Isolation and characterization of photosystem II core complexes with high oxygen evolution activity from spinach using the non-ionic detergent 6-O-(N-heptylcarbamoyl)-methyl-<Emphasis Type="Italic">α</Emphasis>-D-glucopyranoside

Two different kinds of oxygen evolving photosystem II (PSII) core complexes were isolated in the present study by solubilization of PSII enriched thylakoid membranes from spinach with the non-ionic detergent 6-O-(N-heptylcarbamoyl)-methyl-_α-D-glucopyranoside (Hecameg) under different conditions. The PSII core complex isolated at higher ionic strength was similar to that isolated by using octyl-β-D-glucopyranoside (OGP) and lacked the 23 and 17 kDa extrinsic proteins of the oxygen evolving complex but retained the 22 kDa PsbS protein. Solubilization of the PSII membranes with Hecameg at lower ionic strength allowed the isolation of another PSII complex that retained all the three extrinsic proteins (33, 23 and 17 kDa) of the oxygen evolving complex but was depleted of the 22 kDa PsbS protein. This complex exhibited high rates of oxygen evolution and was found to be more sensitive to DCMU indicating a better structural and functional integrity and may be treated as the minimal functional unit required for PSII photochemistry. The detergent Hecameg is relatively inexpensive and the methodology remains simple since it does not require any chromatography or density gradient ultracentrifugation.     

Fast Isolation of Highly Active Photosystem Ⅱ Core Complexes from Spinach

Purification of photosystem Ⅱ (PSII) core complexes is a time-consuming and low-efficiency process. In order to isolate pure and active PSII core complexes in large amounts, we have developed a fast method to isolate highly active monomeric and dimeric PSII core complexes from spinach leaves by using sucrose gradient ultracentrifugation. By using a vertical rotor the process was completed significantly faster compared with a swing-out rotor. In order to keep the core complexes in high activity, the whole isolation procedure was performed in the presence of glycine betain and pH at 6.3. The isolated pigment-protein complexes were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, absorption spectroscopy, 77 K fluorescence spectroscopy and high performance liquid chromatography. Our results show that this method is a better choice for quick and efficient isolation of functionally active PSII core complexes.     

Three-Dimensional Electron Microscopy of the Photosystem II Core Complex

A three-dimensional image of the spinach photosystem II core complex composed of CP47, D1, D2, cytochromeb-559, andpsbI gene product was reconstructed at 20-Å resolution from the two-dimensional crystals negatively stained with phosphotungstate. Confirming the previous proposal, the crystal had ap22₁2₁symmetry. One PSII core complex was measured to be 80 × 80 Å in the membrane plane and 88 Å normal to it. The mass distribution was asymmetric about the lipid bilayer, consistent with predictions from the amino acid sequences. The lumenal mass consisted of three domains forming a characteristic triangular platform with another domain on top of it. Three stromal domains were smaller and linearly arranged. Due to strong stain exclusion in the hydrophobic core part of the lipid bilayer, the transmembrane region appeared to be imaged with a reversed contrast. Inverting the contrast resulted in a reasonable density distribution for that part. Thus, though the information on the transmembrane region is limited, the domain structure of the PSII core complex was revealed and allowed us to propose a model for the arrangement of subunits in the PSII core complex.     

Organisation of Photosystem I and Photosystem II in red alga Cyanidium caldarium: Encounter of cyanobacterial and higher plant concepts

Structure and organisation of Photosystem I and Photosystem II isolated from red alga Cyanidium caldarium was determined by electron microscopy and single particle image analysis. The overall structure of Photosystem II was found to be similar to that known from cyanobacteria. The location of additional 20 kDa (PsbQ′) extrinsic protein that forms part of the oxygen evolving complex was suggested to be in the vicinity of cytochrome c-550 (PsbV) and the 12 kDa (PsbU) protein. Photosystem I was determined as a monomeric unit consisting of PsaA/B core complex with varying amounts of antenna subunits attached. The number of these subunits was seen to be dependent on the light conditions used during cell cultivation. The role of PsaH and PsaG proteins of Photosystem I in trimerisation and antennae complexes binding is discussed.     

