PSII-LHCII Supercomplexes Research Articles

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.

Comparison of the α and β isomeric forms of the detergent n-dodecyl-D-maltoside for solubilizing photosynthetic complexes from pea thylakoid membranes

Mild non-ionic detergents are indispensable in the isolation of intact integral membrane proteins and protein-complexes from biological membranes. Dodecylmaltoside (DM) belongs to this class of detergents being a glucoside-based surfactant with a bulky hydrophilic head group composed of two sugar rings and a non-charged alkyl glycoside chain. Two isomers of this molecule exist, differing only in the configuration of the alkyl chain around the anomeric center of the carbohydrate head group, axial in α-DM and equatorial in β-DM. In this paper, we have investigated the solubilizing properties of α-DM and β-DM on the isolation of photosynthetic complexes from pea thylakoids membranes maintaining their native architecture of stacked grana and stroma lamellae. Exposure of these stacked thylakoids to a single step treatment with increasing concentrations (5–100mM) of α-DM or β-DM resulted in a quick partial or complete solubilization of the membranes. Regardless of the isomeric form used: 1) at the lowest DM concentrations only a partial solubilization of thylakoids was achieved, giving rise to the release of mainly small protein complexes mixed with membrane fragments enriched in PSI from stroma lamellae; 2) at concentrations above 30mM a complete solubilization occurred with the further release of high molecular weight protein complexes identified as dimeric PSII, PSI-LHCI and PSII–LHCII supercomplexes. However, at concentrations of detergent which fully solubilized the thylakoids, the α and β isomeric forms of DM exerted a somewhat different solubilizing effect on the membranes: higher abundance of larger sized PSII–LHCII supercomplexes retaining a higher proportion of LHCII and lower amounts of PSI–LHCI intermediates were observed in α-DM treated membranes, reflecting the mildness of α-DM compared with its isomer. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

The Oligomeric States of the Photosystems and the Light-Harvesting Complexes in the Chl b-Less Mutant

The reversible associations between the light-harvesting complexes (LHCs) and the core complexes of PSI and PSII are essential for the photoacclimation mechanisms in higher plants. Two types of Chls, Chl a and Chl b, both function in light harvesting and are required for the biogenesis of the photosystems. Chl b-less plants have been studied to determine the function of the LHCs because the Chl b deficiency has severe effects specific to the LHCs. Previous studies have shown that the amounts of the LHCs, especially the LHCII trimer, were decreased in the mutants; however, it is still unclear whether Chl b is required for the assembly of the LHCs and for the association of the LHCs with PSI and PSII. Here, to reveal the function of Chl b in the LHCs, we investigated the oligomeric states of the LHCs, PSI and PSII in the Arabidopsis Chl b-less mutant. A two-dimensional blue native-PAGE/SDS-PAGE demonstrated that the PSI-LHCI supercomplex was fully assembled in the absence of Chl b, whereas the trimeric LHCII and PSII-LHCII supercomplexes were not detected. The PSI-NAD(P)H dehydrogenase (NDH) supercomplexes were also assembled in the mutant. Furthermore, we detected two forms of monomeric LHC proteins. The faster migrating forms, which were detected primarily in the mutant, were probably apo-LHC proteins, whereas the slower migrating forms were probably the LHC proteins that contained Chl a. These findings increase our understanding of the Chl b function in the assembly of LHCs and the association of the LHCs with PSI, PSII and NDH.

