A New 4.8-kDa Polypeptide Intrinsic to the PS II Reaction Center, as Revealed by Modified SDS-PAGE with Improved Resolution of Low-Molecular-Weight Proteins

Low-molecular-weight polypeptides in various PS II preparationsfrom spinach and wheat were analyzed by modified SDS-PAGE, whichgave good resolution of low-molecular-weight proteins with minimizedinterference by lipids. PS II membrane fragments contained atleast nine low-molecular-weight polypeptides of between 3.9kDa and 11 kDa, and all of them were identified in thylakoidmembranes. Of these nine polypeptides, the 10-kDa phosphoprotein,the 5-kDa, 4.8-kDa, and 4.1-kDa polypeptides, and the two subunitsof cytochrome b₅₅₉ were commonly found in O₂-evolving core complexesof wheat and spinach. In contrast, PS II reaction center complexesthat consisted of D1, D2 and two cytochrome b₅₅₉ polypeptidesretained only the 4.8- kDa polypeptide. Analysis by Westernblotting revealed that the 4.8-kDa polypeptide is an intrinsiccomponent of the PS II reaction center. (Received May 30, 1988; Accepted August 19, 1988)     

Role of the 33-kDa polypeptide in preserving Mn in the photosynthetic oxygen-evolution system and its replacement by chloride ions

Treatment of Photosystem II particles from spinach chloroplasts with Triton X-100 with 2.6 M urea in the presence of 200 mM NaCl removed 3 polypeptides of 33 kDa, 24 kDa and 18 kDa, but left Mn bound to the particles. The (urea + NaCl)-treated particles could evolve oxygen in 200 mM, but not in 10 mM NaCl. Mn was gradually released with concomitant loss of oxygen-evolution activity in 10 mM NaCl but not in 200 mM Cl^?. The NaCl-treated particles, which contained Mn and the 33-kDa polypeptide but not the 24-kDa and 18-kDa polypeptides, did not lose Mn or oxygen-evolution activity in 10 mM NaCl. These observations suggest that the 33-kDa polypeptide maintains the binding of Mn to the oxygen-evolution system and can be functionally replaced by 200 mM Cl^?.     

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.     

Quantitative Analysis of the Inactivation of Photosynthetic Oxygen Evolution and the Release of Polypeptides and Manganese in the Photosystem II Particles of Spinach Chloroplasts

Photosynthetic oxygen evolution by photosystem II particleswas inactivated by treatment with NaCl, NH₂OH or high pH. Whenthe degree of inactivation was compared with the degree of releasefrom the particles of Mn and three polypeptides having molecularmasses of 33, 24 and 18kdaltons, two types of inactivation werefound: one, brought about with 960 mM NaCl, was related to therelease of the 24 kdalton polypeptide, and the other, broughtabout with 1.5 mM NH₂OH or high pH, seemed to be related tothe release of Mn. ¹Present address: Department of Chemistry, Faculty of Science,Toho University, Miyama 2-2-1, Funabashi 274, Japan. (Received January 31, 1983; Accepted March 28, 1983)     

Mn-preserving extraction of 33-, 24- and 16-kDa proteins from O2-evolving PS II particles by divalent salt-washing

Divalent salt-washing of O₂-evolving PS II particles caused total liberation of 33-, 24- and 16-kDa proteins, but the resulting PS II particles retained almost all amounts of Mn present in initial particles. The retained Mn was EPR-silent when the particles were kept in high concentrations of divalent salt. By divalent salt-washing, the activity of diphenylcarbazide (DPC) photooxidation was not affected at all, neither suppressed nor enhanced, while O₂ evolution was totally inactivated. These results indicate that Mn can be kept associated with PS II particles even after liberation of the 33-kDa protein, and suggest that the 33-kDa protein is probably not responsible for binding Mn onto membranes, but is possibly responsible for maintaining the function of Mn atoms in the O₂-evolving center.     

Partial disintegration and reconstitution of the photosynthetic oxygen evolution system. Binding of 24 kilodalton and 18 kilodalton polypeptides

Treatment with 1 M NaCl almost totally removed two polypeptides of 24 and 18 kDa from the Photosystem II particles of spinach chloroplasts and reduced the oxygen-evolution activity by about half. Both polypeptides were able to rebind to the NaCl-treated particles in a low-salt medium. The rebinding of the 24 kDa polypeptide showed a saturation curve whose maximum level was close to that naturally occurring in the untreated particles. In parallel with the amount of rebound 24 kDa polypeptide, the oxygen-evolution activity was recovered. The 18 kDa polypeptide bound to the NaCl-treated particles without saturation. When the 18 kDa polypeptide was added to the particles previously treated with NaCl and then supplemented with a saturating amount of 24 kDa polypeptide, there appeared, in addition to the binding without saturation, another binding of the 18 kDa polypeptide with saturation to a maximum level close to that naturally occurring in the untreated particles. The 18 kDa polypeptide did not restore the oxygen-evolution activity. These findings suggest that there are specific binding sites; one for the 24 kDa polypeptide located on the Photosystem II particles, and the other for the 18 kDa polypeptide on the 24 kDa polypeptide.     

