The Psb27 assembly factor binds to the CP43 complex of photosystem II in the cyanobacterium Synechocystis sp. PCC 6803

We have investigated the location of the Psb27 protein and its role in photosystem (PS) II biogenesis in the cyanobacterium Synechocystis sp. PCC 6803. Native gel electrophoresis revealed that Psb27 was present mainly in monomeric PSII core complexes but also in smaller amounts in dimeric PSII core complexes, in large PSII supercomplexes, and in the unassembled protein fraction. We conclude from analysis of assembly mutants and isolated histidine-tagged PSII subcomplexes that Psb27 associates with the "unassembled" CP43 complex, as well as with larger complexes containing CP43, possibly in the vicinity of the large lumenal loop connecting transmembrane helices 5 and 6 of CP43. A functional role for Psb27 in the biogenesis of CP43 is supported by the decreased accumulation and enhanced fragmentation of unassembled CP43 after inactivation of the psb27 gene in a mutant lacking CP47. Unexpectedly, in strains unable to assemble PSII, a small amount of Psb27 comigrated with monomeric and trimeric PSI complexes upon native gel electrophoresis, and Psb27 could be copurified with histidine-tagged PSI isolated from the wild type. Yeast two-hybrid assays suggested an interaction of Psb27 with the PsaB protein of PSI. Pull-down experiments also supported an interaction between CP43 and PSI. Deletion of psb27 did not have drastic effects on PSII assembly and repair but did compromise short-term acclimation to high light. The tentative interaction of Psb27 and CP43 with PSI raises the possibility that PSI might play a previously unrecognized role in the biogenesis/repair of PSII.     

Investigating the early stages of photosystem II assembly in Synechocystis sp. PCC 6803: isolation of CP47 and CP43 complexes

Biochemical characterization of intermediates involved in the assembly of the oxygen-evolving Photosystem II (PSII) complex is hampered by their low abundance in the membrane. Using the cyanobacterium Synechocystis sp. PCC 6803, we describe here the isolation of the CP47 and CP43 subunits, which, during biogenesis, attach to a reaction center assembly complex containing D1, D2, and cytochrome b(559), with CP47 binding first. Our experimental approach involved a combination of His tagging, the use of a D1 deletion mutant that blocks PSII assembly at an early stage, and, in the case of CP47, the additional inactivation of the FtsH2 protease involved in degrading unassembled PSII proteins. Absorption spectroscopy and pigment analyses revealed that both CP47-His and CP43-His bind chlorophyll a and β-carotene. A comparison of the low temperature absorption and fluorescence spectra in the Q(Y) region for CP47-His and CP43-His with those for CP47 and CP43 isolated by fragmentation of spinach PSII core complexes confirmed that the spectroscopic properties are similar but not identical. The measured fluorescence quantum yield was generally lower for the proteins isolated from Synechocystis sp. PCC 6803, and a 1-3-nm blue shift and a 2-nm red shift of the 77 K emission maximum could be observed for CP47-His and CP43-His, respectively. Immunoblotting and mass spectrometry revealed the co-purification of PsbH, PsbL, and PsbT with CP47-His and of PsbK and Psb30/Ycf12 with CP43-His. Overall, our data support the view that CP47 and CP43 form preassembled pigment-protein complexes in vivo before their incorporation into the PSII complex.     

Psb28 Protein Is Involved in the Biogenesis of the Photosystem II Inner Antenna CP47 (PsbB) in the Cyanobacterium Synechocystis sp. PCC 6803