The PsbW protein stabilizes the supramolecular organization of photosystem II in higher plants

PsbW, a 6.1-kDa low-molecular-weight protein, is exclusive to photosynthetic eukaryotes, and associates with the photosystem II (PSII) protein complex. In vivo and in vitro comparison of Arabidopsis thaliana wild-type plants with T-DNA insertion knock-out mutants completely lacking the PsbW protein, or with antisense inhibition plants exhibiting decreased levels of PsbW, demonstrated that the loss of PsbW destabilizes the supramolecular organization of PSII. No PSII-LHCII supercomplexes could be detected or isolated in the absence of the PsbW protein. These changes in macro-organization were accompanied by a minor decrease in the chlorophyll fluorescence parameter F(V) /F(M) , a strongly decreased PSII core protein phosphorylation and a modification of the redox state of the plastoquinone (PQ) pool in dark-adapted leaves. In addition, the absence of PsbW protein led to faster redox changes in the PQ pool, i.e. transitions from state 1 to state 2, as measured by changes in stationary fluorescence (F(S) ) kinetics, compared with the wild type. Despite these dramatic effects on macromolecular structure, the transgenic plants exhibited no significant phenotype under normal growth conditions. We suggest that the PsbW protein is located close to the minor antenna of the PSII complex, and is important for the contact and stability between several PSII-LHCII supercomplexes.

Photosystem II organisation in chloroplasts of Arum italicum leaf depends on tissue location

The growth of plants under stable light quality induces long-term acclimation responses of the photosynthetic apparatus. Light can even cause variations depending on the tissue location, as in Arum italicum leaf, where chloroplasts are developed in the lamina and in the entire thickness of the petiole. We addressed the question whether differences in plastids can be characterised in terms of protein-protein interactions in the thylakoid membranes. Thylakoid assembly was studied in the palisade and spongy tissue of the lamina and in the outer parenchyma and inner aerenchyma of the petiole of the mature winter leaf of Arum italicum. The chlorophyll-protein complexes were analysed by means of blue-native-PAGE and fluorescence emission spectra. The petiole chloroplasts differ from those in the lamina in thylakoid composition: (1) reaction centres are scarce, especially photosystem (PS) I in the inner aerenchyma; (2) light-harvesting complex (LHC) II is abundant, (3) the relative amount of LHCII trimers increases, but this is not accompanied by increased levels of PSII-LHCII supercomplexes. Nevertheless, the intrinsic PSII functionality is comparable in all tissues. In Arum italicum leaf, the gradient in thylakoid organisation, which occurs from the palisade tissue to the inner aerenchyma of the petiole, is typical for photosynthetic acclimation to low-light intensity with a high enrichment of far-red light. The results obtained demonstrate a high plasticity of chloroplasts even in an individual plant. The mutual interaction of thylakoid protein complexes is discussed in relation to the photosynthetic efficiency of the leaf parts and to the ecodevelopmental role of light.

Differential turnover of the photosystem II reaction centre D1 protein in mesophyll and bundle sheath chloroplasts of maize

Photoinhibition is caused by an imbalance between the rates of the damage and repair cycle of photosystem II D1 protein in thylakoid membranes. The PSII repair processes include (i) disassembly of damaged PSII–LHCII supercomplexes and PSII core dimers into monomers, (ii) migration of the PSII monomers to the stroma regions of thylakoid membranes, (iii) dephosphorylation of the CP43, D1 and D2 subunits, (iv) degradation of damaged D1 protein, and (v) co-translational insertion of the newly synthesized D1 polypeptide and reassembly of functional PSII complex. Here, we studied the D1 turnover cycle in maize mesophyll and bundle sheath chloroplasts using a protein synthesis inhibitor, lincomycin. In both types of maize chloroplasts, PSII was found as the PSII–LHCII supercomplex, dimer and monomer. The PSII core and the LHCII proteins were phosphorylated in both types of chloroplasts in a light-dependent manner. The rate constants for photoinhibition measured for lincomycin-treated leaves were comparable to those reported for C3 plants, suggesting that the kinetics of the PSII photodamage is similar in C3 and C4 species. During the photoinhibitory treatment the D1 protein was dephosphorylated in both types of chloroplasts but it was rapidly degraded only in the bundle sheath chloroplasts. In mesophyll chloroplasts, PSII monomers accumulated and little degradation of D1 protein was observed. We postulate that the low content of the Deg1 enzyme observed in mesophyll chloroplasts isolated from moderate light grown maize may retard the D1 repair processes in this type of plastids.