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)     

Intermolecular relations of the photosystem II complex in spinach chloroplasts as detected by immunochemical assay

Polyclonal antibodies were prepared to the subunits of the spinach photosystem II fraction (PS II): p47, p43, p27, p33, p24, and p17. (The protein nomenclature refers to Mr). p47 and p43 are the subunits of reaction center complex, and p27 is light-harvesting chlorophyll protein. p33, p24, and p17 are extractable from PS II with 1 M Tris, and p24 and p17 with 1 M NaCl. With untreated PS II fractions, the antibody to p24 inhibited the photosynthetic oxygen-evolving activity, but not the DCPI-photoreduction activity in the presence of DPC, indicating that p24 played an important role in the former activity. Bindings of the respective antibodies to the PS II treated with sodium dodecyl sulfate were regarded as 100%. To untreated PS II, the bindings were 20-30% for p47, p43, and p27, about 50% for p33, and 70-80% for p24 and p17. To NaCl-washed PS II, the binding to p33 increased by 9%, indicating that p33 was adjacent or bound with p24 or/and p17. To Tris-washed PS II, the binding to p43 increased by 7%, indicating that p43 was adjacent or bound with p33. To PS II treated with 3% of Brij 58, only the binding to p27 increased appreciably. To PS II treated with 1% of octyl glucoside, the binding to p47 was still lower than 50%, whereas those to the other subunits were 74-91%. These values could be a measure of the extents to which the subunits were exposed to the aqueous phase, because of the nature of polyclonal antibodies. These results suggest that in intact PS II, p47, p43, and p27 were in most part buried in the inside, p47 being located at the most central and p27 at the outermost part, whereas p33, p24, and p17 were exposed to the outside by 50-75%.     

Partial reconstitution of the photosynthetic oxygen evolution system by rebinding of the 33-kDa polypeptide

Treatment with 2.6 M urea of the Photosystem II particles depleted of two polypeptides of 24 kDa and 18 kDa completely released a polypeptide of 33 kDa and eliminated the oxygen-evolution activity. The 33-kDa polypeptide rebound to the urea-treated particles and partially reactivated the oxygen evolution. A quantitative analysis of the rebinding suggests tha there is a specific binding site for the 33-kDa polypeptide on the membrane surface.     

Biochemical Evidence that the Higher Plant Photosystem II Core Complex is Organized as a Dimer

The organization of the pigmented multiprotein core complexof higher plant PS II has been examined. Oxygen-evolving PSII particles or thylakoid membranes of wild-type and Chi b-lessbarley were extracted with various glycosidic surfactants andelectrophoretically fractionated. The predominant multiproteincore complex II (CC II) fractions had sizes on gel electrophoresisof M_r=230,000 and M_r= 140,000 and were photochemically active.Both fractions had identical absorption spectra, contained thebeta-carotene-chl a-proteins (Cp47 and Cp43), the PS II reactioncenter subunits (Dl and D2), and the two cytochrome b₅₅₉ subunitsin unit stoichiometry. The M_r=230,000 fraction could evolveoxygen in the light and contained an M_r=33,O0O oxygen evolutionenhancer (OEE 33) polypeptide, whereas the M_r= 140,000 fractionlacked OEE 33 and could not evolve oxygen. The apparent sizesof the two fractions were also estimated by gel filtration asM_r=490,000 and M_r=220,000, respectively; the estimates by gelfiltration more accurately reflect their predicted sizes. Furtheranalyses by nondenaturing gel electrophoresis indicated thatCp47, Cp43 and the three OEE gene products probably occur ashomodimers in situ. Our data suggest that phosphorylation ofCC II subunits occurs when they are located in the oligomericform. We propose that the native state of the PS II core complexin higher plants is dimeric, and that this state, which waspreviously observed only in thermophilic cyanobacteria, is probablythe form present in all oxygenic organisms. (Received August 9, 1991; Accepted September 26, 1991)     

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.     

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.     

