Flash-induced consumption of molecular oxygen on the donor side of photosystem II in Mn-depleted subchloroplast membrane fragments: specific effects of manganese and calcium ions

It has been shown that removal of manganese from the water-oxidizing complex (WOC) of photosystem II (PSII) leads to flash-induced oxygen consumption (FIOC) which is activated by low concentration of Mn²⁺ (Yanykin et al., Biochim Biophys Acta 1797:516–523, 2010). In the present work, we examined the effect of transition and non-transition divalent metal ions on FIOC in Mn-depleted PSII (apo-WOC-PSII) preparations. It was shown that only Mn²⁺ ions are able to activate FIOC while other transition metal ions (Fe²⁺, V²⁺ and Cr²⁺) capable of electron donation to the apo-WOC-PSII suppressed the photoconsumption of O₂. Co²⁺ ions with a high redox potential (E ⁰ for Co²⁺/Co³⁺ is 1.8 V) showed no effect. Non-transition metal ions Ca²⁺ by Mg²⁺ did not stimulate FIOC. However, Ca²⁺ (in contrast to Mg²⁺) showed an additional activation effect in the presence of exogenic Mn²⁺. The Ca²⁺ effect depended on the concentration of both Mn²⁺ and Ca²⁺. The Ca effect was only observed when: (1) the activation of FIOC induced by Mn²⁺ did not reach its maximum, (2) the concentration of Ca²⁺ did not exceed 40 μM; at higher concentrations Ca²⁺ inhibited the Mn²⁺-activated O₂ photoconsumption. Replacement of Ca²⁺ by Mg²⁺ led to a suppression of Mn²⁺-activated O₂ photoconsumption; while, addition of Ca²⁺ resulted in elimination of the Mg²⁺ inhibitory effect and activation of FIOC. Thus, only Mn²⁺ and Ca²⁺ (which are constituents of the WOC) have specific effects of activation of FIOC in apo-WOC-PSII preparations. Possible reactions involving Mn²⁺ and Ca²⁺ which could lead to the activation of FIOC in the apo-WOC-PSII are discussed.     

Photoconsumption of molecular oxygen on both donor and acceptor sides of photosystem II in Mn-depleted subchloroplast membrane fragments

Oxygen consumption in Mn-depleted photosystem II (PSII) preparations under continuous and pulsed illumination is investigated. It is shown that removal of manganese from the water-oxidizing complex (WOC) by high pH treatment leads to a 6-fold increase in the rate of O₂ photoconsumption. The use of exogenous electron acceptors and donors to PSII shows that in Mn-depleted PSII preparations along with the well-known effect of O₂ photoreduction on the acceptor side of PSII, there is light-induced O₂ consumption on the donor side of PSII (nearly 30% and 70%, respectively). It is suggested that the light-induced O₂ uptake on the donor side of PSII is related to interaction of O₂ with radicals produced by photooxidation of organic molecules. The study of flash-induced O₂ uptake finds that removal of Mn from the WOC leads to O₂ photoconsumption with maximum in the first flash, and its yield is comparable with the yield of O₂ evolution on the third flash measured in the PSII samples before Mn removal. The flash-induced O₂ uptake is drastically (by a factor of 1.8) activated by catalytic concentration (5-10 μM, corresponding to 2-4 Mn per RC) of Mn²⁺, while at higher concentrations (> 100 μM) Mn²⁺ inhibits the O₂ photoconsumption (like other electron donors: ferrocyanide and diphenylcarbazide). Inhibitory pre-illumination of the Mn-depleted PSII preparations (resulting in the loss of electron donation from Mn²⁺) leads to both suppression of flash-induced O₂ uptake and disappearance of the Mn-induced activation of the O₂ photoconsumption. We assume that the light-induced O₂ uptake in Mn-depleted PSII preparations may reflect not only the negative processes leading to photoinhibition but also possible participation of O₂ or its reactive forms in the formation of the inorganic core of the WOC.     

