EDTA-induced release of manganese and proteins from inside-out thylakoid vesicles and the inhibition of oxygen evolution

Washing of inside-out, but not right-way-round, pea chloroplast thylakoid vesicles with 2 mM EDTA inhibits O2 evolution. Artificial electron donor/acceptor studies indicate that the site of inhibition is on the oxidising side of photosystem two (PS2), a conclusion reinforced by chlorophyll fluorescence measurements. Evidence is presented that the EDTA inhibition of O2 evolution is linked partly to the removal of one Mn atom per PS2 reaction centre and partly to the removal of extrinsic membrane proteins having apparent molecular weights between 58 and 70 kdaltons.     

Photoinactivation of the reactivation capacity of photosystem II in pea subchloroplast particles after a complete removal of manganese

After a complete removal of Mn from pea subchloroplast photosystem-II (PS II) preparations the electron phototransfer and oxygen evolution are restored upon addition of Mn²⁺ and Ca²⁺. Pre-illumination of the sample in the absence of Mn²⁺ leads to photoinhibition (PI) — irreversible loss of the capability of PS II to be reactivated by Mn²⁺. The effect of PI is considerably decreased in the presence of Mn²⁺ (4 Mn atoms per reaction center of PS II) and it is increased in the presence of ferricyanide or p-benzoquinone revealing the oxidative nature of the photoeffect. PI results in suppression of oxygen evolution, variable fluorescence, photoreduction of 2,6-dichlorophenol indophenol from either water or diphenylcarbazide. However, photooxidation of chlorophyll P₆₈₀, the primary electron donor of PS II as well as dark and photoinduced EPR signal II (ascribed to secondary electron donors D ₁ and Z) are preserved. PI is accompanied by photooxidation of 2–3 carotenoid molecules per PS II reaction center (RC) that is accelerated in the presence of ferricyanide and is inhibited upon addition of Mn²⁺ or diuron. The conclusion is made that PI in the absence of Mn leads to irreversible oxidative inactivation of electron transfer from water to RC of PS II which remains photochemically active. A loss of functional interaction of RC with the electron transport chain as a common feature for different types of PS II photoinhibition is discussed.Abbreviations A photoinduced absorbance changes - DPC diphenylcarbazide - DPIP 2,6-dichlorophenol indophenol - F _o constant fluorescence of chlorophyll - F photoinduced changes of Chl fluorescence yield - Mn manganese - P₆₈₀ the primary electron donor in PS II - PI photoinhibition - PS II photosystem II - Q the primary (quinone) electron acceptor in PS II - RC reaction center     

Photoreduction of NADP(+) in photosystem II of higher plants: requirement for manganese

The effects of reversible managanese extraction on NADP(+) photoreduction were studied with higher plant subchloroplast preparations of photosystem II (PS II). Under anaerobic conditions, when the reaction centers (RCs) of PS II are "closed" (i.e. in the state [P680 Pheo] Qā), and in the presence of ferredoxin-ferredoxin-NADP(+) reductase, NADP(+) reduction is observed at a rate of 0.8-1.1 μmol/mg x chlorophyII x h. After complete removal of manganese from PS II, the rate of NADP(+) reduction is reduced 40-5- fold. Upon the addition of Mn at a concentration of approx. 4 Mn atoms per reaction center, the NADP reduction is restored up to 85-90% of the initial value, When half of this amount of Mn is combined with about 40 times of the equivalent concentration of other divalent ions (Ca2?, Sr2?, Mg2? etc) the reaction is also reactivated. Dinoseb (10??M) an inhibitor of electron transfer in PS II prevents NADP(+) photoreduction. It is concluded that under conditions when the first quinone acceptor, Q(A), is in its reduced state (Qā), electrons are transferred from reduced pheophytin (Pheo(-)) to NADP(+), indicating that PS II can reduce NADP(+) without the participation of PS I. On the basis of these and literature data, and alternate pathway for electron phototransfer in PS II reaction centers of higher plants is suggested. Some problems concerning the Z-scheme are discussed.     