Atomic Force Microscopy Studies of Native Photosynthetic Membranes

In addition to providing the earliest surface images of a native photosynthetic membrane at submolecular resolution, examination of the intracytoplasmic membrane (ICM) of purple bacteria by atomic force microscopy (AFM) has revealed a wide diversity of species-dependent arrangements of closely packed light-harvesting (LH) antennae, capable of fulfilling the basic requirements for efficient collection, transmission, and trapping of radiant energy. A highly organized architecture was observed with fused preparations of the pseudocrystalline ICM of Blastochloris viridis, consiting of hexagonally packed monomeric reaction center light-harvesting 1 (RC-LH1) core complexes. Among strains which also form a peripheral LH2 antenna, images of ICM patches from Rhodobacter sphaeroides exhibited well-ordered, interconnected networks of dimeric RC-LH1 core complexes intercalated by rows of LH2, coexisting with LH2-only domains. Other peripheral antenna-containing species, notably Rhodospirillum photometricum and Rhodopseudomonas palustris, showed a less regular organization, with mixed regions of LH2 and RC-LH1 cores, intermingled with large, paracrystalline domains. The ATP synthase and cytochrome bc(1) complex were not observed in any of these topographs and are thought to be localized in the adjacent cytoplasmic membrane or in inaccessible ICM regions separated from the flat regions imaged by AFM. The AFM images have served as a basis for atomic-resolution modeling of the ICM vesicle surface, as well as forces driving segregation of photosynthetic complexes into distinct domains. Docking of atomic-resolution molecular structures into AFM topographs of Rsp. photometricum membranes generated precise in situ structural models of the core complex surrounded by LH2 rings and a region of tightly packed LH2 complexes. A similar approach has generated a model of the highly curved LH2-only membranes of Rba. sphaeroides which predicts that sufficient space exists between LH2 complexes for quinones to diffuse freely. Measurement of the intercomplex distances between adjacent LH2 rings of Phaeospirillum molischianum has permitted the first calculation of the separation of bacteriochlorophyll a molecules in the native ICM. A recent AFM analysis of the organization of green plant photosystem II (PSII) in grana thylakoids revealed the protruding oxygen-evolving complex, crowded together in parallel alignment at three distinct levels of stacked membranes over the lumenal surface. The results also confirmed that PSII-LHCII supercomplexes are displaced relative to one another in opposing grana membranes.

The Mobile Thylakoid Phosphoprotein TSP9 Interacts with the Light-harvesting Complex II and the Peripheries of Both Photosystems

The localization of the plant-specific thylakoid-soluble phosphoprotein of 9 kDa, TSP9, within the chloroplast thylakoid membrane of spinach has been established by the combined use of fractionation, immunoblotting, cross-linking, and mass spectrometry. TSP9 was found to be exclusively confined to the thylakoid membranes, where it is enriched in the stacked grana membrane domains. After mild solubilization of the membranes, TSP9 migrated together with the major light-harvesting antenna (LHCII) of photosystem II (PSII) and with PSII-LHCII supercomplexes upon separation of the protein complexes by either native gel electrophoresis or sucrose gradient centrifugation. Studies with a cleavable cross-linking agent revealed the interaction of TSP9 with both major and minor LHCII proteins as identified by mass spectrometric sequencing. Cross-linked complexes that in addition to TSP9 contain the peripheral PSII subunits CP29, CP26, and PsbS, which form the interface between LHCII and the PSII core, were found. Our observations also clearly suggest an interaction of TSP9 with photosystem I (PSI) as shown by both immunodetection and mass spectrometry. Sequencing identified the peripheral PSI subunits PsaL, PsaF, and PsaE, originating from cross-linked protein complexes of around 30 kDa that also contained TSP9. The distribution of TSP9 among the cross-linked forms was found to be sensitive to conditions such as light exposure. An association of TSP9 with LHCII as well as the peripheries of the photosystems suggests its involvement in regulation of photosynthetic light harvesting.