Development of photosystem-II activity during irradiance of etiolated Helianthus (Asteraceae) seedlings

Light-induced development of photosystem (PS)-II activity was followed during irradiance of etiolated Helianthus annuus (sunflower) cotyledons using chlorophyll a fluorescence. Cotyledons from seedlings grown in continuous darkness for 6 d were exposed to 100 μmol photons·m⁻²·s⁻¹ for time periods of 1, 3, 6, and 12 h. Associated with increased time of irradiance exposure were significant: (1) increases in concentration of PS II, (2) increases in quantum efficiency of PS II, (3) decreases in the ratio of PS-II quinone_B (Q_B)-nonreducing centers to total PS-II centers (PS-II Q_B-nonreducing centers + PS-II Q_B-reducing centers), and (4) decreases in the ratio of slow PS-II Q_B-reducing centers to total PS-II Q_B-reducing centers (fast PS-II Q_B-reducing centers + slow PS-II Q_B-reducing centers). The results support the hypotheses that development of PS II involves assembly of complexes which initially cannot reduce Q_B and that heterogeneous aspects of PS-II pools during chloroplast maturation may represent different developmental states.     

Effects of phospholipase A2 and alkaline pH on photosystem II particles isolated from spinach chloroplasts

An O₂-evolving photosystem II fraction (PS II particles) isolated from spinach ( Spinacia oleracea L.) chloroplasts by Triton X-100 was treated by phospholipase A₂ or by an alkaline pH. Phospholipase A₂ depleted the particles of all phosphatidylcholine and of a part of phosphatidylglycerol containing trans -hexadecenoic acid, and induced a parallel inactivation of the PS II activity. The protein pattern remained similar to that of the control particles. The addition of exogenous polar lipids from thylakoids could not reactivate PS II activity. Treatment of PS II particles by an alkaline pH, known to release the 33, 24 and 18 kdalton polypeptides and to inactivate PS II activity, did not affect the lipid composition. The involvement of lipids in PS II activity is discussed.     

Calcium ions can be substituted for the 24-kDa polypeptide in photosynthetic oxygen evolution

Photosystem II particles were prepared from spinach chloroplasts with Triton X-100, and treated with 1.0 M NaCl to remove polypeptides of 24 kDa and 18 kDa and to reduce the photosynthetic oxygen-evolution activity by about half. Oxygen-evolution activity was restored almost to the original level with 10 mM Ca²⁺, in a similar manner to the rebinding of 24-kDa polypeptide. Other cations such as magnesium, sodium and manganese ions could not restore any oxygen-evolution activity. These observations, together with a kinetic analysis, suggest that Ca²⁺ can be substituted for the 24-kDa polypeptide in photosynthetic oxygen evolution in Photosystem II particles.     

Light-dependent inactivation of photosynthetic oxygen evolution during NaCl treatment of photosystem II particles: The role of the 24-kDa protein

Photosystem (PS) II particles prepared from spinach thylakoids with Triton X-100 were treated with 1.5 M NaCl either in the light or dark. Under both conditions, the 24-kDa and 18-kDa proteins were released from the particles, but rebound to them when the NaCl concentration was reduced to 34 mM by dilution. Oxygen evolution measured after the dilution was inactivated following NaCl treatment in the light, but not following treatment in the dark. The inactivation in the light was suppressed when 5 mM CaCl₂ was added during or after the NaCl treatment. Based on these observations, a scheme is proposed for the mechanism of light-dependent inactivation of oxygen evolution during NaCl treatment of PS II particles and for the function of the 24-kDa protein in regulating the conformation of a supposed Ca²⁺-binding intrinsic protein.Abbreviations Chl chlorophyll - EGTA ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Mes 4-morpholineethanesulphonic acid - PS photosystem - SDS sodium dodecylsulphate     

The roles of the extrinsic subunits in Photosystem II as revealed by EPR

The effects of selective removal of extrinsic proteins on donor side electron transport in oxygen-evolving PS II particles were examined by monitoring the decay time of the EPR signal from the oxidized secondary donor, Z⁺, and the amplitude of the multiline manganese EPR signal. Removal of the 16 and 24 kDa proteins by washing with 1 M NaCl inhibits oxygen evolution, but rapid electron transfer to Z⁺ still occurs as evidenced by the near absence of Signal II_f. The absence of a multiline EPR signal shows that NaCl washing induces a modification of the oxygen-evolving complex which prevents the formation of the S₂ state. This modification is different from the one induced by chloride depletion of PS II particles, since in these a large multiline EPR signal is found. After removal of the 33 kDa protein with 1 M MgCl₂, Signal II_f is generated after a light flash. Readdition of the 33 kDa component to the depleted membranes accelerates the reduction of Z⁺. Added calcium ions show a similar effect. These findings suggest that partial advancement through the oxygen-evolving cycle can occur in the absence of the 16 and 24 kDa proteins. The 33 kDa protein, on the other hand, may be necessary for such reactions to take place.     