Reactivation of oxygen accumulation function after complete removal of manganese from the photosystem II particles

Reactivation of 02 evolution function has been studied in PS-2 particles after complete removal of Mn and water soluble 10, 17, 24, 33 kDa proteins, It has been shown that 02 evolution function in such particles can be reactivated by adding 5 μM Mn2 and 20 mM Ca2(+). Preliminary illumination of the sample is necessary to exhibit the reactivation effect of 02 evolution. The maximum value of the reactivation of 02 evolution rate is about 60% of the control. Upon illumination of the reactivated particles with flashes of 1s duration and at a frequency of 0,1 Hz, 02 evolution occurs according to the mechanism analogous to that in the initial parties of PS 2. Thus the reactivation of water oxidation and 02 evolution after complete removal of Mn and water soluble 10, 17, 24, 33 kDa proteins resulting in the suppression of 02 evolution function has been shown for the first time and it can serve as a basic approach for profound investigation of the mechanism of photosynthetic water oxidation.     

The state of manganese in the photosynthetic apparatus: 4. Structure of the manganese complex in Photosystem II studied using EXAFS spectroscopy. The S1 state of the O2-evolving Photosystem II complex from spinach

The structure of the Mn complex in the oxygen-evolving system and its mechanistic relation to photosynthetic oxygen evolution are poorly understood, though many studies have established that membrane-bound Mn plays an active role. Recently established procedures for isolating oxygen-evolving subchloroplast Photosystem II (PS II) preparations and the discovery of a light-induced multiline EPR signal attributable to the S₂ state of the O₂-evolving complex have facilitated the preparation of samples well characterized in the S₁ and S₂ states. We have used extended X-ray absorption fine structure (EXAFS) spectroscopy to probe the ligand environment of Mn in PS II particles from spinach, and in this report we present our results. The essential feature of the EXAFS results are that at least two Mn atoms per PS II reaction center occur as a binuclear species with a metal-metal distance of approx. 2.7 Å, with low Z atoms, N or O, at a distance of approx. 1.75 Å and at approx. 1.98 Å, which are characteristic of bridging and terminal ligands. These results agree well with those derived from whole chloroplasts that provided the first evidence for a binuclear manganese complex (Kirby, J.A., Robertson, A.S., Smith, J.P., Thompson, A.C., Cooper, S.R. and Klein, M.P. (1981) J. Am. Chem. Soc. 103, 5529–5537).     

Sites of function of manganese within photosystem II. Roles in O2 evolution and system II

The Mn content of spinach chloroplasts has been decreased by growth deficiency, extraction and by ageing at 35°. We studied the effect of subnormal Mn content upon the chloroplasts capacity to evolve O₂ and to photooxidize electron donors other than water via Photosystem II. We observed the following:

1. 1. In fresh chloroplasts ascorbate and other reducing agents, if present in sufficient concentration, fully replace water as the System II oxidant and can sustain maximum rates of 1000–1200 equiv/chlorophyll per h.

2. 2. None of the studied donors proved entirely specific for System II; to a variable extent all could react with the oxidant of System I. We therefore considered only the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-(DCMU)-sensitive fraction of the observed rates as pertinent.

3. 3. Normal fresh chloroplasts contained 3 Mn/200 chlorophylls_II and showed flash yields of approx. 1 O₂/1600 chlorophylls. This indicates that each System II trapping and O₂-evolving center contains three Mn atoms.

4. 4. O₂ evolution capacity is abolished when about 2/3 of the total Mn pool is removed by way of Tris or hydroxylamine extraction, i.e. upon removal of two of the three Mn atoms normally present per reaction center. Between the limits of 1 Mn per trap and 3 Mn per trap O₂ evolution capacity is linear with Mn content.

5. 5. Mn removal affects the rates of O₂ evolution in strong light and in weak light (quantum yield) in the same fashion. This indicates that complete O₂ reaction centers are inactivated.

6. 6. With Mn removal the capacity for donor (ascorbate or p-phenylenediamine) photooxidation in strong light declines in a very similar fashion as the O₂ evolving capacity. However, after removal of 2/3 of the Mn pool (by Tris or hydroxylamine extraction) 15–20% of the maximum rate remains (100–250 equiv/chlorophyll per h) as previously noticed by other workers. Secondly, the rate in weak light (quantum yield) of these photooxidations remains unaffected by Mn removal. This shows that for donor photooxidation the larger of the two Mn pools is not essential.

7. 7. Complete removal of Mn (< 1 Mn/4000 chlorophylls) led to 90–95% loss of donor photooxidation in strong light.

