Organization of transmembrane helices in photosystem II: comparison of plants and cyanobacteria

Electron microscopy and X-ray crystallography are revealing the structure of photosystem II. Electron crystallography has yielded a 3D structure at sufficient resolution to identify subunit positioning and transmembrane organization of the reaction-centre core complex of spinach. Single-particle analyses are providing 3D structures of photosystem II-light-harvesting complex II supercomplexes that can be used to incorporate high-resolution structural data emerging from electron and X-ray crystallography. The positions of the chlorins and metal centres within photosystem II are now available. It can be concluded that photosystem II is a dimeric complex with the transmembrane helices of CP47/D2 proteins related to those of the CP43/D1 proteins by a twofold axis within each monomer. Further, both electron microscopy and X-ray analyses show that P(680) is not a 'special pair' and that cytochrome b559 is located on the D2 side of the reaction centres some distance from P(680). However, although comparison of the electron microscopy and X-ray models for spinach and Synechococcus elongatus show considerable similarities, there seem to be differences in the number and positioning of some small subunits.     

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 structure, function and dynamics of photosystem two

One of the greatest challenges in modern photosynthesis research is to elucidate fully the structural and functional properties of photosystem two (PSII). This water-plasto-quinone oxidoreductase is located in a membrane complex composed of more than 25 subunits. The primary and secondary structures of all known subunits which constitute the central core of PSII are reviewed. How these subunits interact with each other to produce the tertiary and quaternary structure of PSII in vivo is not fully understood. However, electron microscopy is helping to fill this gap in our knowledge both by single particle analysis and electron crystallography. These studies suggest that active PSII is dimeric, although the functional significance of this oligomeric state is not yet understood. Moreover, the elucidation of the structure of photosystem one (PSI) by X-ray crystallography has revealed features which are likely to be relevant to PSII structure. It seems highly likely that the D1 protein with CP43 and D2 protein with CP47 (summing 11 transmembrane helices in each case) will have structural similarities to the organisation of PsaA and PsaB. It is likely that the turnover of the D1 protein is aided by the relatively easy removal of CP43 from this arrangement of the PSII core.     

Phosphatidylglycerol is involved in the dimerization of photosystem II

Photosystem II core dimers (450 kDa) and monomers (230 kDa) consisting of CP47, CP43, the D1 and D2 proteins, the extrinsic 33-kDa subunit, and the low molecular weight polypeptides PsbE, PsbF, PsbH, PsbI, PsbK, PsbL, PsbTc, and PsbW were isolated by sucrose density gradient centrifugation. The photosystem II core dimers were treated with phospholipase A2 (PL-A2), which cuts phosphatidylglycerol (PG) and phosphatidylcholine molecules at the sn-2 position. The PL-A2-treated dimers dissociated into two core monomers and further, yielding a CP47-D1-D2 subcomplex and CP43. Thin layer chromatography showed that photosystem II dimers contained four times more PG than their monomeric counterparts but with similar levels of phosphatidylcholine. Consistent with this was the finding that, compared with monomers, the dimers contained a higher level of trans-hexadecanoic fatty acid (C16:1Delta3tr), which is specific to PG of the thylakoid membrane. Moreover, treatment of dimers with PL-A2 increased the free level of this fatty acid specific to PG compared with untreated dimers. Further evidence that PG is involved in stabilizing the dimeric state of photosystem II comes from reconstitution experiments. Using size exclusion chromatography, it was shown that PG containing C16:1Delta3tr, but not other lipid classes, induced significant dimerization of isolated photosystem II monomers. Moreover, this dimerization was observed by electron crystallography when monomers were reconstituted into thylakoid lipids containing PG. The unit cell parameters, p2 symmetry axis, and projection map of the reconstituted dimer was similar to that observed for two-dimensional crystals of the native dimer.     