pH dependence of the donor side reactions in Ca2+-depleted photosystem II

We have studied how low pH affects the water-oxidizing complex in Photosystem II when depleted of the essential Ca(2+) ion cofactor. For these samples, it was found that the EPR signal from the Y(Z)(*) radical decays faster at low pH than at high pH. At 20 degrees C, Y(Z)(*) decays with biphasic kinetics. At pH 6.5, the fast phase encompasses about 65% of the amplitude and has a lifetime of approximately 0.8 s, while the slow phase has a lifetime of approximately 22 s. At pH 3.9, the kinetics become totally dominated by the fast phase, with more than 90% of the signal intensity operating with a lifetime of approximately 0.3 s. The kinetic changes occurred with an approximate pK(a) of 4.5. Low pH also affected the induction of the so-called split radical EPR signal from the S(2)Y(Z)(*) state that is induced in Ca(2+)-depleted PSII membranes because of an inability of Y(Z)(*) to oxidize the S(2) state. At pH 4.5, about 50% of the split signal was induced, as compared to the amplitude of the signal that was induced at pH 6.5-7, using similar illumination conditions. Thus, the split-signal induction decreased with an apparent pK(a) of 4.5. In the same samples, the stable multiline signal from the S(2) state, which is modified by the removal of Ca(2+), was decreased by the illumination to the same extent at all pHs. It is proposed that decreased induction of the S(2)Y(Z)(*) state at lower pH was not due to inability to oxidize the modified S(2) state induced by the Ca(2+) depletion. Instead, we propose that the low pH makes Y(Z)(*) able to oxidize the S(2) state, making the S(2) --> S(3) transition available in Ca(2+)-depleted PSII. Implications of these results for the catalytic role of Ca(2+) and the role of proton transfer between the Mn cluster and Y(Z) during oxygen evolution is discussed.     

Photoreduction of 2,6-dichlorophenolindophenol by diphenylcarbazide: a photosystem 2 reaction catalyzed by tris-washed chloroplasts and subchloroplast fragments

The use of diphenylcarbazide as an electron donor coupled to the photoreduction of 2,6-dichlorophenolindophenol by tris-washed chloroplasts or subchloroplast fragments provides a simple and sensitive assay for photosystem 2 of chloroplasts. By varying the concentration of tris buffer at pH 8.0 during an incubation period it is shown that the destruction of oxygen evolution activity is accompanied by a corresponding emergence of an ability to photooxidize diphenylcarbazide, as evidenced by absorbance changes due to diphenylcarbazide at 300 nm. The temperature-sensitive oxidation of diphenylcarbazide is inhibited by DCMU and by high ionic strengths. This activity appears to measure the primary photochemical reaction of photosystem 2.     

Effects of pH on reactions on the donor side of photosystem II.

The effects of pH on the increase of fluorescence yield measured in the microsecond range, and on the microsecond delayed fluorescence have been studied in dark adapted chloroplasts as a function of flash number. (1) At pH 7, the amplitude of the fast-phase of the microsecond fluorescence yield rise oscillated as a function of flash number with period 4 and with maxima on flashes 1 and 5, and minima on flashes 3 and 7. The damped oscillations were apparent over the range between 6 and 8, although the absolute amplitude of the fast phase was diminished at the lower end of the range. At pH 4, there was no fast phase in the rise and, at pH 9, an enhanced fast-phase occurred only for the first flash. (2) The decay of microsecond delayed fluorescence was described by the sum of exponentials with half-times of 10--15 mus and 40--50 mus. Over the pH range 6- less than 8, the extrapolated initial amplitude and the proportion of the change due to the faster component showed oscillations which were opposite in phase to those observed for the prompt fluorescence yield rise; the slower component showed weaker oscillations of the same phase. At pH 4, there were no oscillations and the slow phase predominated. At pH 9, the delayed fluorescence intensity was diminished on the first flash, and high on subsequent flashes. (3) The results are interpreted in terms of a model in which protons are released during all transitions of the S-states with the exception of S1 leads to S2, and in which ther are two sites of inhibition on the donor side of the photo-system at extreme pH values. At pH 4, electron donation to P+ occurs with a half-time approx. 135 mus, either by a back reaction from Q-, or from D; electron transport is interrupted between Z1 and P. At pH 9, electron transport is inhibited between Z1 and Z2; rapid re-reduction of P+ by Z1 occurs after 1 flash, and on subsequent flashes electrons from D, an alternative donor reduce P+. The location of the positive charge on states S2 and S3 is discussed.     

Effects of the lack of phosphatidylglycerol on the donor side of photosystem II

Our previous studies with the pgsA mutant of the cyanobacterium Synechocystis sp. PCC6803 (hereafter termed pgsA mutant), which is defective for the biosynthesis of phosphatidylglycerol (PG), revealed an important role for PG in the electron acceptor side of photosystem II (PSII), especially in the electron transport between plastoquinones Q(A) and Q(B). This study now shows that PG also plays an important role in the electron donor side of PSII, namely, the oxygen-evolving system. Analyses of purified PSII complexes indicated that PSII from PG-depleted pgsA mutant cells sustained only approximately 50% of the oxygen-evolving activity compared to wild-type cells. Dissociation of the extrinsic proteins PsbO, PsbV, and PsbU, which are required for stabilization of the manganese (Mn) cluster, followed by the release of a Mn atom, was observed in PSII of the PG-depleted mutant cells. The released PsbO rebound to PSII when PG was added back to the PG-depleted mutant cells, even when de novo protein synthesis was inhibited. Changes in photosynthetic activity of the PG-depleted pgsA mutant cells induced by heat treatment or dark incubation resembled those of DeltapsbO, DeltapsbV, and DeltapsbU mutant cells. These results suggest that PG plays an important role in binding extrinsic proteins required for sustaining a functional Mn cluster on the donor side of PSII.     