8. 8. Removal of 2/3 of the Mn left a low fluorescence yield (variable fraction = 0) which could be fully restored by adding DCMU. After complete removal of Mn (< 1 Mn/4000 chlorophylls) DCMU enhanced the yield of the variable fluorescence to only 1/2 the maximum level but the full maximum could be restored by chemical reduction. This indicates that fluorescence quencher of System II, Q, is not affected by Mn removal.

9. 9. Of the three Mn associated with each trapping center, one is linked more closely to the center than the other two. While all three are essential for O₂ evolution, artificial donors can enter with various rate constants at several loci on the oxidant side of System II.

A ligand field chemistry of oxygen generation by the oxygen-evolving complex and synthetic active sites

Oxygen-oxygen bond formation and O2 generation occur from the S4 state of the oxygen-evolving complex (OEC). Several mechanistic possibilities have been proposed for water oxidation, depending on the formal oxidation state of the Mn atoms. All fall under two general classifications: the AB mechanism in which nucleophilic oxygen (base, B) attacks electrophilic oxygen (acid, A) of the Mn4Ca cluster or the RC mechanism in which radical-like oxygen species couple within OEC. The critical intermediate in either mechanism involves a metal oxo, though the nature of this oxo for AB and RC mechanisms is disparate. In the case of the AB mechanism, assembly of an even-electron count, high-valent metal-oxo proximate to a hydroxide is needed whereas, in an RC mechanism, two odd-electron count, high-valent metal oxos are required. Thus the two mechanisms give rise to very different design criteria for functional models of the OEC active site. This discussion presents the electron counts and ligand geometries that support metal oxos for AB and RC O-O bond-forming reactions. The construction of architectures that bring two oxygen functionalities together under the purview of the AB and RC scenarios are described.     

Chemical modification of amine groups on PS II protein(s) retards photoassembly of the photosynthetic water-oxidizing complex

Four Mn atoms function as catalysts in the water-oxidizing complex located on the oxidizing side of PS II. We have studied the involvement of amine groups of the PS II proteins in photoligation of Mn2+ to the apo water-oxidizing complex, using the combined techniques of photoactivation and chemical modification with the modifiers methyl acetimidate (MAI), acetic acid N-hydroxysuccinimide ester (NHS), and 2,4,6-trinitrobenzenesulfonic acid (TNBS). Chemical modification of hydroxylamine-treated PS II core complexes decreased their capacity for restoration of oxygen evolution and photoligation of Mn2+ to the apo water-oxidizing complex (WOC), but did not affect their electron transfer activity in the vicinity of PS II. The number of functional high-affinity Mn-binding sites, but not of low-affinity sites, was significantly modulated by chemical modification. Kinetic analysis of photoactivation with the repetitive flashes revealed that the intermediate generated during a photoactivation process was destabilized by the chemical modification. To identify which proteins possess the amine groups involved in ligation of functional Mn, we examined the difference in NHS biotinylation between PS II core complexes with and without the Mn cluster. NHS biotinylation resulting in altered ligation of functional Mn apparently occurred on three proteins: an antenna chlorophyll binding protein (CP47), a light-harvesting chlorophyll protein (CP29), and another chlorophyll binding protein (PS II-S). Of these proteins, only the Mn-dependent biotinylation of CP47 was found to occur independently of the application of an NHS-masking concentration before removal of the functional Mn. These results suggest that lysyl residues of CP47, and perhaps also CP29 and PS II-S, function in direct photoligation of Mn2+ to the apo WOC.     

Fat type in phytosterol products influence their cholesterol-lowering potential: A systematic review and meta-analysis of RCTs

The most common form of phytosterol (PS) fortified foods are fat spreads and dairy products. The predominant fats used are soybean/sunflower (SS) or rapeseed/canola (RC) oils and animal fat (D) in dairy products. This review aimed to investigate whether the carrier fat is a determinant of the hypocholesterolaemic effects of PS fortified foods. Databases were searched using relevant keywords and published RCTs from 1990 investigating the effects of dietary PS intervention (≥ 1.5 g per day) on total cholesterol and LDL-C were included. After methodological quality assessment and data extraction, a total of 32 RCTs (RC, n = 15; SS, n = 9; D, n = 8) were included. As expected, all fat groups significantly reduced TC and LDL-C (p < 0.01). When compared across different carrier fats, RC as the main carrier fat, reduced LDL-C significantly more than the SS spreads (p = 0.01). Therefore, a combination of monounsaturated fatty acid rich spread with adequate amounts of omega-3 fatty acids (as evident in RC spreads) may be the superior carrier fat for the delivery of PS for optimal blood cholesterol-lowering. The findings of this research provide useful evidence for optimising the hypocholesterolaemic effects of PS and support further investigation into the possible mechanisms behind these findings.     