Characterization of a Photosystem II core and its three-dimensional crystals

A photosystem II core from spinach containing the chlorophyll-binding proteins 47 kDa, 43 kDa, the reaction center proteins D1, D2 and cytochromeb ₅₅₉ and three low molecular weight polypeptides (MW < 10 kDa) was isolated, its three-dimensional crystals were prepared, and both core and crystals were studied by spectroscopic techniques and electron microscopy. The absorption spectra of the crystallized form of the core indicate a specific orientation of the various pigments within the crystal.     

Presence in Photosystem II Core Complexes of a 34-Kilodalton Polypeptide Required for Water Photolysis

Photosystem II (PSII) reaction center core complexes have been isolated and characterized from wild type (WT) Scenedesmus obliquus and from its LF-1 mutant. LF-1 thylakoids are blocked on the oxidizing side of PSII and have a reduced Mn content. Visible absorption and low temperature fluorescence spectra of both core complexes are identical and resemble those reported for spinach (Satoh, Butler 1978 Plant Physiol 61: 373-379). Lithium dodecyl sulfate-polycrylamide gel electrophoresis reveals that a protein alteration, originally observed in thylakoid membranes (Metz, Wong, Bishop 1980 FEBS Lett 114: 61-66), is retained in the PSII core particles. That is, a 34-kilodalton (kD) polypeptide, present in the WT core complex, is missing in the mutant, and the core complex of the mutant contains a 36-kD protein not present in the WT. The 34-kD intrinsic protein is also observed in O₂-evolving PSII preparations and PSII core complexes from spinach. It is distinct from the 33-kD extrinsic protein first reported by T. Kuwabara and N. Murata (1979 Biochim Biophys Acta 581: 228-236). We suggest that the 34-kD protein is a site of Mn binding in the PSII membrane.     

Steady-state polarized light spectroscopy of isolated Photosystem I complexes

Monomeric and trimeric Photosystem I core complexes from the cyanobacterium Synechocystis PCC 6803 and LHC-I containing Photosystem I (PS I-200) complexes from spinach have been characterized by steady-state, polarized light spectroscopy at 77 K. The absorption spectra of the monomeric and trimeric core complexes from Synechocystis were remarkably similar, except for the amplitude of a spectral component at long wavelength, which was about twice as large in the trimeric complexes. This spectral component did not contribute significantly to the CD-spectrum. The (77 K) steady-state emission spectra showed prominent peaks at 724 nm (for the Synechocystis core complexes) and at 735 nm (for PS I-200). A comparison of the excitation spectra of the main emission band and the absorption spectra suggested that a significant part of the excitations do not pass the red pigments before being trapped by P-700. Polarized fluorescence excitation spectra of the monomeric and trimeric core complexes revealed a remarkably high anisotropy (0.3) above 705 nm. This suggested one or more of the following possibilities: 1) there is one red-most pigment to which all excitations are directed, 2) there are more red-most pigments but with (almost) parallel orientations, 3) there are more red-most pigments, but they are not connected by energy transfer. The high anisotropy above 705 nm of the trimeric complexes indicated that the long-wavelength pigments on different monomers are not connected by energy transfer. In contrary to the Synechocystis core complexes, the anisotropy spectrum of the LHC I containing complexes from spinach was not constant in the region of the long-wavelength pigments, and decreased significantly below 720 nm, the wavelength where the long-wavelength pigments on the core complexes start to absorb. These results suggested that in spinach the long-wavelength pigments on core and LHC-I are connected by energy transfer and have a non-parallel average Q_y(0-0) transitions.Abbreviations PS Photosystem - P Primary donor - Chl chlorophyll - LHC light-harvesting complex - CD circular dichroism - LD linear dichroism - BisTris 2-[bis(2-hydroxyethyl)amino]-2-hydroxy-methylpropane-1,3-diol - RC reaction center     

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.     