The role of the Psb28 protein in the structure and function of the photosystem II (PSII) complex has been studied in the cyanobacterium Synechocystis sp. PCC 6803. The protein was localized in the membrane fraction and, whereas most of the protein was detected as an unassembled protein, a small portion was found in the PSII core complex lacking the CP43 antenna (RC47). The association of Psb28 with RC47 was further confirmed by preferential isolation of RC47 from the strain containing a histidine-tagged derivative of Psb28 using nickel-affinity chromatography. However, the affinity-purified fraction also contained a small amount of the unassembled PSII inner antenna CP47 bound to Psb28-histidine, indicating a structural relationship between Psb28 and CP47. A psb28 deletion mutant exhibited slower autotrophic growth than wild type, although the absence of Psb28 did not affect the functional properties of PSII. The mutant showed accelerated turnover of the D1 protein, faster PSII repair, and a decrease in the cellular content of PSI. Radioactive labeling revealed a limitation in the synthesis of both CP47 and the PSI subunits PsaA/PsaB in the absence of Psb28. The mutant cells contained a high level of magnesium protoporphyrin IX methylester, a decreased level of protochlorophyllide, and released large quantities of protoporphyrin IX into the medium, indicating inhibition of chlorophyll (Chl) biosynthesis at the cyclization step yielding the isocyclic ring E. Overall, our results show the importance of Psb28 for synthesis of Chls and/or apoproteins of Chl-binding proteins CP47 and PsaA/PsaB.PSII is a multisubunit pigment-protein complex of plants, algae, and cyanobacteria, which is responsible for oxidation of water and reduction of plastoquinone during oxygenic photosynthesis (Barber, 2006). In the heart of the complex, there are two similar membrane-spanning proteins, D1 and D2, that bind the cofactors involved in primary charge separation (Nanba and Satoh, 1987) and subsequent electron transfer within PSII (for review, see Barber, 2006). Peripherally to the D1-D2 heterodimer, there are two chlorophyll (Chl)-binding inner antenna proteins, CP47 and CP43, that deliver energy to the reaction center (RC), driving electron transfer. In addition, CP43 also provides important ligands to the Mn₄Ca cluster, the site of water oxidation (Ferreira et al., 2004; Loll et al., 2005). These four large proteins are surrounded by a number of smaller membrane polypeptides (for review, see Shi and Schröder, 2004). One of them, the so-called PsbW, was originally detected in the isolated RC complex from spinach (Spinacia oleracea; Irrgang et al., 1995; Lorković et al., 1995). The mature protein with a predicted one-transmembrane α-helix in the central hydrophobic region seems to have (unlike most of PSII membrane proteins) the N terminus oriented into the lumen in close vicinity to the extrinsic, nuclear-encoded 33-kD PsbO protein. Cross-linking experiments also indicated a close association of PsbW with D1, D2, and the α-subunit of cytochrome (cyt) b-559 in the isolated RC complex (Irrgang et al., 1995; Lorković et al., 1995). At variance with these results, Rokka et al. (2005) located PsbW predominantly in PSII-light-harvesting complex II (LHCII) supercomplexes and only minor amounts were found in PSII core dimers and monomers. In transgenic plants of Arabidopsis (Arabidopsis thaliana) lacking the PsbW protein, the stability of the dimeric PSII was diminished and the PSII-LHCII supercomplexes could not be detected. It has been suggested that PsbW functions as a linker for LHCII binding to the PSII complex (Shi et al., 2000). Because LHCII is absent in cyanobacteria, it was intelligible that the PsbW was not detected in these oxygenic autotrophs. Nevertheless, N-terminal sequencing and mass spectrometric analyses of protein subunits in the purified His-tagged PSII from Synechocystis sp. PCC 6803 (Synechocystis 6803) revealed the presence of an unknown protein with 16% sequence identity to PsbW from Arabidopsis (Kashino et al., 2002). This protein was designated as Psb28 (also Psb13 or ycf79). Its amino acid sequence suggests that it is a rather hydrophilic protein without a transmembrane helix and is larger than PsbW (about 13 kD). In the recent crystal structures of the cyanobacterial PSII (Ferreira et al., 2004; Loll et al., 2005), this protein was not identified and it remains an issue of contention whether the protein is a true PSII subunit, a transiently associated assembly factor, or just an impurity of the preparation. The relatively low content of this protein in the isolated preparation suggested that the two latter possibilities are more probable. Very recently, the protein has been detected as a component of PSII complexes in Synechocystis depleted of phosphatidylglycerol (Sakurai et al., 2007). It has been proposed that the protein may play a regulatory role during the assembly of PSII. A gene encoding a similar soluble protein has also been found in the genome of Arabidopsis and the protein was designated PsbW-like.Here, we present a detailed analysis of the role of Psb28 in the structure and function of PSII in Synechocystis 6803. The results showed that Psb28 is not a component of the fully assembled dimeric PSII core complex, but it is preferentially bound to PSII assembly intermediates containing the inner antenna CP47. The results support the role of the protein in biogenesis of certain Chl-binding proteins via regulating synthesis of their apoproteins or Chls.     