Comparative study of effects of artificial electron donors on the AT-band of photosystem II thermoluminescence

Extraction of the Mn-cluster from photosystem II (PS II) inhibits the main bands of thermoluminescence and induces a new A_T-band at –20°C. This band is attributed to the charge recombination between acceptor Q_A ^– and a redoxactive histidine residue on the donor side of PS II. The effect of Mn(II) and Fe(II) cations as well as the artificial donors diphenylcarbazide and hydroxylamine on the A_T-band of thermoluminescence was studied to elucidate the role of the redoxactive His residue in binding to the Mn(II) and Fe(II). At the Mn/PS II reaction center (RC) ratio of 90 : 1 and Fe/PS II RC ratio of 120 : 1, treatment with Mn(II) and Fe(II) causes only 60% inhibition of the A_T-band. Preliminary exposure of Mn-depleted PS II preparations to light in the presence of Mn(II) and Fe(II) causes binding of the cations to the high-affinity Mn-binding site, thereby inhibiting oxidation of the His residue involved in the AT -band formation. The efficiency of the A_T-band quenching induced by diphenylcarbazide and hydroxylamine is almost an order of magnitude higher than the quenching efficiency of Mn(II) and Fe(II). Our results suggest that the redox-active His is not a ligand of the high-affinity site and does not participate in the electron transport from Mn(II) and Fe(II) to YZ . The concentration dependences of the A_T-band inhibition by Mn(II) and Fe(II) coincide with each other, thereby implying specific interaction of Fe(II) with the donor side of PS II.     

The basic properties of the electronic structure of the oxygen-evolving complex of photosystem II are not perturbed by Ca2+ removal

Ca(2+) is an integral component of the Mn(4)O(5)Ca cluster of the oxygen-evolving complex in photosystem II (PS II). Its removal leads to the loss of the water oxidizing functionality. The S(2)' state of the Ca(2+)-depleted cluster from spinach is examined by X- and Q-band EPR and (55)Mn electron nuclear double resonance (ENDOR) spectroscopy. Spectral simulations demonstrate that upon Ca(2+) removal, its electronic structure remains essentially unaltered, i.e. that of a manganese tetramer. No redistribution of the manganese valence states and only minor perturbation of the exchange interactions between the manganese ions were found. Interestingly, the S(2)' state in spinach PS II is very similar to the native S(2) state of Thermosynechococcus elongatus in terms of spin state energies and insensitivity to methanol addition. These results assign the Ca(2+) a functional as opposed to a structural role in water splitting catalysis, such as (i) being essential for efficient proton-coupled electron transfer between Y(Z) and the manganese cluster and/or (ii) providing an initial binding site for substrate water. Additionally, a novel (55)Mn(2+) signal, detected by Q-band pulse EPR and ENDOR, was observed in Ca(2+)-depleted PS II. Mn(2+) titration, monitored by (55)Mn ENDOR, revealed a specific Mn(2+) binding site with a submicromolar K(D). Ca(2+) titration of Mn(2+)-loaded, Ca(2+)-depleted PS II demonstrated that the site is reversibly made accessible to Mn(2+) by Ca(2+) depletion and reconstitution. Mn(2+) is proposed to bind at one of the extrinsic subunits. This process is possibly relevant for the formation of the Mn(4)O(5)Ca cluster during photoassembly and/or D1 repair.     