Revealing the structure of the photosystem II chlorophyll binding proteins, CP43 and CP47

A review of the structural properties of the photosystem II chlorophyll binding proteins, CP47 and CP43, is given and a model of the transmembrane helical domains of CP47 has been constructed. The model is based on (i) the amino acid sequence of the spinach protein, (ii) an 8 A three-dimensional electron density map derived from electron crystallography and (iii) the structural homology which the membrane spanning region of CP47 shares with the six N-terminal transmembrane helices of the PsaA/PsaB proteins of photosystem I. Particular emphasis has been placed on the position of chlorophyll molecules assigned in the 8 A three-dimensional map of CP47 (K.-H. Rhee, E.P. Morris, J. Barber, W. Kühlbrandt, Nature 396 (1998) 283-286) relative to histidine residues located in the transmembrane regions of this protein which are likely to form axial ligands for chlorophyll binding. Of the 14 densities assigned to chlorophyll, the model predicted that five have their magnesium ions within 4 A of the imidazole nitrogens of histidine residues. For the remaining seven histidine residues the densities attributed to chlorophylls were within 4-8 A of the imidazole nitrogens and thus too far apart for direct ligation with the magnesium ion within the tetrapyrrole head group. Improved structural resolution and reconsiderations of the orientation of the porphyrin rings will allow further refinement of the model.     

Protein composition of the photosystem II core complex in genetically engineered mutants of the cyanobacterium Synechocystis sp. PCC 6803

The presence of four photosystem II proteins, CP47, CP43, D1 and D2, was monitored in mutants of Synechocystis sp. PCC 6803 that have modified or inactivated genes for CP47, CP43, or D2. It was observed that: (1) thylakoids from mutants without a functional gene encoding CP47 are also depleted in D1 and D2; (2) inactivation of the gene for CP43 leads to decreased but significant levels of CP47, D1 and D2; (3) deletion of part of both genes encoding D2, together with deletion of part of the CP43-encoding gene causes a complete loss of CP47 and D1; (4) thylakoids from a site-directed mutant in which the His-214 residue of D2 has been replaced by asparagine do not contain detectable photosystem II core proteins. However, in another site-directed mutant, in which His-197 has been replaced by tyrosine, some CP47 as well as breakdown products of CP43, but no D1 and D2, can be detected. These data could indicate a central function of CP47 and D2 in stable assembly of the photosystem II complex. CP43, however, is somewhat less critical for formation of the core complex, although CP43 is required for a physiologically functional photosystem II unit. A possible model for the assembly of the photosystem II core complex is proposed.     

Localisation of CP43 in single molecules of photosystem 2 using protein deletion and scanning tunnelling microscopy

Scanning tunnelling microscopy of intact D1/D2/CP47/CP43 photosystem 2 (PS2) core complexes and CP43-deleted D1/D2/CP47 core complexes shows definitively that the CP43 subunits reside at the ends of the dimeric core complex. The CP43-removal procedure produces CP43-deleted cores with minimal conformational distortion to the D1/D2/CP47 residual core complex. There was excellent agreement between the X-ray and STM structures for the intact core complex, and between the STM image for the CP43-deleted core complex and the X-ray model with the components assigned to CP43 omitted.     

Nomenclature for membrane-bound light-harvesting complexes of cyanobacteria

Accessory chlorophyll-binding proteins (CBP) in cyanobacteria have six transmembrane helices and about 11 conserved His residues that might participate in chlorophyll binding. In various species of cyanobacteria, the CBP proteins bind different types of chlorophylls, including chlorophylls a, b, d and divinyl-chlorophyll a, b. The CBP proteins do not belong to the light-harvesting complexes (LHC) superfamily of plant and algae. The proposed new name of CBP for this class of proteins, which is a unique accessory light-harvesting superfamily in cyanobacteria, clarifies the confusion of names of prochlorophytes chlorophyll binding protein (Pcb), PSII-like light-harvesting proteins and iron-stress-induced protein A (IsiA). The CBP complexes are a member of a larger family that includes the chlorophyll a-binding proteins CP43 and CP47 that function as core antennas of photosystem II.     

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.     

On the molecular mechanism of light-induced D1 protein degradation in photosystem II core particles.

