Effect of linolenic acid on release of the 43 kDa polypeptide and manganese from photosystem II membranes depleted of the 33 kDa extrinsic polypeptide

The effect of linolenic acid (18:3) on release of the 43 kDa polypeptide and manganese from photosystem II ( PS II ) membranes depleted of extrinsic polypeptides was studied. In both control and NaCl-washed particles which were depleted of the extrinsic 23 and 16 kDa polypeptides, the 18:3 treatment caused a 20% release of the 33 and 43 kDa polypeptides. In CaCl₂, (or urea + NaCl)-washed particles, which were depleted of the 33 kDa polypeptide in addition to the 23 and 16 kDa polypeptides, the release of the 43 kDa polypeptide increased to 70%, whereas only 25% of the 47 kDa polypeptide was removed. These findings suggest (i) that the 33 and the 43 kDa polypeptides are neighbows in the photosynthetic membrane and (ii) that the 33 kDa polypeptide shields the 43 kDa polypeptide against the action of 18:3. Incubation of CaCl₂, or (urea + NaCI)-treated PSII particles in the presence or absence of 18:3 resulted in the loss of only 2 of the 4 Mn atoms present per reaction center. this indicates that the 2 Mn atoms more firmly associated with PSII are not affected by the removal of the extrinsic 16, 23 and 33 kDa polypeptides, and the intrinsic 43 kDa polypeptide. nor by the treatment with linolenic acid.     

Effect of the manganese complex on the binding of the extrinsic proteins (17, 23 and 33 kDa) of Photosystem II

Selective extraction-reconstitution experiments with the extrinsic Photosystem II polypeptides (33 kDa, 23 kDa and 17 kDa) have demonstrated that the manganese complex and the 33 kDa polypeptide are both necessary structural elements for the tight binding of the water soluble 17 and 23 kDa species. When the manganese complex is intact the 33 kDa protein interacts strongly with the rest of the photosynthetic complex. Destruction of the Mn-complex has two dramatic effects: i) The binding of the 33 kDa polypeptide is weaker, since it can be removed by exposure of the PS II system to 2 M NaCl, and ii) the 17 and 23 kDa species do not rebind to Mn-depleted Photosystem II membranes that retain the 33 kDa protein.Abbreviations Chl chlorophyll - HQ hydroquinone - MES 2(N-morpholino)ethanesulfonic acid - PS II Photosystem II - Tris 2-amino-2-hydroxymethylpropane-1,3-diol     

Structural and catalytic properties of the oxygen-evolving complex. Correlation of polypeptide and manganese release with the behavior of Z+ in chloroplasts and a highly resolved preparation of the PS II complex

Treatment of intact thylakoid membranes with Triton X-100 at pH 6 produces a preparation of the PS II complex capable of high rates of O₂ evolution. The preparation contains four managanese, one cytochrome b-559, one Signal II_f and one Signal II_s per 250 chlorophylls. By selective manipulation of the preparation polypeptides of approximate molecular weights of 33, 23 and 17 kDa can be removed from the complex. Release of 23 and 17 kDa polypeptides does not release functional manganese. Under these conditions Z⁺ is not readily and directly accessible to an added donor (benzidine) and it appears as if at least some of the S-state transitions occur. Evidence is presented which indicates that benzidine does have increased access to the oxygen-evolving complex in these polypeptide depleted preparations. Conditions which release the 33 kDa species along with Mn and the 23 and 17 kDa polypeptides generate an alteration in the structure of the oxidizing side of PS II, which becomes freely accessible to benzidine. These findings are examined in relationship to alterations of normal S-state behavior (induced by polypeptide release) and a model is proposed for the organization of functional manganese and polypeptides involved in the oxygen-evolving reaction.     

Stabilisation of PS-II-mediated electron transport in oxygen-evolving PS II core preparations by the addition of compatible co-solutes.