Vectorial charge transfer reactions on the donor side of manganese-depleted and reconstituted photosystem 2 core complexes

The light-induced functioning of photosystem 2 (PS 2) is directly linked to the translocation of both electrons and protons across the membrane, which results in the formation of transmembrane electric potential difference (ΔΨ). Generation of ΔΨ due to S-state transitions of the water oxidation complex was demonstrated for the first time in Mn-depleted and reconstituted PS 2 core complexes incorporated into liposomes. The kinetics and relative amplitudes of the electrogenic reactions in dark-adapted samples during S₁→S₂, S₂→S₃, and S₄→S₀ transitions in response to the first, second and third laser flashes were comparable to those obtained in the intact PS 2 core particles. These results expand current understanding of the nature and mechanisms of electrogenic (vectorial) reactions due to a charge transfer on the donor side of PS 2.     

EPR studies on the photosystem II donor side in salt-washed and reconstituted inside-out thylakoids

EPR measurements on inside-out thylakoids revealed that salt-washing, known to inhibit oxygen evolution and release a 23 and a 16 kDa protein, induced a Signal II_f and decreased the EPR signal from state S₂. Readdition of the released 23 kDa protein restored the oxygen evolution and decreased the Signal II_f, but did not relieve the decrease in the state S₂ signal. It is suggested that salt-washing inhibits the electron transfer from the oxygen-evolving site to Z, the physiological donor to P680. In inhibited photosystem II units lacking Signal II_f, Z⁺ is rapidly reduced, possibly by a modified S-cycle unable to evolve oxygen.     

Efficiency of hydrogen photoproduction by photosystem I-enriched subchloroplast vesicles combined with Proteus mirabilis cells. Effects of some exogenous electron donors

High rates of hydrogen photoproduction are obtained when glutaraldehyde-fixed Photosystem I-enriched vesicles (Photosystem II-depleted) are added to hydrogenase-containing cells of Proteus mirabilis in the presence of the mediator methylviologen and a suitable electron donating system. This donor system includes ascorbate, dithioerythritol (DTE) and the mediator tetramethylphenylene-diamine (TMPD) and reduces the photosynthetic electron transfer chain at the level of plastocyanin. Both DTE and ascorbate are required for hydrogen photoproduction, DTE being the ultimate electron donor and ascorbate only having a catalytic function. Whereas the aerobic photoreduction of methylviologen is similar in the presence of DTE, ascorbate or both, under anaerobic conditions only combination of both compounds results in a high and stable amount of reduced methylviologen that can be utilized by the hydrogenase. It is concluded that oxidation reactions of reduced methylviologen, competing with the hydrogenase, rather than methylviologen photoreduction, limit hydrogen photoproduction in the presence of either DTE or ascorbate. These oxidation reactions are suggested to involve back reactions to the oxidized form(s) of ascorbate and DTE but backflow to the photosynthetic electron transfer chain (i.e. cyclic electron transfer) can not be excluded.Abbreviations Tes N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid - DTE 1,4-dithioerythritol - TMPD, N,N,N N-tetramethyl-p-phenylenediamine - DCMU 3-(3, 4-dichlorophenyl)-1, 1,-dimethylureum - EDAC 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide - DNP-INT 2-iodo-6-isopropyl-3-methyl-2, 4, 4-trinitrodiphenyl ether - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone - PS photosystem - Chl chlorophyll     

Interaction of 2-n-heptyl-4-hydroxyquinoline-N-oxide with photosystem II in chloroplasts and subchloroplast particles

The effects of 2-n-heptyl-4-hydroxyquinoline-N-oxide on electron transport in thylakoids and oxygen-evolving photosystem II particles has been examined. Kinetic fluorescence studies reveal that the site of inhibition for alkyl derivatives of hydroxyquinoline-N-oxide (I50 approximately equal to 2 microM) is located between Q and plastoquinone. Studies with thylakoids isolated from atrazine-resistant pigweed plants indicate that the modification in the Q/B membrane complex that confers increased resistance to inhibition by atrazine also results in decreased sensitivity to inhibition by 2-n-heptyl-4-hydroxyquinoline-N-oxide (resistant/ sensitive ratio = 11). From the results of tetramethylphenylenediamine by-pass experiments, determinations of inhibitor sensitivity in trypsin-treated thylakoids and competitive displacement experiments made with [14C]metribuzin in thylakoids and photosystem II particles, it is suggested that 2-n-heptyl-4-hydroxyquinoline-N-oxide binds in a region of the Q/B complex that is distinct from the 3-(3,4-dichloro)-1,1-dimethyl urea and atrazine binding sites.     