Removal of both Ycf48 and Psb27 in Synechocystis sp. PCC 6803 disrupts Photosystem II assembly and alters QA oxidation in the mature complex

The Photosystem II (PS II) assembly factors Psb27 and Ycf48 are transiently associated with PS II during its biogenesis and repair pathways. We investigated the function of these proteins by constructing knockout mutants in Synechocystis sp. PCC 6803. In ΔYcf48 cells, PS II electron transfer and stable oxygen evolution were perturbed. Additionally, Psb27 was required for photoautotrophic growth of cells lacking Ycf48 and assembly beyond the RC47 assembly complex in ΔYcf48:ΔPsb27 cells was impeded. Our results suggest the RC47 complex formed in ΔYcf48 cells is defective and that this deficiency is exacerbated if CP43 binds in the absence of Psb27.     

Role of the PsbI protein in photosystem II assembly and repair in the cyanobacterium Synechocystis sp. PCC 6803

The involvement of the PsbI protein in the assembly and repair of the photosystem II (PSII) complex has been studied in the cyanobacterium Synechocystis sp. PCC 6803. Analysis of PSII complexes in the wild-type strain showed that the PsbI protein was present in dimeric and monomeric core complexes, core complexes lacking CP43, and in reaction center complexes containing D1, D2, and cytochrome b-559. In addition, immunoprecipitation experiments and the use of a histidine-tagged derivative of PsbI have revealed the presence in the thylakoid membrane of assembly complexes containing PsbI and either the precursor or mature forms of D1. Analysis of PSII assembly in the psbI deletion mutant and in strains lacking PsbI together with other PSII subunits showed that PsbI was not required for formation of PSII reaction center complexes or core complexes, although levels of unassembled D1 were reduced in its absence. However, loss of PsbI led to a dramatic destabilization of CP43 binding within monomeric and dimeric PSII core complexes. Despite the close structural relationship between D1 and PsbI in the PSII complex, PsbI turned over much slower than D1, whereas high light-induced turnover of D1 was accelerated in the absence of PsbI. Overall, our results suggest that PsbI is an early assembly partner for D1 and that it plays a functional role in stabilizing the binding of CP43 in the PSII holoenzyme.     

LPA2 is required for efficient assembly of photosystem II in Arabidopsis thaliana

To elucidate the molecular mechanism of photosystem II (PSII) assembly, we characterized the low psii accumulation2 (lpa2) mutant of Arabidopsis thaliana, which is defective in the accumulation of PSII supercomplexes. The levels and processing patterns of the RNAs encoding the PSII subunits are unaltered in the mutant. In vivo protein-labeling experiments showed that the synthesis of CP43 (for chlorophyll a binding protein) was greatly reduced, but CP47, D1, and D2 were synthesized at normal rates in the lpa2-1 mutant. The newly synthesized CP43 was rapidly degraded in lpa2-1, and the turnover rates of D1 and D2 were higher in lpa2-1 than in wild-type plants. The newly synthesized PSII proteins were assembled into PSII complexes, but the assembly of PSII was less efficient in the mutant than in wild-type plants. LPA2 encodes an intrinsic thylakoid membrane protein, which is not an integral subunit of PSII. Yeast two-hybrid assays indicated that LPA2 interacts with the PSII core protein CP43 but not with the PSII reaction center proteins D1 and D2. Moreover, direct interactions of LPA2 with Albino3 (Alb3), which is involved in thylakoid membrane biogenesis and cell division, were also detected. Thus, the results suggest that LPA2, which appears to form a complex with Alb3, is involved in assisting CP43 assembly within PSII.     

Deletion of psbJ leads to accumulation of Psb27-Psb28 photosystem II complexes in Thermosynechococcus elongatus

The life cycle of Photosystem II (PSII) is embedded in a network of proteins that guides the complex through biogenesis, damage and repair. Some of these proteins, such as Psb27 and Psb28, are involved in cofactor assembly for which they are only transiently bound to the preassembled complex. In this work we isolated and analyzed PSII from a ΔpsbJ mutant of the thermophilic cyanobacterium Thermosynechococcus elongatus. From the four different PSII complexes that could be separated the most prominent one revealed a monomeric Psb27-Psb28 PSII complex with greatly diminished oxygen-evolving activity. The MALDI-ToF mass spectrometry analysis of intact low molecular weight subunits (<10kDa) depicted wild type PSII with the absence of PsbJ. Relative quantification of the PsbA1/PsbA3 ratio by LC-ESI mass spectrometry using (15)N labeled PsbA3-specific peptides indicated the complete replacement of PsbA1 by the stress copy PsbA3 in the mutant, even under standard growth conditions (50μmol photons m(-2) s(-1)). This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Isolation and characterization of photosystem II subcomplexes from cyanobacteria lacking photosystem I.