Addition of high concentrations of compatible co-solutes such as sugars, sugar alcohols and polyols has recently been shown to lead to marked increases in the thermal stability of oxygen-evolution in chloroplasts (Williams et al. (1992) Biochim. Biophys. Acta 1099, 137-144). In this paper, a similar stabilisation is demonstrated for oxygen-evolving PS II core preparations. The presence of such co-solutes appears, however, to have no ability to stabilise PS II reaction-centre preparations against heat-induced changes in their absorption spectrum. Nor do they protect electron transport from artificial electron donors in PS II core preparations lacking the extrinsic 33 kDa polypeptide of the oxygen-evolution system. Measurements performed on core preparations retaining the 33 kDa polypeptide but lacking the 17 kDa and 23 kDa polypeptides indicate that the co-solutes protect PS-II-mediated electron transport by stabilising the binding of the 33 kDa polypeptide to the core complexes. These findings are discussed in terms of an extension of the general principles underlying the Hofmeister effect observed for soluble proteins to the stabilisation of photosynthetic membrane preparations.     

Degradation and release from the thylakoid membrane of Photosystem II subunits after UV-B irradation of the liverwort Conocephalum conicum

The effect of ultraviolet-B (UV-B) radiation on the amount of various Photosystem (PS) II subunits has been studied in the thalloid liverwort Conocephalum conicum. UV-B irradiation led to a drastic decrease of the reaction center proteins D1 and D2 and the outer light harvesting antenna (LHC II). A minor reduction was found in the levels of the CP 43 polypeptide of the inner antenna and the 33, 23 and 16 kDa extrinsic polypeptides of PS II. During UV-B irradiation, the extrinsic polypeptides accumulated in the soluble protein fraction, but D1 and D2 were not dedectable. Streptomycin, but not cycloheximide inhibited the repair process of PS II, indicating that only protein synthesis in the chloroplast is necessary for recovery. This indicates that the extrinsic proteins of PS II dissociate from the membrane during UV-B treatment and reassociate with PS II in the course of the repair process. We conclude that the reaction center core is a target of UV-B radiation in C. concicum. The extrinsic proteins of PS II are not directly affected by UV-B, but their release is the consequence of UV-B-induced degradation of the D1 and D2 proteins.     

An Oxygen-Evolving Photosystem II Complex Associated with the Core Substructure of the Phycobilisome from Synechococcus elongatus

Oxygen-evolving photosystem II (PS II) particles isolated fromthe thermophilic cyanobacterium Synechococcus elongatus consistedof about twenty polypeptides. Six polypeptides were identifiedby reaction with specific antisera as constituent subunit polypeptidesof oxygen-evolving PS II reaction center complexes. The mostabundant polypeptides were the and ß subunits ofallophycocyanin. Comparison with the polypeptide profile ofisolated phycobilisomes, as well as immunoblotting with an antiserumagainst the large linker polypeptide, showed that the largelinker polypeptide or some proteolytic fragments of it werepresent in the preparation. Thus, each PS II particle is, inessence, an oxygen-evolving PS II complex that is associatedwith the core substructure of the phycobilisome. Cross-linkingexperiments indicated that fragments of the large linker polypeptidesare closely associated with one another and that the Chl-carrying47- kDa polypeptide is located in close proximity to the D2protein and the extrinsic 33-kDa protein. (Received November 12, 1991; Accepted January 23, 1992)     

Photosystem 2 Efficiency and Thylakoid Protein Pattern in DCMU-Treated Wheat Seedlings during Senescence

Changes in various components of photosynthetic apparatus during the 6-d dark incubation at 25 °C of detached control and DCMU-treated Triticum aestivum L. leaves were examined. The rate of photosystem 2 (PS2) activity was decreased with increase of the time of dark incubation in control leaves. In contrast to this, DCMU-treated leaves demonstrated high stability by slowing down the inactivation processes. Diphenyl carbazide and NH₂OH restored the PS2 activity more in control leaves than in DCMU-treated leaves. Mn²⁺ failed to restore the PS2 activity in both control and DCMU-treated samples. Similar results were obtained when F_v/F_m was evaluated by chlorophyll fluorescence measurements. The marked loss of PS2 activity in dark incubated control leaves was primarily due to the loss of D1, 33, and 23 kDa extrinsic polypeptides and 28-25 kDa LHCP2 polypeptides.     