Deletion of PsbM in Tobacco Alters the QB Site Properties and the Electron Flow within Photosystem II

Photosystem II, the oxygen-evolving complex of photosynthetic organisms, includes an intriguingly large number of low molecular weight polypeptides, including PsbM. Here we describe the first knock-out of psbM using a transplastomic, reverse genetics approach in a higher plant. Homoplastomic Delta psbM plants exhibit photoautotrophic growth. Biochemical, biophysical, and immunological analyses demonstrate that PsbM is not required for biogenesis of higher order photosystem II complexes. However, photosystem II is highly light-sensitive, and its activity is significantly decreased in Delta psbM, whereas kinetics of plastid protein synthesis, reassembly of photosystem II, and recovery of its activity are comparable with the wild type. Unlike wild type, phosphorylation of the reaction center proteins D1 and D2 is severely reduced, whereas the redox-controlled phosphorylation of photosystem II light-harvesting complex is reversely regulated in Delta psbM plants because of accumulation of reduced plastoquinone in the dark and a limited photosystem II-mediated electron transport in the light. Charge recombination in Delta psbM measured by thermoluminescence oscillations significantly differs from the 2/6 patterns in the wild type. A simulation program of thermoluminescence oscillations indicates a higher Q(B)/Q(-)(B) ratio in dark-adapted mutant thylakoids relative to the wild type. The interaction of the Q(A)/Q(B) sites estimated by shifts in the maximal thermoluminescence emission temperature of the Q band, induced by binding of different herbicides to the Q(B) site, is changed indicating alteration of the activation energy for back electron flow. We conclude that PsbM is primarily involved in the interaction of the redox components important for the electron flow within, outward, and backward to photosystem II.

Structure, function and assembly of Photosystem II and its light-harvesting proteins

Photosystem II (PSII) is a multisubunit chlorophyll-protein complex that drives electron transfer from water to plastoquinone using energy derived from light. In green plants, the native form of PSII is surrounded by the light-harvesting complex (LHCII complex) and thus it is called the PSII-LHCII supercomplex. Over the past several years, understanding of the structure, function, and assembly of PSII and LHCII complexes has increased considerably. The unicellular green alga Chlamydomonas reinhardtii has been an excellent model organism to study PSII and LHCII complexes, because this organism grows heterotrophically and photoautotrophically and it is amenable to biochemical, genetic, molecular biological and recombinant DNA methodology. Here, the genes encoding and regulating components of the C. reinhardtii PSII-LHCII supercomplex have been thoroughly catalogued: they include 15 chloroplast and 20 nuclear structural genes as well as 13 nuclear genes coding for regulatory factors. This review discusses these molecular genetic data and presents an overview of the structure, function and assembly of PSII and LHCII complexes.

Resonance Raman spectroscopy of xanthophylls in pigment mutant thylakoid membranes of pea.

Low-temperature resonance Raman spectroscopy was used to study the changes in the molecular structure and configuration of the major xanthophylls in thylakoid membranes isolated from mutants of pea with modified pigment content and altered structural organization of their pigment-protein complexes. The Raman spectra contained four known groups of bands, nu(1)-nu(4), which could be assigned to originate mainly from the long wavelength absorbing lutein and neoxanthin upon 514.5 nm and at 488 nm excitations, respectively. The overall configuration of these bound xanthophyll molecules in the mutants appeared to be similar to the wild type, and the configuration in the wild type was almost identical with that in the isolated main chlorophyll a/b light harvesting protein complex of photosystem II (LHCII). Significant differences were found mainly in the region of nu(4) (around 960 cm(-1)), which suggest that the macroorganization of PS II-LHCII supercomplexes and/or of the LHCII-only domains are modified in the mutants compared to the wild type.