A photosystem II (PSII) core complex lacking the internal antenna CP43 protein was isolated from the photosystem II of Synechocystis PCC6803, which lacks photosystem I (PSI). CP47-RC and reaction centre (RCII) complexes were also obtained in a single procedure by direct solubilization of whole thylakoid membranes. The CP47-RC subcore complex was characterized by SDS/PAGE, immunoblotting, MALDI MS, visible and fluorescence spectroscopy, and absorption detected magnetic resonance. The purity and functionality of RCII was also assayed. These preparations may be useful for mutational analysis of PSII RC and CP47-RC in studying primary reactions of oxygenic photosynthesis.     

Magneto-optical measurements of the pigments in fully active photosystem II core complexes from plants

Preparation of a minimum PSII core complex from spinach is described, containing four Mn per reaction center (RC) and exhibiting high O2 evolving activity [approximately 4000 micromol of O2 (mg of chl)(-1) x h(-1)]. The complex consists of the CP47 and CP43 chlorophyll binding proteins, the RC D1/D2 pair, the cytochrome b559 subunits, and the Mn-stabilizing psbO (33 kDa) protein, all present in the same stoichiometric amounts found in the parent PSII membranes. Several small subunits are also present. The cyt b559 content is 1.0 per RC in core complexes and PSII membranes. The total chlorophyll content is 32 chl a and <1 chl b per RC, the lowest yet reported for any active PSII preparation. The core complex exhibits the characteristic EPR signals seen in the S2 state of higher plant PSII. A procedure for preparing low-temperature samples of very high optical quality is developed, allowing detailed optical studies in the S1 and S2 states of the system to be made. Optical absorption, CD, and MCD spectra reveal unprecedented detail, including a prominent, well-resolved feature at 683.5 nm (14630 cm(-1)) with a weaker partner at 187 cm(-1) to higher energy. On the basis of band intensity, CD, and MCD arguments, these features are identified as the exciton split components of P680 in an intact, active reaction center special pair. Comparisons are made with solubilized D1/D2/cyt b559 material and cyanobacterial PSII.     

The Arabidopsis thylakoid protein PAM68 is required for efficient D1 biogenesis and photosystem II assembly

Photosystem II (PSII) is a multiprotein complex that functions as a light-driven water:plastoquinone oxidoreductase in photosynthesis. Assembly of PSII proceeds through a number of distinct intermediate states and requires auxiliary proteins. The photosynthesis affected mutant 68 (pam68) of Arabidopsis thaliana displays drastically altered chlorophyll fluorescence and abnormally low levels of the PSII core subunits D1, D2, CP43, and CP47. We show that these phenotypes result from a specific decrease in the stability and maturation of D1. This is associated with a marked increase in the synthesis of RC (the PSII reaction center-like assembly complex) at the expense of PSII dimers and supercomplexes. PAM68 is a conserved integral membrane protein found in cyanobacterial and eukaryotic thylakoids and interacts in split-ubiquitin assays with several PSII core proteins and known PSII assembly factors. Biochemical analyses of thylakoids from Arabidopsis and Synechocystis sp PCC 6803 suggest that, during PSII assembly, PAM68 proteins associate with an early intermediate complex that might contain D1 and the assembly factor LPA1. Inactivation of cyanobacterial PAM68 destabilizes RC but does not affect larger PSII assembly complexes. Our data imply that PAM68 proteins promote early steps in PSII biogenesis in cyanobacteria and plants, but their inactivation is differently compensated for in the two classes of organisms.     