Orientation of calcium in the Mn4Ca cluster of the oxygen-evolving complex determined using polarized strontium EXAFS of photosystem II membranes

The oxygen-evolving complex of photosystem II (PS II) in green plants and algae contains a cluster of four Mn atoms in the active site, which catalyzes the photoinduced oxidation of water to dioxygen. Along with Mn, calcium and chloride ions are necessary cofactors for proper functioning of the complex. The current study using polarized Sr EXAFS on oriented Sr-reactivated samples shows that Fourier peak II, which fits best to Mn at 3.5 A rather than lighter atoms (C, N, O, or Cl), is dichroic, with a larger magnitude at 10 degrees (angle between the PS II membrane normal and the X-ray electric field vector) and a smaller magnitude at 80 degrees . Analysis of the dichroism of the Sr EXAFS yields a lower and upper limit of 0 degrees and 23 degrees for the average angle between the Sr-Mn vectors and the membrane normal and an isotropic coordination number (number of Mn neighbors to Sr) of 1 or 2 for these layered PS II samples. The results confirm the contention that Ca (Sr) is proximal to the Mn cluster and lead to refined working models of the heteronuclear Mn(4)Ca cluster of the oxygen-evolving complex in PS II.     

The reaction centre from green sulphur bacteria: progress towards structural elucidation

The reaction centre (RC) of green sulphur bacteria is a FeS-type RC, as are the RCs of Photosystems I (PS I) of oxygenic photosynthetic organisms and of heliobacteria. The core domains of both green sulphur bacterial and heliobacterial RCs are considered to be homodimeric, in contrast to those of purple bacteria, PS I and Photosystem II (PS II). This paper briefly describes the techniques of electron microscopy and image processing suited to investigate the structure of these proteins. Recent advances in the study of the structure of the green sulphur bacterial RC, primarily achieved by the application of scanning transmission electron microscopy, are reviewed.This revised version was published online in October 2005 with corrections to the Cover Date.     

Purification of Photosystem II particles from Phormidium laminosum using the detergent dodecyl-β-d-maltoside. Properties of the purified complex

The purification and properties of a new oxygen-evolving Photosystem (PS) II particle from the thermophilic blue-green alga Phormidium laminosum are described. The activity of the lauryldimethylamine N-oxide PS II-enriched supernatant described previously (Stewart, A.C. and Bendall, D.S. (1979) FEBS Lett. 107, 308–312) was found to be stabilized for several days at 4°;C by the addition of a second detergent, dodecyl-β-d-maltoside (lauryl maltoside). The lauryl maltoside/lauryldimethylamine N-oxide extract could be fractionated by sucrose density gradient centrifugation. Very high rates of oxygen evolution, typically 1900–2400 μmol O₂/mg chlorophyll a per h at pH 7 with dimethylbenzoquinone and ferricyanide as acceptors, were observed for the lowest green band from the gradient. This fraction contained cytochromes b-559 (high-potential) and c-549, but was completely devoid of P-700 and cytochromes b-563 and f. The purified oxygen-evolving particles comprised seven major polypeptides (M_r 58 900, 52 400, 43 200, 33 900, 30 000, 16 000 and 15 000) and approximately five minor polypeptides. The particles contained 3–4 Mn atoms per reaction centre and had a chlorophyll antenna of approx. 50 chlorophyll a. The fast phase of fluorescence induction curves in the presence of hydroxylamine and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) could be described by an exponential, suggesting that no energy transfer was occurring between the PS II units responsible for this phase. Comparison of the area above the fluorescence induction curves in the absence and presence of DCMU suggested an acceptor pool size of 2–3 equivalents per centre.     

Photosystem I complexes associated with fucoxanthin-chlorophyll-binding proteins from a marine centric diatom, Chaetoceros gracilis

Diatoms occupy a key position as a primary producer in the global aquatic ecosystem. We developed methods to isolate highly intact thylakoid membranes and the photosystem I (PS I) complex from a marine centric diatom, Chaetoceros gracilis. The PS I reaction center (RC) was purified as a super complex with light-harvesting fucoxanthin-chlorophyll (Chl)-binding proteins (FCP). The super complex contained 224 Chl a, 22 Chl c, and 55 fucoxanthin molecules per RC. The apparent molecular mass of the purified FCP-PS I super complex (approximately 1000 kDa) indicated that the super complex was composed of a monomer of the PS I RC complex and about 25 copies of FCP. The complex contained menaquinone-4 as the secondary electron acceptor A1 instead of phylloquinone. Time-resolved fluorescence emission spectra at 77 K indicated that fast (16 ps) energy transfer from a Chl a band at 685 nm on FCP to Chls on the PS I RC complex occurs. The ratio of fucoxanthin to Chl a on the PS I-bound FCP was lower than that of weakly bound FCP, suggesting that PS I-bound FCP specifically functions as the mediator of energy transfer between weakly bound FCPs and the PS I RC.     