Lumen-side topography of the alpha-subunit of the chloroplast cytochrome b-559

Removal of the extrinsic 33 kDa polypeptide increased the accessibility to trypsin of a COOH-terminal tridecapeptide epitope of the alpha subunit of cytochrome b-559 (psbE gene product). The sensitivity of the cytochrome epitope to trypsin was not measurably affected by removal of the 16 and 23 kDa extrinsic polypeptides, nor increased by removal of the OEC manganese along with the 33 kDa protein. While protecting alpha-cytochrome b-559 against trypsin, the 33 kDa protein is also proteolyzed, suggesting the possibility of an additional protein component involved in the shielding of the cytochrome. Shielding of the COOH-terminal epitope of alpha-cytochrome b-559 by the OEC 33 kDa protein implies that these COOH-terminal chains of the cytochrome are part of a protein network in the lumen space near the photosystem II reaction center. This network may contain residues that are involved in the binding of essential OEC metal ions.     

The 16 and 23 kDa extrinsic polypeptides and the associated Ca2+ and Cl- modify atrazine interaction with the photosystem II core complex

The oxygen evolving complex of photosystem II (PS II) contains three extrinsic polypeptides of approximate molecular weights 16, 23 and 33 kDa. These polypeptides are associated with the roles of Cl^-, Ca²⁺ and Mn²⁺ in oxygen evolution. We have shown that selective removal of 16 and 23 kDa polypeptides from the above complex by NaCl washing of PS II enriched membrane fragments renders the PS II core complex more susceptible to the herbicide atrazine. On the other hand, when both native and depleted preparations were resupplied with exogenous Ca²⁺ and Cl^-, we obtained a reduction of atrazine inhibition which was much stronger in the depleted preparations than in the native ones. It is concluded that removal of 16 and 23 kDa polypeptides in general, and disorganization of associated Ca²⁺ and Cl^- in particular, enhances atrazine penetration to its sites of action in the vicinity of the PS II complex. The above could be interpreted if we assume a reduced plastoquinone affinity at the Q_B (secondary plastoquinone electron acceptor) pocket of D1 polypeptide following transmembranous modifications caused by the depletion of these polypeptides.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - Chl chlorophyll - DCIP 2,6-dichlorophenolindophenol - MES 2-(N-morpholino)ethanesulfonic acid - PMSF phenylmethylsul-phonyfluoride - PS II photosystem II - PAGE polyacrilamide gel electrophoresis     

Identification of polypeptides associated with the 23 and 33 kDa proteins of photosynthetic oxygen evolution

An immunological approach was used for nearest-neighbor analyses for the 23 and 33 kDA proteins of the oxygen-evolving complex. Functional Photosystem II particles with a simple polypeptide composition were partly solubilized with detergent and incubated with monospecific antibodies against either the 23 or the 33 kDa protein. SDS-polyacrylamide gel electrophoresis revealed that the immunoprecipitates, apart from the antigenic proteins, also contained polypeptides at 24, 22 and 10 kDa. In contrast, polypeptides of the light-harvesting and Photosystem II core complexes showed very poor coprecipitation with the 23 and 33 kDa proteins. The 24, 22 and 10 kDa polypeptides were not precipitated by the antibodies if the 23 and 33 kDa proteins had been removed from the particles prior to solubilization. These observations demonstrate a close association between the 24, 22 and 10 kDa polypeptides and the 23 and 33 kDa proteins of the oxygen-evolving complex. None of these precipitated polypeptides contained any manganese. It is suggested that the 24, 22 and 10 kDa polypeptides are subunits of the oxygen-evolving complex and involved in the binding of the extrinsic 23 and 33 kDa proteins to the inner thylakoid surface.     