The cyanobacterial homologue of HCF136/YCF48 is a component of an early photosystem II assembly complex and is important for both the efficient assembly and repair of photosystem II in Synechocystis sp. PCC 6803

The role of the slr2034 (ycf48) gene product in the assembly and repair of photosystem II (PSII) has been studied in the cyanobacterium Synechocystis PCC 6803. YCF48 (HCF136) is involved in the assembly of Arabidopsis thaliana PSII reaction center (RC) complexes but its mode of action is unclear. We show here that YCF48 is a component of two cyanobacterial PSII RC-like complexes in vivo and is absent in larger PSII core complexes. Interruption of ycf48 slowed the formation of PSII complexes in wild type, as judged from pulse-labeling experiments, and caused a decrease in the final level of PSII core complexes in wild type and a marked reduction in the levels of PSII assembly complexes in strains lacking either CP43 or CP47. Absence of YCF48 also led to a dramatic decrease in the levels of the COOH-terminal precursor (pD1) and the partially processed form, iD1, in a variety of PSII mutants and only low levels of unassembled mature D1 were observed. Yeast two-hybrid analyses using the split ubiquitin system showed an interaction of YCF48 with unassembled pD1 and, to a lesser extent, unassembled iD1, but not with unassembled mature D1 or D2. Overall our results indicate a role for YCF48 in the stabilization of newly synthesized pD1 and in its subsequent binding to a D2-cytochrome b559 pre-complex, also identified in this study. Besides a role in assembly, we show for the first time that YCF48 also functions in the selective replacement of photodamaged D1 during PSII repair.     

Accumulation of the D2 protein is a key regulatory step for assembly of the photosystem II reaction center complex in Synechocystis PCC 6803

Accumulation of monomer and dimer photosystem (PS) II reaction center core complexes has been analyzed by two-dimensional Blue-native/SDS-PAGE in Synechocystis PCC 6803 wild type and in mutant strains lacking genes psbA, psbB, psbC, psbDIC/DII, or the psbEFLJ operon. In vivo pulse-chase radiolabeling experiments revealed that mutant cells assembled PSII precomplexes only. In DeltapsbC and DeltapsbB, assembly of reaction center cores lacking CP43 and reaction center complexes was detected, respectively. In DeltapsbA, protein subunits CP43, CP47, D2, and cytochrome b559 were synthesized, but proteins did not assemble. Similarly, in DeltapsbD/C lacking D2, and CP43, the de novo synthesized proteins D1, CP47, and cytochrome b559 did not form any mutual complexes, indicating that assembly of the reaction center complex is a prerequisite for assembly with core subunits CP47 and CP43. Finally, although CP43 and CP47 accumulated in DeltapsbEFLJ, D2 was neither expressed nor accumulated. We, furthermore, show that the amount of D2 is high in the strain lacking D1, whereas the amount of D1 is low in the strain lacking D2. We conclude that expression of the psbEFLJ operon is a prerequisite for D2 accumulation that is the key regulatory step for D1 accumulation and consecutive assembly of the PSII reaction center complex.     

Light harvesting in photosystem II

Water oxidation in photosynthesis takes place in photosystem II (PSII). This photosystem is built around a reaction center (RC) where sunlight-induced charge separation occurs. This RC consists of various polypeptides that bind only a few chromophores or pigments, next to several other cofactors. It can handle far more photons than the ones absorbed by its own pigments and therefore, additional excitations are provided by the surrounding light-harvesting complexes or antennae. The RC is located in the PSII core that also contains the inner light-harvesting complexes CP43 and CP47, harboring 13 and 16 chlorophyll pigments, respectively. The core is surrounded by outer light-harvesting complexes (Lhcs), together forming the so-called supercomplexes, at least in plants. These PSII supercomplexes are complemented by some “extra” Lhcs, but their exact location in the thylakoid membrane is unknown. The whole system consists of many subunits and appears to be modular, i.e., both its composition and organization depend on environmental conditions, especially on the quality and intensity of the light. In this review, we will provide a short overview of the relation between the structure and organization of pigment-protein complexes in PSII, ranging from individual complexes to entire membranes and experimental and theoretical results on excitation energy transfer and charge separation. It will become clear that time-resolved fluorescence data can provide invaluable information about the organization and functioning of thylakoid membranes. At the end, an overview will be given of unanswered questions that should be addressed in the near future.     