Cyanobacterial Acclimation to Photosystem I or Photosystem II Light

The organization and function of the photochemical apparatus of Synechococcus 6301 was investigated in cells grown under yellow and red light regimes. Broadband yellow illumination is absorbed preferentially by the phycobilisome (PBS) whereas red light is absorbed primarily by the chlorophyll (Chl) pigment beds. Since PBSs are associated exclusively with photosystem II (PSII) and most of the Chl with photosystem I (PSI), it follows that yellow and red light regimes will create an imbalance of light absorption by the two photosystems. The cause and effect relationship between light quality and photosystem stoichiometry in Synechococcus was investigated. Cells grown under red light compensated for the excitation imbalance by synthesis/assembly of more PBS-PSII complexes resulting in high PSII/PSI = 0.71 and high bilin/Chl = 1.30. The adjustment of the photosystem stoichiometry in red light-grown cells was necessary and sufficient to establish an overall balanced absorption of red light by PSII and PSI. Cells grown under yellow light compensated for this excitation imbalance by assembly of more PSI complexes, resulting in low PSII/PSI = 0.27 and low bilin/Chl = 0.42. This adjustment of the photosystem stoichiometry in yellow light-grown cells was necessary but not quite sufficient to balance the absorption of yellow light by the PBS and the Chl pigment beds. A novel excitation quenching process was identified in yellow light-grown cells which dissipated approximately 40% of the PBS excitation, thus preventing over-excitation of PSII under yellow light conditions. It is hypothesized that State transitions in O₂ evolving photosynthetic organisms may serve as the signal for change in the stoichiometry of photochemical complexes in response to light quality conditions.     

Revisiting the Supramolecular Organization of Photosystem II in Chlamydomonas reinhardtii

Photosystem II (PSII) is a multiprotein complex that splits water and initiates electron transfer in photosynthesis. The central part of PSII, the PSII core, is surrounded by light-harvesting complex II proteins (LHCIIs). In higher plants, two or three LHCII trimers are seen on each side of the PSII core whereas only one is seen in the corresponding positions in Chlamydomonas reinhardtii, probably due to the absence of CP24, a minor monomeric LHCII. Here, we re-examined the supramolecular organization of the C. reinhardtii PSII-LHCII supercomplex by determining the effect of different solubilizing detergents. When we solubilized the thylakoid membranes with n-dodecyl-β-d-maltoside (β-DM) or n-dodecyl-α-d-maltoside (α-DM) and subjected them to gel filtration, we observed a clear difference in molecular mass. The α-DM-solubilized PSII-LHCII supercomplex bound twice more LHCII than the β-DM-solubilized supercomplex and retained higher oxygen-evolving activity. Single-particle image analysis from electron micrographs of the α-DM-solubilized and negatively stained supercomplex revealed that the PSII-LHCII supercomplex had a novel supramolecular organization, with three LHCII trimers attached to each side of the core.     

Organisation of Photosystem I and Photosystem II in red alga Cyanidium caldarium: encounter of cyanobacterial and higher plant concepts

Structure and organisation of Photosystem I and Photosystem II isolated from red alga Cyanidium caldarium was determined by electron microscopy and single particle image analysis. The overall structure of Photosystem II was found to be similar to that known from cyanobacteria. The location of additional 20 kDa (PsbQ') extrinsic protein that forms part of the oxygen evolving complex was suggested to be in the vicinity of cytochrome c-550 (PsbV) and the 12 kDa (PsbU) protein. Photosystem I was determined as a monomeric unit consisting of PsaA/B core complex with varying amounts of antenna subunits attached. The number of these subunits was seen to be dependent on the light conditions used during cell cultivation. The role of PsaH and PsaG proteins of Photosystem I in trimerisation and antennae complexes binding is discussed.     

Size,shape and mass of the oxygen-evolving photosystem II complex from the thermophilic cyanobacterium Synechococcus sp.

