manganese-stabilizing protein, PsbO has been found to bind calcium and GTP and possess a carbonic anhydrase activity. In addition, specific roles have been suggested for the two isoforms of the PsbO protein in Arabidopsis thaliana. PsbP and PsbQ seem to play an additional role in the formation of PSII supercomplexes and in grana stacking, besides their originally recognized role in providing a proper calcium and chloride ion concentration for water splitting.     

Three-dimensional structure of Chlamydomonas reinhardtii and Synechococcus elongatus photosystem II complexes allows for comparison of their oxygen-evolving complex organization

Electron microscopy and single-particle analyses have been carried out on negatively stained photosystem II (PSII) complexes isolated from the green alga Chlamydomonas reinhardtii and the thermophilic cyanobacterium Synechococcus elongatus. The analyses have yielded three-dimensional structures at 30-A resolution. Biochemical analysis of the C. reinhardtii particle suggested it to be very similar to the light-harvesting complex II (LHCII).PSII supercomplex of spinach, a conclusion borne out by its three-dimensional structure. Not only was the C. reinhardtii LHCII.PSII supercomplex dimeric and of comparable size and shape to that of spinach, but the structural features for the extrinsic OEC subunits bound to the lumenal surface were also similar thus allowing identification of the PsbO, PsbP, and PsbQ OEC proteins. The particle isolated from S. elongatus was also dimeric and retained its OEC proteins, PsbO, PsbU, and PsbV (cytochrome c(550)), which were again visualized as protrusions on the lumenal surface of the complex. The overall size and shape of the cyanobacterial particle was similar to that of a PSII dimeric core complex isolated from spinach for which higher resolution structural data are known from electron crystallography. By building the higher resolution structural model into the projection maps it has been possible to relate the positioning of the OEC proteins of C. reinhardtii and S. elongatus with the underlying transmembrane helices of other major intrinsic subunits of the core complex, D1, D2, CP47, and CP43 proteins. It is concluded that the PsbO protein is located over the CP47 and D2 side of the reaction center core complex, whereas the PsbP/PsbQ and PsbV/PsbU are positioned over the lumenal surface of the N-terminal region of the D1 protein. However, the mass attributed to PsbV/PsbU seems to bridge across to the PsbO, whereas the PsbP/PsbQ proteins protrude out more from the lumenal surface. Nevertheless, within the resolution and quality of the data, the relative positions of the center of masses for OEC proteins of C. reinhardtii and S. elongatus are similar and consistent with those determined previously for the OEC proteins of spinach.     

Proteomic analysis of Arabidopsis thaliana leaves in response to acute boron deficiency and toxicity reveals effects on photosynthesis,carbohydrate metabolism,and protein synthesis

Boron (B) stress (deficiency and toxicity) is common in plants, but as the functions of this essential micronutrient are incompletely understood, so too are the effects of B stress. To investigate mechanisms underlying B stress, we examined protein profiles in leaves of Arabidopsis thaliana plants grown under normal B (30 μM), compared to plants transferred for 60 and 84 h (i.e., before and after initial visible symptoms) in deficient (0 μM) or toxic (3 mM) levels of B. B-responsive polypeptides were sequenced by mass spectrometry, following 2D gel electrophoresis, and 1D gels and immunoblotting were used to confirm the B-responsiveness of some of these proteins. Fourteen B-responsive proteins were identified, including: 9 chloroplast proteins, 6 proteins of photosynthetic/carbohydrate metabolism (rubisco activase, OEC23, photosystem I reaction center subunit II-1, ATPase δ-subunit, glycolate oxidase, fructose bisphosphate aldolase), 6 stress proteins, and 3 proteins involved in protein synthesis (note that the 14 proteins may fall into multiple categories). Most (8) of the B-responsive proteins decreased under both B deficiency and toxicity; only 3 increased with B stress. Boron stress decreased, or had no effect on, 3 of 4 oxidative stress proteins examined, and did not affect total protein. Hence, our results indicate relatively early specific effects of B stress on chloroplasts and protein synthesis.     

Exploring the energetics of water permeation in photosystem II by multiple steered molecular dynamics simulations

The Mn(4)Ca cluster of the oxygen-evolving complex (OEC) of photosynthesis catalyzes the light-driven splitting of water into molecular oxygen, protons, and electrons. The OEC is buried within photosystem II (PSII), a multisubunit integral membrane protein complex, and water must find its way to the Mn(4)Ca cluster by moving through protein. Molecular dynamics simulations were used to determine the energetic barriers for water permeation though PSII extrinsic proteins. Potentials of mean force (PMFs) for water were derived by using the technique of multiple steered molecular dynamics (MSMD). Calculation of free energy profiles for water permeation allowed us to characterize previously identified water channels, and discover new pathways for water movement toward the Mn(4)Ca cluster. Our results identify the main constriction sites in these pathways which may serve as selectivity filters that restrict both the access of solutes detrimental to the water oxidation reaction and loss of Ca(2+) and Cl(-) from the active site.