Charge separation and energy transfer in the photosystem II core complex studied by femtosecond midinfrared spectroscopy

The core of photosystem II (PSII) of green plants contains the reaction center (RC) proteins D1D2-cytb559 and two core antennas CP43 and CP47. We have used time-resolved visible pump/midinfrared probe spectroscopy in the region between 1600 and 1800 cm(-1) to study the energy transfer and charge separation events within PSII cores. The absorption difference spectra in the region of the keto and ester chlorophyll modes show spectral evolution with time constants of 3 ps, 27 ps, 200 ps, and 2 ns. Comparison of infrared (IR) difference spectra obtained for the isolated antennas CP43 and CP47 and the D1D2-RC with those measured for the PSII core allowed us to identify the features specific for each of the PSII core components. From the presence of the CP43 and CP47 specific features in the spectra up to time delays of 20-30 ps, we conclude that the main part of the energy transfer from the antennas to the RC occurs on this timescale. Direct excitation of the pigments in the RC evolution associated difference spectra to radical pair formation of PD1+PheoD1- on the same timescale as multi-excitation annihilation and excited state equilibration within the antennas CP43 and CP47, which occur within approximately 1-3 ps. The formation of the earlier radical pair ChlD1+PheoD1-, as identified in isolated D1D2 complexes with time-resolved mid-IR spectroscopy is not observed in the current data, probably because of its relatively low concentration. Relaxation of the state PD1+PheoD1-, caused by a drop in free energy, occurs in 200 ps in closed cores. We conclude that the kinetic model proposed earlier for the energy and electron transfer dynamics within the D1D2-RC, plus two slowly energy-transferring antennas C43 and CP47 explain the complex excited state and charge separation dynamics in the PSII core very well. We further show that the time-resolved IR-difference spectrum of PD1+PheoD1- as observed in PSII cores is virtually identical to that observed in the isolated D1D2-RC complex of PSII, demonstrating that the local structure of the primary reactants has remained intact in the isolated D1D2 complex.     

Impact of PsbTc on forward and back electron flow, assembly, and phosphorylation patterns of photosystem II in tobacco

Photosystem II (PSII) of oxygen-evolving cyanobacteria, algae, and land plants mediates electron transfer from the Mn₄Ca cluster to the plastoquinone pool. It is a dimeric supramolecular complex comprising more than 30 subunits per monomer, of which 16 are bitopic or peripheral, low-molecular-weight components. Directed inactivation of the plastid gene encoding the low-molecular-weight peptide PsbTc in tobacco (Nicotiana tabacum) does not prevent photoautotrophic growth. Mutant plants appear normal green, and levels of PSII proteins are not affected. Yet, PSII-dependent electron transport, stability of PSII dimers, and assembly of PSII light-harvesting complexes (LHCII) are significantly impaired. PSII light sensitivity is moderately increased and recovery from photoinhibition is delayed, leading to faster D1 degradation in ΔpsbTc under high light. Thermoluminescence emission measurements revealed alterations of midpoint potentials of primary/secondary electron-accepting plastoquinone of PSII interaction. Only traces of CP43 and no D1/D2 proteins are phosphorylated, presumably due to structural changes of PSII in ΔpsbTc. In striking contrast to the wild type, LHCII in the mutant is phosphorylated in darkness, consistent with its association with PSI, indicating an increased pool of reduced plastoquinone in the dark. Finally, our data suggest that the secondary electron-accepting plastoquinone of PSII site, the properties of which are altered in ΔpsbTc, is required for oxidation of reduced plastoquinone in darkness in an oxygen-dependent manner. These data present novel aspects of plastoquinone redox regulation, chlororespiration, and redox control of LHCII phosphorylation.     

Revealing the structure of the oxygen-evolving core dimer of photosystem II by cryoelectron crystallography.

Here we present cryoelectron crystallographic analysis of an isolated dimeric oxygen-evolving complex of photosystem II (at a resolution of approximately 0.9 nm), revealing that the D1-D2 reaction center (RC) proteins are centrally located between the chlorophyll-binding proteins, CP43 and CP47. This conclusion supports the hypothesis that photosystems I and II have similar structural features and share a common evolutionary origin. Additional density connecting the two halves of the dimer, which was not observed in a recently described CP47-RC complex that did not include CP43, may be attributed to the small subunits that are involved in regulating secondary electron transfer, such as PsbH. These subunits are possibly also required for stabilization of the dimeric photosystem II complex. This complex, containing at least 29 transmembrane helices in its asymmetric unit, represents one of the largest membrane protein complexes studied at this resolution.     