Two different, highly active O₂-evolving photosystem II complexes were purified from the cyanobacterium Synechococcus sp. in the presence of the non-ionic detergent β-dodecyl-D-maltoside. Both complexes are homogeneous and have molecular masses of approx. 300 and 500 kDa, respectively. By electron microscopy it was found that both complexes have the shape of an elliptical disk, with a thickness of about 6.5 nm and top view dimensions of 10.5 × 15.5 nm for the 300 kDa particle and 18.5 × 15 nm for the 500 kDa particle. It is concluded that the particles represent monomeric and dimeric forms of photosystem II.     

Proteolytic activity against the light-harvesting complex and the D1/D2 core proteins of Photosystem II in close association to the light-harvesting complex II trimer

Light-harvesting complex II (LHCII) prepared from isolated thylakoids of either broken or intact chloroplasts by three independent methods, exhibits proteolytic activity against LHCII. This activity is readily detectable upon incubation of these preparations at 37 °C (without addition of any chemicals or prior pre-treatment), and can be monitored either by the LHCII immunostain reduction on Western blots or by the Coomassie blue stain reduction in substrate-containing “activity gels”. Upon SDS-sucrose density gradient ultracentrifugation of SDS-solubilized thylakoids, a method which succeeds in the separation of the pigment-protein complexes in their trimeric and monomeric forms, the protease activity copurifies with the LHCII trimer, its monomer exhibiting no activity. This LHCII trimer, apart from being “self-digested”, also degrades the Photosystem II (PSII) core proteins (D1, D2) when added to an isolated PSII core protein preparation containing the D1/D2 heterodimer. Under our experimental conditions, 50% of LHCII or the D1, D2 proteins are degraded by the LHCII-protease complex within 30 min at 37 °C and specific degradation products are observed. The protease is light-inducible during chloroplast biogenesis, stable in low concentrations of SDS, activated by Mg²⁺, and inhibited by Zn²⁺, Cd²⁺, EDTA and p-hydroxy-mercury benzoate (pOHMB), suggesting that it may belong to the cysteine family of proteases. Upon electrophoresis of the LHCII trimer on substrate-containing “activity gels” or normal Laemmli gels, the protease is released from the complex and runs in the upper part of the gel, above the LHCII trimer. A polypeptide of 140 kDa that exhibits proteolytic activity against LHCII, D1 and D2 has been identified as the protease. We believe that this membrane-bound protease is closely associated to the LHCII complex in vivo, as an LHCII-protease complex, its function being the regulation of the PSII unit assembly and/or adaptation.     

Structural characterization of photosystem II complex from red alga Porphyridium cruentum retaining extrinsic subunits of the oxygen-evolving complex.

The structure of photosystem II (PSII) complex isolated from thylakoid membranes of the red alga Porphyridium cruentum was investigated using electron microscopy followed by single particle image analysis. The dimeric complexes observed contain all major PSII subunits (CP47, CP43, D1 and D2 proteins) as well as the extrinsic proteins (33 kDa, 12 kDa and the cytochrome c(550)) of the oxygen-evolving complex (OEC) of PSII, encoded by the psbO, psbU and psbV genes, respectively. The single particle analysis of the top-view projections revealed the PSII complex to have maximal dimensions of 22 x 15 nm. The analysis of the side-view projections shows a maximal thickness of the PSII complex of about 9 nm including the densities on the lumenal surface that has been attributed to the proteins of the OEC complex. These results clearly demonstrate that the red algal PSII complex is structurally very similar to that of cyanobacteria and to the PSII core complex of higher plants. In addition, the arrangement of the OEC proteins on the lumenal surface of the PSII complex is consistent to that obtained by X-ray crystallography of cyanobacterial PSII.     