Molecular mechanism of action of Pb and Zn on water oxidizing complex of photosystem II

Pb²⁺ and Zn²⁺ inhibition of photosystem II (PSII) activity was reported to be mediated via displacement of native inorganic cofactors (Cl⁻, Ca²⁺ and Mn²⁺) from the oxygen evolving complex, OEC [Rashid and Popovic (1990) FEBS Lett. 271, 181–184; Rashid et al. (1991) Photosynth. Res. 30, 123–130]. Since the binding sites of these cofactors are protected by a shield of three extrinsic polypeptides (17, 23 and 33 kDa), we investigated whether these metal ions affect the extrinsic polypeptide shield of OEC. By immunoblotting with antibodies recognizing the 23 and 33 kDa polypeptides, we showed that both the metal ions significantly dissociated the 23 kDa (+17 kDa) polypeptide, and partially dissociated the 33 kDa. Ca²⁺, one of the important inorganic cofactors of oxygen evolution, strongly prevented the dissociating action of Pb²⁺ but did not prevent the action of Zn²⁺. The probable molecular mechanism of action of Pb²⁺ and Zn²⁺ on PSII OEC is discussed.     

Effects of NaCl stress on photochemical activity and thylakoid membrane polypeptide composition of a salt-tolerant and a salt-sensitive rice cultivar

NaCl stress (200 mM) inhibited the electron transport activity of photosystem 2 (PS2) more than that of PS1. The degree of electron transport activity inhibition was lower in the salt-tolerant cultivar Pokkali than in the salt-sensitive cultivar Peta. The polypeptide composition of the thylakoid membrane and PS2 particles did not change after NaCl treatment but there was a difference in polypeptide compositions of thylakoid membrane and PS2 particles between the two cultivars. PS2 particles of cv. Pokkali contained more 33-kDa and 43-kDa polypeptides than cv. Peta. Additionally, PS2 particles after NaCl treatment showed deficiency of 23-kDa outside polypeptides of PS2.     

Selective extraction of 22 kDa and 10 kDa polypeptides from Photosystem II without removal of 23 kDa and 17 kDa extrinsic proteins

Selective solubilization of Photosystem II membranes with the non-ionic detergent octyl thioglucopyranoside has allowed the isolation of a PS II system which has been depleted of the 22 and 10 kDa polypeptides but retains all three extrinsic proteins (33, 23 and 17 kDa). The PS II membranes which have been depleted of the 22 and 10 kDa species show high rates of oxygen evolution activity, external calcium is not required for activity and the manganese complex is not destroyed by exogenous reductants. When we compared this system to control PS II membranes, we observed a minor modification of the reducing side, and a conversion of the high-potential to the low-potential form of cytochrome b ₅₅₉.Abbreviations Chl- chlorophyll - DCBQ- 2,5-dichloro-p-benzoquinone - DCMU- 3-(3,4-dichlorophenyl)-1,1-dimethylurea - ESR- electron spin resonance - MES- 2-(N-morpholino)ethanesulfonic acid - OTG- octyl--d-thioglucopyranoside - PS II- Photosystem II - PEG- polyethylene glycol, M_r=6000 - Tris- 2-amino-2-hydroxyethylpropane-1,3-diol     

Evidence for the ultraviolet-B (280–320 nm) radiation induced structural reorganization and damage of photosystem II polypeptides in isolated chloroplasts

Direct evidence for the possible loss of photosystem II (PS II) activity in chloroplasts of Vigna sinensis L. cv. Walp after ultraviolet-B (UV-B, 280–320 nm) radiation treatment was provided by polyacrylamide gel electrophoretic analysis of PS II polypeptides. A 30 min UV-B treatment of chloroplasts caused a 50% loss of PS II activity. The artificial electron donor. Mn²⁺ failed to restore UV-B radiation induced loss of PS II activity, while diphenyl carbazide (DPC) and NH₂OH only partially restored activity. Such a loss in PS II activity was found to be primarily due to a loss of 23 and 33 kDa extrinsic polypeptides. UV-B treatment induced the synthesis of a few polypeptides and a 29 kDa light-harvesting chlorophyll protein.     