The Psb27 protein facilitates manganese cluster assembly in photosystem II

Photosystem II (PSII) is a large membrane protein complex that uses light energy to convert water to molecular oxygen. This enzyme undergoes an intricate assembly process to ensure accurate and efficient positioning of its many components. It has been proposed that the Psb27 protein, a lumenal extrinsic subunit, serves as a PSII assembly factor. Using a psb27 genetic deletion strain (Deltapsb27) of the cyanobacterium Synechocystis sp. PCC 6803, we have defined the role of the Psb27 protein in PSII biogenesis. While the Psb27 protein was not essential for photosynthetic activity, various PSII assembly assays revealed that the Deltapsb27 mutant was defective in integration of the Mn(4)Ca(1)Cl(x) cluster, the catalytic core of the oxygen-evolving machinery within the PSII complex. The other lumenal extrinsic proteins (PsbO, PsbU, PsbV, and PsbQ) are key components of the fully assembled PSII complex and are important for the water oxidation reaction, but we propose that the Psb27 protein has a distinct function separate from these subunits. We show that the Psb27 protein facilitates Mn(4)Ca(1)Cl(x) cluster assembly in PSII at least in part by preventing the premature association of the other extrinsic proteins. Thus, we propose an exchange of lumenal subunits and cofactors during PSII assembly, in that the Psb27 protein is replaced by the other extrinsic proteins upon assembly of the Mn(4)Ca(1)Cl(x) cluster. Furthermore, we show that the Psb27 protein provides a selective advantage for cyanobacterial cells under conditions such as nutrient deprivation where Mn(4)Ca(1)Cl(x) cluster assembly efficiency is critical for survival.     

Cooperation of LPA3 and LPA2 Is Essential for Photosystem II Assembly in Arabidopsis