The Effect of Zeaxanthin as the Only Xanthophyll on the Structure and Function of the Photosynthetic Apparatus in Arabidopsis thaliana

In green plants, the xanthophyll carotenoid zeaxanthin is synthesized transiently under conditions of excess light energy and participates in photoprotection. In the Arabidopsis lut2 npq2 double mutant, all xanthophylls were replaced constitutively by zeaxanthin, the only xanthophyll whose synthesis was not impaired. The relative proportions of the different chlorophyll antenna proteins were strongly affected with respect to the wild-type strain. The major antenna, LHCII, did not form trimers, and its abundance was strongly reduced as was CP26, albeit to a lesser extent. In contrast, CP29, CP24, LHCI proteins, and the PSI and PSII core complexes did not undergo major changes. PSII-LHCII supercomplexes were not detectable while the PSI-LHCI supercomplex remained unaffected. The effect of zeaxanthin accumulation on the stability of the different Lhc proteins was uneven: the LHCII proteins from lut2 npq2 had a lower melting temperature as compared with the wild-type complex while LHCI showed increased resistance to heat denaturation. Consistent with the loss of LHCII, light-state 1 to state 2 transitions were suppressed, the photochemical efficiency in limiting light was reduced and photosynthesis was saturated at higher light intensities in lut2 npq2 leaves, resulting in a photosynthetic phenotype resembling that of high light-acclimated leaves. Zeaxanthin functioned in vivo as a light-harvesting accessory pigment in lut2 npq2 chlorophyll antennae. As a whole, the in vivo data are consistent with the results obtained by using recombinant Lhc proteins reconstituted in vitro with purified zeaxanthin. While PSII photoinhibition was similar in wild type and lut2 npq2 exposed to high light at low temperature, the double mutant was much more resistant to photooxidative stress and lipid peroxidation than the wild type. The latter observation is consistent with an antioxidant and lipid protective role of zeaxanthin in vivo.

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

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

Supermolecular organization of photosystem II and its associated light‐harvesting antenna in Arabidopsis thaliana

The organization of Arabidopsis thaliana photosystem II (PSII) and its associated light-harvesting antenna (LHCII) was studied in isolated PSII-LHCII supercomplexes and native membrane-bound crystals by transmission electron microscopy and image analysis. Over 4000 single-particle projections of PSII-LHCII supercomplexes were analyzed. In comparison to spinach supercomplexes [Boekema, E.J., van Roon, H., van Breemen, J.F.L. & Dekker, J.P. (1999) Eur. J. Biochem. 266, 444-452] some striking differences were revealed: a much larger number of supercomplexes from Arabidopsis contain copies of M-type LHCII trimers. M-type trimers can also bind in the absence of the more common S-type trimers. No binding of l-type trimers could be detected. Analysis of native membrane-bound PSII crystals revealed a novel type of crystal with a unit cell of 25.6 x 21.4 nm (angle 77 degrees ), which is larger than any of the PSII lattices observed before. The data show that the unit cell is built up from C2S2M2 supercomplexes, rather than from C2S2M supercomplexes observed in native membrane crystals from spinach [Boekema, E.J., Van Breemen, J.F.L., Van Roon, H. & Dekker, J.P. (2000) J. Mol. Biol. 301, 1123-1133]. It is concluded from both the single particle analysis and the crystal analysis that the M-type trimers bind more strongly to PSII core complexes in Arabidopsis than in spinach.

Light-induced short-term adaptation mechanisms under redox control in the PS II-LHCII supercomplex: LHC II state transitions and PS II repair cycle.