The mechanism of D1 protein degradation was investigated during photoinhibitory illumination of isolated photosystem II core preparations. The studies revealed that a proteolytic activity resides within the photosystem II core complex. A relationship between the inhibition of D1 protein degradation and the binding of the highly specific serine protease inhibitor diisopropyl fluorophosphate to isolated complexes of photosystem II was observed, evidence that this protease is of the serine type. Using radiolabeled inhibitor, it was shown that the binding site, representing the active serine of the catalytic site, is located on a 43-kDa polypeptide, probably the chlorophyll a protein CP43. The protease is apparently active in darkness, with the initiation of breakdown being dependent on high light-induced substrate activation. The proteolysis, which has an optimum at pH 7.5, gives rise to primary degradation fragments of 23 and 16 kDa. In addition, D1 protein fragments of 14, 13, and 10 kDa were identified. Experiments with phosphate-labeled D1 protein and sequence-specific antisera showed that the 23- and 16-kDa fragments originate from the N- and C-termini, respectively, suggesting a primary cleavage of the D1 protein at the outer thylakoid surface in the region between transmembrane helices D and E.     

Further identification of the exoplasmic face particles on the freeze-fractured thylakoid membranes: a study using double and triple mutants from Chlamydomonas reinhardtii lacking various photosystem II subunits and the cytochrome b6/f complex.

About 20% of the exoplasmic face (EF) particles present in the freeze-fractured thylakoid membranes of the wild type strain of Chlamydomonas reinhardtii remain in mutants lacking photosystem II (PSII) because of the absence of either one of the two PSII subcomplexes CP43 or D1/D2/CP47. We show that about half of these residual EF particles can be accounted for by PSII subcomplexes still present in such mutants, and by cytochrome (cyt) b6/f complexes. Analysis of double mutants lacking both types of protein complexes points to an association of cyt b6/f complexes with PSII subcomplexes in some of these EF particles and to a requirement in cyt b6/f complexes for the translocation of each of the two PSII subcomplexes (the CP43 subunit and the D1/D2/CP47 subcomplex) from the unstacked to the stacked regions of the thylakoid membranes.     

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.     

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.     

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.     

Electron crystallographic study of photosystem II of the cyanobacterium Synechococcus elongatus

The determination of the structure of PSII at high resolution is required in order to fully understand its reaction mechanisms. Two-dimensional crystals of purified highly active Synechococcus elongatus PSII dimers were obtained by in vitro reconstitution. Images of these crystals were recorded by electron cryo-microscopy, and their analysis revealed they belong to the two-sided plane group p22(1)2(1), with unit cell parameters a = 121 A, b = 333 A, and alpha = 90 degrees. From these crystals, a projection map was calculated to a resolution of approximately 16 A. The reliability of this projection map is confirmed by its close agreement with the recently presented three-dimensional model of the same complex obtained by X-ray crystallography. Comparison of the projection map of the Synechococcus elongatus PSII complex with data obtained by electron crystallography of the spinach PSII core dimer reveals a similar organization of the main transmembrane subunits. However, some differences in density distribution between the cyanobacterial and higher plant PSII complexes exist, especially in the outer region of the complex between CP43 and cytochrome b(559) and adjacent to the B-helix of the D1 protein. These differences are discussed in terms of the number and organization of some of the PSII low molecular weight subunits.     

D1 protein of photosystem II: The light sensor in chloroplasts

Light, controls the “blueprint” for chloroplast development, but at high intensities is toxic to the chloroplast. Excessive light intensities inhibit primarily photosystem II electron transport. This results in generation of toxic singlet oxygen due to impairment of electron transport on the acceptor side between pheophytin and Q_B -the secondary electron acceptor. High light stress also impairs electron transport on the donor side of photosystem II generating highly oxidizing species Z⁺ and P680⁺. A conformationsl change in the photosystem II reaction centre protein Dl affecting its Q_B-binding site is involved in turning the damaged protein into a substrate for proteolysis. The evidence indicates that the degradation of D1 is an enzymatic process and the protease that degrades D1 protein has been shown to be a serine protease Although there is evidence to indicate that the chlorophyll a-protein complex CP43 acts as a serine-type protease degrading Dl, the observed degradation of Dl protein in photosystem II reaction centre particlesin vitro argues against the involvement of CP43 in Dl degradation. Besides the degradation during high light stress of Dl, and to a lesser extent D2-the other reaction centre protein, CP43 and CP29 have also been shown to undergo degradation. In an oxygenic environment, Dl is cleaved from its N-and C-termini and the disassembly of the photosystem II complex involves simultaneous release of manganese and three extrinsic proteins involved in oxygen evolution. It is known that protein with PEST sequences are subject to degradation; D1 protein contains a PEST sequence adjacent to the site of cleavage on the outer side of thylakoid membrane between helices IV and V. The molecular processes of “triggering” of Dl for proteolytic degradation are not clearly understood. The changes in structural organization of photosystem II due to generation of oxy-radicals and other highly oxidizing species have also not been resolved. Whether CP43 or a component of the photosystem II reaction centre itself (Dl. D2 or cy1 b559 subunits), which may be responsible for degradation of Dl, is also subject to light modification to become an active protease, is also not known. The identity of proteases degrading Dl, LHCII and CP43 and C29 remains to be established     