Photosystem II (PSII) is a multisubunit membrane protein complex that is assembled in a sequence of steps. However, the molecular mechanisms responsible for the assembly of the individual subunits into functional PSII complexes are still largely unknown. Here, we report the identification of a chloroplast protein, Low PSII Accumulation3 (LPA3), which is required for the assembly of the CP43 subunit in PSII complexes in Arabidopsis (Arabidopsis thaliana). LPA3 interacts with LPA2, a previously identified PSII CP43 assembly factor, and a double mutation of LPA2 and LPA3 is more deleterious for assembly than either single mutation, resulting in a seedling-lethal phenotype. Our results indicate that LPA3 and LPA2 have overlapping functions in assisting CP43 assembly and that cooperation between LPA2 and LPA3 is essential for PSII assembly. In addition, we provide evidence that LPA2 and LPA3 interact with Albino3 (Alb3), which is essential for thylakoid protein biogenesis. Thus, the function of Alb3 in some PSII assembly processes is probably mediated through interactions with LPA2 and LPA3.Oxygenic photosynthesis, in which oxygen and organic carbon are produced from water and carbon dioxide using sunlight, provides energy for nearly all living organisms on Earth. Four major multiprotein complexes, located in thylakoid membranes, are responsible for the capture of light and its conversion to chemical energy in eukaryotic photosynthetic organisms: PSI, PSII, cytochrome b₆/f, and ATP synthase (Wollman et al., 1999; Nelson and Yocum, 2006). PSII catalyzes one of the most important of all biochemical reactions, the light-induced transfer of electrons from water to plastoquinone, which generates most of the oxygen in the Earth’s atmosphere. PSII consists of more than 20 subunits in higher plants (Wollman et al., 1999; Iwata and Barber, 2004; Nelson and Yocum, 2006). The PSII reaction center consists of the D1 and D2 proteins, the α- and β-subunits of cytochrome b₅₅₉, and the PsbI protein, and the D1 and D2 heterodimers bind all the redox components essential for the primary charge separation (Nanba and Satoh, 1987). The PSII core complex additionally contains CP47, CP43, the oxygen-evolving complex, and several low molecular mass proteins (Wollman et al., 1999; Nelson and Yocum, 2006). CP47 and CP43, two inner chlorophyll a-binding proteins, are closely associated with, and located on opposite sides of, the PSII reaction center (Hankamer et al., 1999). The functional form of PSII cores in thylakoid membranes is dimeric and is associated with light-harvesting complex (LHC). In PSII-LHCII supercomplexes, PSII core dimers are surrounded by LHCII trimers, which consist of Lhcb1 and Lhcb2 proteins (Wollman et al., 1999; Iwata and Barber, 2004; Nelson and Yocum, 2006).Our knowledge of the molecular mechanisms involved in the biogenesis and assembly of PSII in the thylakoid membranes is still limited, although the structure and function of PSII have been extensively studied. Genetic and biochemical studies have elucidated several distinct steps that occur in PSII assembly. D2 and cytochrome b₅₅₉ form an initial complex, which serves as a receptor for the cotranslational assembly of D1 (Adir et al., 1990; van Wijk et al., 1997; Müller and Eichacker, 1999; Zhang et al., 1999). The next step involves the association of CP47 with the PSII reaction center (Zhang et al., 1999; Rokka et al., 2005), while CP43 is synthesized independently and then continuously associates and dissociates with PSII (de Vitry et al., 1989; Zhang et al., 2000). The biogenesis of PSII involves “a control by epistasy of synthesis” process (Minai et al., 2006). D2 is required for D1 synthesis, which itself is needed for CP47 synthesis. However, many aspects of the processes involved in the oligomerization and coordination of the various PSII subunits are still unclear (Rochaix, 2001). Due to the structural complexity of PSII, its assembly consists of multiple assembly steps, which is likely to require the participation of a number of assembly factors.Several assembly factors involved in the biosynthesis and assembly of the PSII complex have been identified recently. For instance, the thylakoid lumen protein HCF136 is known to be required for the formation of PSII, since the hcf136 mutant is capable of synthesizing plastid-encoded proteins, but it does not appear to accumulate any stable PSII complexes, due to blockage of the assembly of the PSII reaction center (Meurer et al., 1998; Plücken et al., 2002). Alb3.1, a homolog of Arabidopsis (Arabidopsis thaliana) Albino3 (Alb3), is essential for the efficient assembly of PSII in Chlamydomonas reinhardtii, probably through interactions with D1 following its insertion (Ossenbühl et al., 2004), and another Alb3 homolog, Alb3.2, appears to be required for photosystem assembly in Chlamydomonas (Göhre et al., 2006). Coimmunoprecipitation analysis has shown that Alb3.1 and Alb3.2 interact directly, while Alb3.2 reportedly interacts with the PSI and PSII reaction centers proteins (Göhre et al., 2006). The lumenal immunophilins, AtCYP38 and FKBP20-2, have also been shown to be involved in PSII assembly (Lima et al., 2006; Fu et al., 2007; Sirpiö et al., 2008). In addition, we recently identified two PSII assembly factors, Low PSII Accumulation1 (LPA1) and LPA2, involved in PSII assembly. The LPA1 protein appears to be an integral membrane chaperone required for efficient assembly of the PSII core complex, probably through direct interaction with D1 (Peng et al., 2006). LPA2, which interacts with Alb3, forms a protein complex that assists CP43 assembly within PSII (Ma et al., 2007). These findings suggest that each stage of the PSII assembly process is assisted by one or more specific assembly factors, most of which have not yet been identified.Here, we report the identification of a lpa3 mutant with reduced levels of PSII. Functional characterization points to the possible role of LPA3 in assisting CP43 assembly within PSII. In addition, biochemical and genetic analyses indicate that an assembly complex of LPA3 and LPA2 is essential for PSII assembly.     

Interaction of methylamine with extrinsic and intrinsic subunits of photosystem II

The interaction of methylamine with chloroplasts' photosystem II (PSII) was studied in isolated thylakoid membranes. Low concentration of methylamine (mM range) was shown to affect water oxidation and the advancement of the S-states. Modified kinetics of chlorophyll fluorescence rise and thermoluminescence in the presence of methylamine indicated that the electron transfer was affected at both sides of PSII, and in particular the electron transfer between Y_Z and P680⁺. As the concentration of methylamine was raised above 10 mM, the extrinsic polypeptides associated with the oxygen-evolving complex were lost and energy transfer between PSII antenna complexes and reaction centers was impaired. It was concluded that methylamine is able to affect both extrinsic and intrinsic subunits of PSII even at the lowest concentrations used where the extrinsic polypeptides of the OEC are still associated with the luminal side of the photosystem. As methylamine concentration increases, the extrinsic polypeptides are lost and the interaction with intrinsic domains is amplified resulting in an increased F₀.