Correlation of the cytochrome c 2 content of cyanobacterial Photosystem II with the EPR properties of the oxygen-evolving complex

The Mn₄ cluster of PS II advances through a series of oxidation states (S states) that catalyze the breakdown of water to dioxygen in the oxygen-evolving complex. The present study describes the engineering and purification of highly active PS II complexes from mesophilic His-tagged Synechocystis PCC 6803 and purification of PS II core complexes from thermophilic wild-type Synechococcus lividus with high levels of the extrinsic polypeptide, cytochrome c ₅₅₀. The g = 4.1 S₂ state EPR signal, previously not characterized in untreated cyanobacterial PS II, is detected in high yields in these PS II preparations. We present a complete characterization of the g = 4.1 state in cyanobacterial His-tagged Synechocystis PCC 6803 PS II and S. lividus PS II. Also presented are a determination of the stoichiometry of cytochrome c ₅₅₀ bound to His-tagged Synechocystis PCC 6803 PS II and analytical ultracentrifugation results which indicate that cytochrome c ₅₅₀ is a monomer in solution. The temperature-dependent multiline to g = 4.1 EPR signal conversion observed for the S₂ state in cyanobacterial PS II with high cytochrome c ₅₅₀ content is very similar to that previously found for spinach PS II. In spinach PS II, the formation of the S₂ state g = 4.1 EPR signal has been found to correlate with the binding of the extrinsic 17 and 23 kDa polypeptides. The finding of a similar correlation in cyanobacterial PS II with the binding of cytochrome c ₅₅₀ suggests a functional homology between cytochrome c ₅₅₀ and the 17 and 23 kDa extrinsic proteins of spinach PS II. This revised version was published online in June 2006 with corrections to the Cover Date.     

Nitrogen ligation to the manganese cluster of Photosystem II in the absence of the extrinsic proteins and as a function of pH

Three extrinsic proteins (PsbO, PsbP and PsbQ), with apparent molecular weights of 33, 23 and 17 kDa, bind to the lumenal side of Photosystem II (PS II) and stabilize the manganese, calcium and chloride cofactors of the oxygen evolving complex (OEC). The effect of these proteins on the structure of the tetramanganese cluster, especially their possible involvement in manganese ligation, is investigated in this study by measuring the reported histidine-manganese coupling [Tang et al. (1994) Proc Natl Acad Sci USA 91: 704–708] of PS II membranes depleted of none, two or three of these proteins using ESEEM (electron spin echo envelope modulation) spectroscopy. The results show that neither of the three proteins influence the histidine ligation of manganese. From this, the conserved histidine of the 23 kDa protein can be ruled out as a manganese ligand. Whereas the 33 and 17 kDa proteins lack conserved histidines, the existence of a 33 kDa protein-derived carboxylate ligand has been posited; our results show no evidence for a change of the manganese co-ordination upon removal of this protein. Studies of the pH-dependence of the histidine–manganese coupling show that the histidine ligation is present in PS II centers showing the S₂ multiline EPR signal in the pH-range 4.2–9.5. This revised version was published online in June 2006 with corrections to the Cover Date.     

Influence of the 33 kDa manganese-stabilizing protein on the structure and substrate accessibility of the oxygen-evolving complex of photosystem II

The 33 kDa manganese-stabilizing extrinsic protein binds to the lumenal side of photosystem II (PS II) close to the Mn(4)Ca cluster of the oxygen-evolving complex, where it limits access of small molecules to the metal site. Our previous finding that the removal of this protein did not alter the magnetic coupling regime within the manganese cluster, measured by electron spin-echo envelope modulation [Gregor, W., and Britt, R. D. (2000) Photosynth. Res. 65, 175-185], prompted us to examine whether this accessibility control is also true for substrate water, using the same pulsed EPR technique. Comparing the deuteron modulation of the S(2)-state multiline signal of PS II membranes, equilibrated with deuterated water (D(2)O) after removal or retention of the 33 kDa protein, we observed no change in the number and the distance of deuterons magnetically coupled to manganese, indicating that the number and distance of water molecules bound to the manganese cluster are independent of bound 33 kDa protein in the S(1) state, in which the sample was poised prior to cryogenic illumination. A simple modulation depth analysis revealed a distance of 2.5-2.6 A between the closest deuteron and manganese. These results are in agreement with our refined X-ray absorption analysis. The manganese K-edge positions, reflecting their oxidation states, and the extended X-ray absorption fine structure amplitudes and distances between the manganese ions and their oxygen and nitrogen ligands (1.8, 2.7, and 3.3-3.4 A) were independent of bound 33 kDa protein.     