Identification of Oxidized Amino Acid Residues in the Vicinity of the Mn(4)CaO(5) Cluster of Photosystem II: Implications for the Identification of Oxygen Channels within the Photosystem

As a light-driven water-plastoquinone oxidoreductase, Photosystem II produces molecular oxygen as an enzymatic product. Additionally, under a variety of stress conditions, reactive oxygen species are produced at or near the active site for oxygen evolution. In this study, Fourier-transform ion cyclotron resonance mass spectrometry was used to identify oxidized amino acid residues located in several core Photosystem II proteins (D1, D2, CP43, and CP47) isolated from spinach Photosystem II membranes. While the majority of these oxidized residues (81%) are located on the oxygenated solvent-exposed surface of the complex, several residues on the CP43 protein ((354)E, (355)T, (356)M, and (357)R) which are in close proximity (<15 ?) to the Mn(4)CaO(5) active site are also modified. These residues appear to be associated with putative oxygen/reactive oxygen species exit channel(s) in the photosystem. These results are discussed within the context of a number of computational studies which have identified putative oxygen channels within the photosystem.     

Pathways for energy transfer in the core light-harvesting complexes CP43 and CP47 of photosystem II

The pigment-protein complexes CP43 and CP47 transfer excitation energy from the peripheral antenna of photosystem II toward the photochemical reaction center. We measured the excitation dynamics of the chlorophylls in isolated CP43 and CP47 complexes at 77 K by time-resolved absorbance-difference and fluorescence spectroscopy. The spectral relaxation appeared to occur with rates of 0.2-0.4 ps and 2-3 ps in both complexes, whereas an additional relaxation of 17 ps was observed only in CP47. Using the 3.8-A crystal structure of the photosystem II core complex from Synechococcus elongatus (A. Zouni, H.-T. Witt, J. Kern, P. Fromme, N. Krauss, W. Saenger, and P. Orth, 2001, Nature, 409:739-743), excitation energy transfer kinetics were calculated and a Monte Carlo simulation of the absorption spectra was performed. In both complexes, the rate of 0.2-0.4 ps can be ascribed to excitation energy transfer within a layer of chlorophylls near the stromal side of the membrane, and the slower 2-3-ps process to excitation energy transfer to the calculated lowest excitonic state. We conclude that excitation energy transfer within CP43 and CP47 is fast and does not contribute significantly to the well-known slow trapping of excitation energy in photosystem II.     

Low-molecular-mass proteins in cyanobacterial photosystem II: identification of psbH and psbK gene products by N-terminal sequencing

The O2-evolving photosystem II core complex was isolated from a thermophilic cyanobacterium, Synechococcus vulcanus Copeland. Analysis by SDS-polyacrylamide gel electrophoresis revealed that the complex contained at least seven low-molecular-mass proteins in addition to the well characterized CP47 apoprotein, CP43 apoprotein, 33 kDa extrinsic protein, D1 protein, D2 protein and large subunit of cytochrome b-559. The separation of these low-molecular-mass proteins were very similar between cyanobacterial and higher plant PS II. N-terminal sequences of the 6.5 kDa and 3.9 kDa proteins of cyanobacterial core complex were determined after blotting to a polyvinylidene difluoride membrane. The sequence of the 6.5 kDa protein showed high homology with an internal sequence of plant psbH gene product, so-called 10 kDa phosphoprotein, but did not conserve the Thr residue which is specifically phosphorylated in plants. The sequence of the 3.9 kDa protein corresponded to the K protein of higher plants (mature form of psbK gene product). These results indicate that the products of both psbH and psbK genes are present in cyanobacterial PS II as well as being associated with the O2-evolving core complex.     

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.