Oxygenic photosynthesis takes place in the thylakoid membranes of cyanobacteria, algae and higher plants. While cyanobacteria have adapted to relatively constant environments, higher plants had to evolve mechanisms to adapt to continuous environmental changes. These include changes in light intensity, temperature and availability of water. One of the great challenges in plant cell biology is therefore to determine the regulatory mechanisms employed by higher plants and some algae to adapt to these constant environmental changes. The particular emphasis of this review is the description and characterisation of light-induced redox-controlled processes regulating the photosynthetic reactions, which involves maintaining maximal electron transport flow through the PS II-Cytb6f-PS I-F0F1ATPase electron transport chain and minimising light-induced oxidative damage to PS II which drives the highly oxidising water-splitting reaction. Two of the mechanisms involved in such short-term regulation processes are known as light harvesting complex II (LHC II) state transitions and photosystem II (PS II) repair cycle. They are followed by, and indeed may be a precondition in order to establish, the onset of the subsequent long-term mechanisms of regulation. In particular, the redox control of LHC II state transitions by reversible phosphorylation has been in the focus of many investigations, leading to many new results demonstrating the complexity of thylakoid-associated redox control mechanisms.

The Chloroplast Gene ycf9 Encodes a Photosystem II (PSII) Core Subunit, PsbZ, That Participates in PSII Supramolecular Architecture

We have characterized the biochemical nature and the function of PsbZ, the protein product of a ubiquitous open reading frame, which is known as ycf9 in Chlamydomonas and ORF 62 in tobacco, that is present in chloroplast and cyanobacterial genomes. After raising specific antibodies to PsbZ from Chlamydomonas and tobacco, we demonstrated that it is a bona fide photosystem II (PSII) subunit. PsbZ copurifies with PSII cores in Chlamydomonas as well as in tobacco. Accordingly, PSII mutants from Chlamydomonas and tobacco are deficient in PsbZ. Using psbZ-targeted gene inactivation in tobacco and Chlamydomonas, we show that this protein controls the interaction of PSII cores with the light-harvesting antenna; in particular, PSII-LHCII supercomplexes no longer could be isolated from PsbZ-deficient tobacco plants. The content of the minor chlorophyll binding protein CP26, and to a lesser extent that of CP29, also was altered substantially under most growth conditions in the tobacco mutant and in Chlamydomonas mutant cells grown under photoautotrophic conditions. These PsbZ-dependent changes in the supramolecular organization of the PSII cores with their peripheral antennas cause two distinct phenotypes in tobacco and are accompanied by considerable modifications in (1) the pattern of protein phosphorylation within PSII units, (2) the deepoxidation of xanthophylls, and (3) the kinetics and amplitude of nonphotochemical quenching. The role of PsbZ in excitation energy dissipation within PSII is discussed in light of its proximity to CP43, in agreement with the most recent structural data on PSII.

The chloroplast gene ycf9 encodes a photosystem II (PSII) core subunit, PsbZ, that participates in PSII supramolecular architecture.

We have characterized the biochemical nature and the function of PsbZ, the protein product of a ubiquitous open reading frame, which is known as ycf9 in Chlamydomonas and ORF 62 in tobacco, that is present in chloroplast and cyanobacterial genomes. After raising specific antibodies to PsbZ from Chlamydomonas and tobacco, we demonstrated that it is a bona fide photosystem II (PSII) subunit. PsbZ copurifies with PSII cores in Chlamydomonas as well as in tobacco. Accordingly, PSII mutants from Chlamydomonas and tobacco are deficient in PsbZ. Using psbZ-targeted gene inactivation in tobacco and Chlamydomonas, we show that this protein controls the interaction of PSII cores with the light-harvesting antenna; in particular, PSII-LHCII supercomplexes no longer could be isolated from PsbZ-deficient tobacco plants. The content of the minor chlorophyll binding protein CP26, and to a lesser extent that of CP29, also was altered substantially under most growth conditions in the tobacco mutant and in Chlamydomonas mutant cells grown under photoautotrophic conditions. These PsbZ-dependent changes in the supramolecular organization of the PSII cores with their peripheral antennas cause two distinct phenotypes in tobacco and are accompanied by considerable modifications in (1) the pattern of protein phosphorylation within PSII units, (2) the deepoxidation of xanthophylls, and (3) the kinetics and amplitude of nonphotochemical quenching. The role of PsbZ in excitation energy dissipation within PSII is discussed in light of its proximity to CP43, in agreement with the most recent structural data on PSII.