Mechanism of Interaction of Al3+ with the Proteins Composition of Photosystem II

The inhibitory effect of Al3+on photosystem II (PSII) electron transport was investigated using several biophysical and biochemical techniques such as oxygen evolution, chlorophyll fluorescence induction and emission, SDS-polyacrylamide and native green gel electrophoresis, and FTIR spectroscopy. In order to understand the mechanism of its inhibitory action, we have analyzed the interaction of this toxic cation with proteins subunits of PSII submembrane fractions isolated from spinach. Our results show that Al ³⁺, especially above 3 mM, strongly inhibits oxygen evolution and affects the advancement of the S states of the Mn₄O₅Ca cluster. This inhibition was due to the release of the extrinsic polypeptides and the disorganization of the Mn4O5Ca cluster associated with the oxygen evolving complex (OEC) of PSII. This fact was accompanied by a significant decline of maximum quantum yield of PSII (F_v/F_m) together with a strong damping of the chlorophyll a fluorescence induction. The energy transfer from light harvesting antenna to reaction centers of PSII was impaired following the alteration of the light harvesting complex of photosystem II (LHCII). The latter result was revealed by the drop of chlorophyll fluorescence emission spectra at low temperature (77 K), increase of F₀ and confirmed by the native green gel electrophoresis. FTIR measurements indicated that the interaction of Al ³⁺ with the intrinsic and extrinsic polypeptides of PSII induces major alterations of the protein secondary structure leading to conformational changes. This was reflected by a major reduction of α-helix with an increase of β-sheet and random coil structures in Al ³⁺-PSII complexes. These structural changes are closely related with the functional alteration of PSII activity revealed by the inhibition of the electron transport chain of PSII.     

Highly purified thermo-stable oxygen-evolving photosystem II core complex from the thermophilic cyanobacterium Synechococcus elongatus having His-tagged CP43

The carboxyl terminus of the CP43 subunit of photosystem II (PSII) in the thermophilic cyanobacterium, Synechococcus elongatus, was genetically tagged with six consecutive histidine residues to create a metal binding site on the PSII supramolecular complex. The histidine-tagging enabled rapid isolation of an intact cyanobacterial PSII core complex from dodecyl maltoside-solubilized thylakoids by a simple one-step Ni(2+)-affinity column chromatography. The isolated core complex was in a dimeric form with a molecular mass of about 580 kDa, consisting of five major intrinsic membrane proteins (CP47, CP43, D1, D2 and cytochrome b-559), three extrinsic proteins (33 kDa, 12 kDa, and cytochrome c-550), and a few low molecular mass membrane proteins, and evolved oxygen at a rate as high as 3,400 mumol (mg Chl)-1 h-1 at 45 degrees C with ferricyanide as an electron acceptor. The core complex emitted thermoluminescence B2-, B1- and Q-bands arising from S2QB-, S3QB- and S2QA- charge recombinations at respective emission temperatures of 45, 38 and 20 degrees C, all of which were higher by about 15 degrees C as compared with those in mesophilic spinach BBY membranes. These results indicated that the isolated core complex well retained the intact properties of thermoluminescence of thermophilic cyanobacterial cells, the deeper stabilization of PSII charge pairs. The isolated complex was extremely stable in terms of both protein composition and function, exhibiting no release of extrinsic proteins, no proteolytic degradation in any of its subunits, accompanied by only a slight (less than 10%) loss in oxygen evolution, after dark-incubation at 20 degrees C for 8 d. These properties of the thermophilic PSII core complex are highly useful for various types of studies on PSII.     

Association of chlorophyll a/c(2) complexes to photosystem I and photosystem II in the cryptophyte Rhodomonas CS24

Photosynthetic supercomplexes from the cryptophyte Rhodomonas CS24 were isolated by a short detergent treatment of membranes from the cryptophyte Rhodomonas CS24 and studied by electron microscopy and low-temperature absorption and fluorescence spectroscopy. At least three different types of supercomplexes of photosystem I (PSI) monomers and peripheral Chl a/c(2) proteins were found. The most common complexes have Chl a/c(2) complexes at both sides of the PSI core monomer and have dimensions of about 17x24 nm. The peripheral antenna in these supercomplexes shows no obvious similarities in size and/or shape with that of the PSI-LHCI supercomplexes from the green plant Arabidopsis thaliana and the green alga Chlamydomonas reinhardtii, and may be comprised of about 6-8 monomers of Chl a/c(2) light-harvesting complexes. In addition, two different types of supercomplexes of photosystem II (PSII) dimers and peripheral Chl a/c(2) proteins were found. The detected complexes consist of a PSII core dimer and three or four monomeric Chl a/c(2) proteins on one side of the PSII core at positions that in the largest complex are similar to those of Lhcb5, a monomer of the S-trimer of LHCII, Lhcb4 and Lhcb6 in green plants.     