The function of 33-kDa protein in the photosynthetic oxygen-evolution system studied by reconstitution experiments

Treatment of Photosystem II particles with 1.2 M CaCl₂ released three proteins of 33, 24 and 18 kDa of the photosynthetic oxygen evolution system, but left Mn bound to the particles as demonstrated by Ono and Inoue (Ono, T. and Inoue, Y. (1983) FEBS Lett. 164, 252–260). Oxygen-evolution activity of the CaCl₂-treated particles was very low in a medium containing 10 mM NaCl as a salt, but could be restored by the 33-kDa protein. When the particles were incubated in 10 mM NaCl at 0°C, two of the four Mn atoms per oxygen-evolution system were released with concomitant loss of oxygen-evolution activity. The 33-kDa protein suppressed the release of Mn and the inactivation during the incubation. These findings from reconstitution experiments suggest that the 33-kDa protein acts to preserve Mn atoms in the oxygen-evolution system. The 33-kDa protein could be partially substituted by 100 or 150 mM Cl⁻ for the preservation of the Mn and oxygen-evolution activity. The Mn in Photosystem II particles enhanced rebinding of the 33-kDa protein to the particles.     

Analysis of photosystem II using particle II preparation. I. Experimental evidence supporting the idea of the involvement of two light reactions in photosystem II of green plant photosynthesis

(1) To analyze the photoelectron flow related to photosystemII, particle II preparation, i.e., the chloroplast fragmenthaving only photosystem II activity, proved to be far betterthan the generally used chloroplast preparations having activitiesof both PS-I and PS-II. (2) By simultaneous measurements ofthe activities of O₂ evolution and DPIP- and ferricyanide photoreductionusing variously-treated particle II preparations, it was foundthat a noticeable activity of ferricyanide photoreduction wasstill observed, though the former two activities were completelylost in the course of treatments such as Tris-treatment, pre-illuminationand aging. (3) Besides this, differences were found betweenferricyanide- and DPIP-photoreduction in respect to susceptibilityto CCCP, availability of artificial electron donor, and theeffect of chloride addition. However, both photo-reductionswere equally inactivated by heat-treatment and addition of DCMU.(4) To explain the observed distinctions between DPIP and ferricyanidein their mode of action as electron acceptor for PS-II, a schemesuggesting the involvement of two light reactions in PS-II isproposed and the electron flow near PS-II is discussed. ¹ This work has been supported by Grants from the Ministry ofEducation (Nos. 8425- 70-'71; 4970l4-'69-'71), which are gratefullyacknowledged here. (Received January 12, 1972; )     

Long-lived primary radical pair state detected by time-resolved fluorescence and absorption spectroscopy in an isolated Photosystem two core

A Photosystem two (PS II) core preparation containing the chlorophyll a binding proteins CP 47, CP 43, D1 and D2, and the non-chlorophyll binding cytochrome-b559 and 33 kDA polypeptides, has been isolated from PS II-enriched membranes of peas using the non-ionic detergent heptylthioglucopyranoside and elevated ionic strengths. The primary radical pair state, P680⁺Pheo^-, was studied by time-resolved absorption and fluorescence spectroscopy, under conditions where quinone reduction and water-splitting activities were inhibited. Charge recombination of the primary radical pair in PS II cores was found to have lifetimes of 17.5 ns measured by fluorescence and 21 ns measured by transient decay kinetics under anaerobic conditions. Transient absorption spectroscopy demonstrated that the activity of the particles, based on primary radical pair formation, was in excess of 70% (depending on the choice of kinetic model), while time-resolved fluorescence spectroscopy indicated that the particles were 91% active. These estimates of activity were further supported by steady-state measurements which quantified the amount of photoreducible pheophytin. It is concluded that the PS II core preparation we have isolated is ideal for studying primary radical pair formation and recombination as demonstrated by the correlation of our absorption and fluorescence transient data, which is the first of its kind to be reported in the literature for isolated PS II core complexes from higher plants.Abbreviations CP 43 and CP 47 chlorophyll binding proteins of PS II having apparent molecular weights on SDS-PAGE of 43 kDa and 47 kDa, respectively - D1 and D2 polypeptides PS II reaction centre polypeptides encoded by the psbA and psbD genes, respectively - HPLC high performance liquid chromatography - PS II Photosystem two - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - P680 primary electron donor of PS II - Pheo phenophytin a - SPC single photon counting - PBQ phenyl-p-benzoquinone - DPC 1,5-diphenylcarbazide AFRC Photosynthesis Research Group, Department of Biochemistry