Magnetic interaction of manganese with pheophytin anion-radical and chlorophyll cation-radical in reaction centers of the photosystem II

The influence of Mn on saturation curves of ESR spectra of Ph(-) and P(+)(680) at 1-200K in samples with different content of Mn has been studied. An analysis of these data and those on photoinduced changes of fluorescence yield of chlorophyll leads to the conclusion that the Mn-containing centre in Photosystem 2 is a cluster of 4 Mn atoms, two of which can be replaced by Mg(2+) or any other divalent metal. The distances between Mn Na Ph as well as between Mn and P(680) have been estimated.     

Isolation of PS II reaction centre and its relationship to the minor chlorophyll-protein complexes

Evidence is presented for the identification of the chlorophyll- protein complex CPa-1 (CP 47) as the reaction centre of photosystem II (PS II). We have developed a simple, rapid method using octyl glucoside solubilization to obtain preparations from spinach and barley that are highly enriched in PS II reaction centre activity (measured as the light-driven reduction of diphenylcarbazide by 2,6-dichlorophenolindophenol). These preparations contain only the two minor chlorophyll-protein complexes CPa-1 and CPa-2. During centrifugation on a sucrose density gradient, there is a partial separation of the two CPa complexes from each other, and a complete separation from other chlorophyll-protein complexes. The PS II activity comigrates with CPa-1 but not CPa-2, strongly suggesting that the former is the reaction centre complex of PS II. Reaction centre preparations are sensitive to the herbicide 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), but only at much higher concentrations than those required to inhibit intact thylakoid membranes. A model of PS II incorporating our current knowledge of the chlorophyll-protein complexes is presented. It is proposed that CPa-2 and the chlorophyll a + b complex CP 29 may function as internal antenna complexes surrounding the reaction centre, with the addition of variable amounts of the major chlorophyll a + b light-harvesting complex.     

The release of polypeptides and manganese from oxygen-evolving photosystem II preparations following zinc-treatment

Inhibition of oxygen evolution in photosystem II membrane fragments from pea chloroplasts by washing with Zn₂₊ causes appearance of the EPR signal of Mn(H₂O)₆²⁺. This Mn²⁺ remains associated with the membrane fraction. Release of Mn²⁺ into the medium was correlated with the amount of the 23 kDa protein removed from the membrane. This suggests that this protein may function as a ‘gate’ to an aqueous compartment into which Mn²⁺ is released. Inhibition by Zn²⁺ correlated with the release of 1 Mn²⁺ per reaction centre, out of a total stoichiometry of 4 Mn atoms per reaction centre. By comparing the release of Mn following Zn-treatment of NaCI or CaC1₂ washed membranes, it is concluded that the 33 kDa protein is involved in binding of 2 Mn.     

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.     

Predominant Protection of D2-Protein against Photodestruction in Isolated D1/D2/Cytochrome b 559 by K15, a Phenolic-Type Inhibitor of Electron Transfer in Photosystem 2

A protective action of K15 (4-[methoxy-bis(trifluoromethyl)methyl]-2,6-dinitrophenylhydrazone methyl ketone), an inhibitor of electron transport in photosystem 2 (PS 2), against photoinactivation of the PS 2 reaction center (RC) D1/D2/cytochrome b(559) complex, isolated from pea chloroplasts, by red light (0.7 mmol photons/sec per m(2)) has been investigated under aerobic conditions. The inhibitor K15 causing cyclic electron transfer around PS 2 and thus prohibiting stabilization of separated charges has been shown to effectively protect RC both against the loss of photochemical activity (measured as reversible photoinduced absorbance changes related to photoreduction of pheophytin) and aggregation and degradation of the proteins D2 and D1 during photoinactivation. Comparison of the protective action of K15 and of another inhibitor of electron transfer in PS 2, diuron, against light-induced destruction of proteins D1 and D2 shows that diuron stabilizes protein D1 and K15 stabilizes protein D2. The preferential protection of D2 against photoinduced destruction revealed in our work is in accord with the concept of a specific binding of K15 with this protein. It is proposed that this binding site may be that of the primary quinone electron acceptor Q(A) located on the D2 protein (in contrast to diuron, which is known to replace the secondary electron acceptor Q(B) from its binding site on D1).