Fitting of a two-domain model for PsbO into a 3D map of the PSII-LHCII supercomplex obtained by electron cryo-microscopy

Spinach PSII-LHCII supercomplexes were prepared by sucrose density gradient centrifugation of detergent solubilised PSII-enriched membranes (BBYs). SDS-PAGE revealed all major PSII subunits to be present, including the PsbO, P and Q extrinsic proteins, an

Control of the photosynthetic electron transport by PQ diffusion microdomains in thylakoids of higher plants

We investigate the role of plastoquinone (PQ) diffusion in the control of the photosynthetic electron transport. A control analysis reveals an unexpected flux control of the whole chain electron transport by photosystem (PS) II. The contribution of PSII to the flux control of whole chain electron transport was high in stacked thylakoids (control coefficient, CJ(PSII)=0.85), but decreased after destacking (CJ(PSII)=0.25). From an ‘electron storage’ experiment, we conclude that in stacked thylakoids only about 50 to 60% of photoreducable PQ is involved in the light-saturated linear electron transport. No redox equilibration throughout the membrane between fixed redox groups at PSII and cytochrome (cyt) bf complexes, and the diffusable carrier PQ is achieved. The data support the PQ diffusion microdomain concept by Lavergne et al. [J. Lavergne, J.-P. Bouchaud, P. Joliot, Biochim. Biophys. Acta 1101 (1992) 13–22], but we come to different conclusions about size, structure and size distribution of domains. From an analysis of cyt b 6 reduction, as a function of PSII inhibition, we conclude that in stacked thylakoids about 70% of PSII is located in small domains, where only 1 to 2 PSII share a local pool of a few PQ molecules. Thirty percent of PSII is located in larger domains. No small domains were found in destacked thylakoids. We present a structural model assuming a hierarchy of specific, strong and weak interactions between PSII core, light harvesting complexes (LHC) II and cyt bf. Peripheral LHCII’s may serve to connect PSII–LHCII supercomplexes to a flexible protein network, by which small closed lipid diffusion compartments are formed. Within each domain, PQ moves rapidly and shuttles electrons between PSII and cyt bf complexes in the close vicinity. At the same time, long range diffusion is slow. We conclude, that in high light, cyt bf complexes located in distant stromal lamellae (20 to 30%) are not involved in the linear electron transport.

Supramolecular organization of photosystem II and its light-harvesting antenna in partially solubilized photosystem II membranes.

We present an extended analysis of the organization of green plant photosystem II and its associated light-harvesting antenna using electron microscopy and image analysis. The analysis is based on a large dataset of 16 600 projections of negatively stained PSII-LHCII supercomplexes and megacomplexes prepared by means of three different pretreatments. In addition to our previous work on this system [Boekema, E.J., van Roon, H., Calkoen, F., Bassi, R. and Dekker, J.P. (1999) Biochemistry 38, 2233-2239], the following results were obtained. The rotational orientation of trimeric LHCII at the S, M and L binding positions was determined. It was found that compared to the S trimer, the M and L trimers are rotationally shifted by about -20 degrees and -50 degrees, respectively. The number of projections with empty CP29, CP26 and CP24 binding sites was found to be about 0, 18 and 4%, respectively. We suggest that CP26 and CP24 are not required for the binding of trimeric LHCII at any of the three binding positions. A new type of megacomplex was observed with a characteristic windmill-like shape. This type III megacomplex consists of two C2S2 supercomplexes connected at their CP26 tips. Structural variation in the region of the central dimeric photosystem II complex was found to occur at one specific position near the periphery of the complex. We attribute this variation to the partial absence of an extrinsic polypeptide or one or more small intrinsic membrane proteins.