Structural Changes of the Oxygen-evolving Complex in Photosystem II during the Catalytic Cycle

The oxygen-evolving complex (OEC) in the membrane-bound protein complex photosystem II (PSII) catalyzes the water oxidation reaction that takes place in oxygenic photosynthetic organisms. We investigated the structural changes of the Mn₄CaO₅ cluster in the OEC during the S state transitions using x-ray absorption spectroscopy (XAS). Overall structural changes of the Mn₄CaO₅ cluster, based on the manganese ligand and Mn-Mn distances obtained from this study, were incorporated into the geometry of the Mn₄CaO₅ cluster in the OEC obtained from a polarized XAS model and the 1.9-Å high resolution crystal structure. Additionally, we compared the S₁ state XAS of the dimeric and monomeric form of PSII from Thermosynechococcus elongatus and spinach PSII. Although the basic structures of the OEC are the same for T. elongatus PSII and spinach PSII, minor electronic structural differences that affect the manganese K-edge XAS between T. elongatus PSII and spinach PSII are found and may originate from differences in the second sphere ligand atom geometry.     

Probing tyrosine Z oxidation in photosystem II core complex isolated from spinach by EPR at liquid helium temperatures

Tyrosine Z (Tyr_Z) oxidation observed at liquid helium temperatures provides new insights into the structure and function of Tyr_Z in active Photosystem II (PSII). However, it has not been reported in PSII core complex from higher plants. Here, we report Tyr_Z oxidation in the S₁ and S₂ states in PSII core complex from spinach for the first time. Moreover, we identified a 500 G-wide symmetric EPR signal (peak position g = 2.18, trough position g = 1.85) together with the g = 2.03 signal induced by visible light at 10 K in the S₁ state in the PSII core complex. These two signals decay with a similar rate in the dark and both disappear in the presence of 6% methanol. We tentatively assign this new feature to the hyperfine structure of the S₁Tyr_Z ^• EPR signal. Furthermore, EPR signals of the S₂ state of the Mn-cluster, the oxidation of the non-heme iron, and the S₁Tyr_Z ^• in PSII core complexes and PSII-enriched membranes from spinach are compared, which clearly indicate that both the donor and acceptor sides of the reaction center are undisturbed after the removal of LHCII. These results suggest that the new spinach PSII core complex is suitable for the electron transfer study of PSII at cryogenic temperatures.     

Isolation and spectral characterization of Photosystem II reaction center from Synechocystis sp. PCC 6803

We isolated highly-purified photochemically active photosystem (PS) II reaction center (RC) complexes from the cyanobacterium Synechocystis sp. PCC 6803 using a histidine-tag introduced to the 47 kDa chlorophyll protein, and characterized their spectroscopic properties. Purification was carried out in a one-step procedure after isolation of PS II core complex. The RC complexes consist of five polypeptides, the same as in spinach. The pigment contents per two molecules of pheophytin a were 5.8 +/- 0.3 chlorophyll (Chl) a and 1.8 +/- 0.1 beta-carotene; one cytochrome b(559) was found per 6.0 Chl a molecules. Overall absorption and fluorescence properties were very similar to those of spinach PS II RCs; our preparation retains the best properties so far isolated from cyanobacteria. However, a clear band-shift of pheophytin a and beta-carotene was observed. Reasons for these differences, and RC composition, are discussed on the basis of the three-dimensional structure of complexes.