The mechanism of photosynthetic water oxidation

Photosynthetie water oxidation is unique to plants and cyanobacteria, it occurs in thylakoid membranes. The components associated with this process include: a reaction center polypeptide, having a molecular weight (Mr) of 47–50 kilodaltons (kDa), containing a reaction center chlorophyll a labeled as P680, a plastoquinol(?)-electron donor Z, a primary electron acceptor pheophytin, and a quinone electron acceptor Q_A; three ‘extrinsic’ polypeptides having Mr of approximately 17 kDa, 23 kDa, and 33 kDa; and, in all likelihood, an approximately 34 kDa ‘intrinsic’ polypeptide associated with manganese (Mn) atoms. In addition, chloride and calcium ions appear to be essential components for water oxidation. Photons, absorbed by the so-called photosystem II, provide the necessary energy for the chemical oxidation-reduction at P680; the oxidized P680 (P680⁺), then, oxidizes Z, which then oxidizes the water-manganese system contained, perhaps, in a protein matrix. The oxidation of water, leading to O₂ evolution and H⁺ release, requires four such independent acts, i.e., there is a charge accumulating device (the so-called S-states). In this minireview, we have presented our current understanding of the reaction center P680, the chemical nature of Z, a possible working model for water oxidation, and the possible roles of manganese atoms, chloride ions, and the various polypeptides, mentioned above. A comparison with cytochrome c oxidase, which is involved in the opposite process of the reduction of O₂ to H₂O, is stressed. This minireview is a prelude to the several minireviews, scheduled to be published in the forthcoming issues of Photosynthesis Research, including those on photosystem II (by H.J. van Gorkom); polypeptides of the O₂-evolving system (by D.F. Ghanotakis and C.F. Yocum); and the role of chloride in O₂ evolution (by S. Izawa).     

Temperature and Light Dependence of Photosynthetic Activities in Wheat Seedlings Grown in the Presence of DCMU [3-(3,4-Dichlorophenyl)-1,1-Dimethylurea]

Photosynthetic electron transfer was studied in thylakoids isolated from control and DCMU-grown wheat (Triticum aestivum L.) seedlings. When exposed to high temperature (HT) and high iradiance (HI), thylakoids showed large variations in the photosynthetic electron transport activities and thylakoid membrane proteins. A drastic reduction in the rate of whole electron transport chain (H₂O MV) was envisaged in control thylakoids when exposed to HT and HI. Such reduction was mainly due to the loss of photosystem 2, PS2 (H₂O DCBQ) activity. The thylakoids isolated from seedlings grown in the presence of DCMU showed greater resistance to HT and HI treatment. The artificial exogenous electron donors MnCl₂, DPC, and NH₂OH failed to restore the HI induced loss of PS2 activity in both control and DCMU thylakoids. In contrast, addition of DPC and NH₂OH significantly restored the HT induced loss of PS2 activity in control thylakoids and partially in DCMU thylakoids. Similar results were obtained when F_v/F_m was evaluated by chlorophyll fluorescence measurements. The marked loss of PS2 activity in control thylakoids was evidently due to the loss of 33, 23, and 17 kDa extrinsic polypeptides and 28-25 kDa LHCP polypeptides.     

The action of mercury on the binding of the extrinsic polypeptides associated with the water oxidizing complex of photosystem II

Mercury (Hg²⁺), a sulfhydryl group reactant, wasused to probe structure-function relationships in photosystem II (PSII). In the present work, we investigated the impact of mercury on the polypeptide composition of PSII submembrane preparations. Electrophoretic analysis revealed that the incubation of the membranes in the presence of mercury produces the depletion of a polypeptide of molecular weight of 33 kDa. This polypeptide corresponds to the extrinsic protein EP33 of the oxygen evolving complex removed following urea treatment. However, the two closely related extrinsic polypeptides of 16 and 23 kDa, usually removed concomitantly after urea treatment, remained unaffected after the mercury treatment. These data demonstrated the existence of an intrinsic binding site for EP23. The molecular mode of action of mercury in the oxygen evolving complex of PSII is discussed.