On the inhibitory action of cadmium on the donor side of photosystem II in isolated chloroplasts.

The inhibitory effect of the Cd2+ in the electron transport of the isolated chloroplasts has been observed by measuring the oxygen uptake from the solution and the fluorescence induction. Cd2+ is found to be an inhibitor on the donor side of Photosystem II and its action site, as determined by experiments using hydroxylamine and exogenous Mn, is supposed to be on the water-splitting enzyme itself. Moreover, physicochemical and physiological studies indicate that only the ionic form of Cd is acting at the level of the manganoprotein. It is not possible, from this work, to define precisely in which form Cd is taken up through the thylakoid membranes.     

Structure of donor side components in photosystem II predicted by computer modelling.

Thirty-one and eleven sequences for the photosystem II reaction centre proteins D1 and D2 respectively, were compared to identify conserved single amino acid residues and regions in the sequences. Both proteins are highly conserved. One important difference is that the lumenal parts of the D1 protein are more conserved than the corresponding parts in the D2 protein. The three-dimensional structures around the electron donors tyrosineZ and tyrosineD on the oxidizing side of photosystem II have been predicted by computer modelling using the photosynthetic reaction centre from purple bacteria as a framework. In the model the tyrosines occupy two cavities close to the lumenal surface of the membrane. They are symmetrically arranged around the primary donor P680 and the distances between the centre of the tyrosines and the closest Mg ion in P680 are around 14 A. Both tyrosineZ and tyrosineD are suggested to form a hydrogen bond with histidine 190 from the loop connecting helices C and D in the D1 and D2 proteins, respectively. The Mn cluster in the oxygen evolving complex has been localized by using known and estimated distances from the tyrosine radicals. It is suggested that a binding region for the Mn cluster is constituted by the lumenal ends of helices A and B and the loop connecting them in the D1 protein. This part of the D1 protein contains a large number of strictly conserved carboxylic acid residues and histidines which could participate in the Mn binding. There is little probability that the Mn cluster binds on the lumenal surface of the D2 protein.     

Regulation of excitation energy distribution in subchloroplast particles: photosystem I

Mono- and divalent cations were found to increase the transfer of excitation energy within Photosystem I from the light-harvesting chlorophyll a molecules to P700. The P700-chlorophyll a protein of Shiozawa et al. (J. A. Shiozawa, R. S. Alberte, and J. P. Thornber, 1974, Arch. Biochem. Biophys.165, 388–397) was used for these studies. Cations stimulated the quantum yields for electron transport when the light-harvesting chlorophyll a molecules were irradiated. They also decreased chlorophyll a fluorescence. Half-maximal effects were observed at 0.5–0.6 mm for divalent cations and at 5–6 mm for monovalent cations. Triton X-100, 0.02%, also increased energy transfer. The increases in energy transfer are due to an intramolecular conformational change in the protein. A structural change is involved, since there is a correlation between the cation-induced changes in energy transfer and increases in 90 ° light scattering. However, there was no change in the molecular weight upon the addition of MgCl₂. The molecular weight, as determined by gel filtration, was 105,000 in the presence of 0.05% Triton X-100. On the other hand, circular dichroism measurements showed an increase in the α-helical content from 51 to 63% when 5 mm MgCl₂ was added. Changes in the absorption spectra were also observed. We believe that the cation regulation of Photosystem I activity provides a fine-tuning mechanism for the regulation of energy transfer.     

Localization of polylysine inhibition in a photosystem I subchloroplast particle.

Polylysine was found to increase the efficiency of electron donation from plastocyanin to P700⁺ in highly resolved Photosystem I subchloroplast particles. This increase in efficiency is due to a decrease in the Km for plastocyanin in the presence of polylysine and is similar to results obtained with divalent cations. Cytochrome f photooxidation is observed in the presence of plastocyanin and divalent cations but not in the presence of plastocyanin and polylysine. The results indicate that the binding of polylysine to plastocyanin prevents the reduction of plastocyanin by cytochrome f.     

Two sites of primary degradation of the D1-protein induced by acceptor or donor side photo-inhibition in photosystem II core complexes.

Depending on experimental conditions we have found that photo-inhibitory treatment of photosystem II (PSII) core complexes, isolated from wheat, can generate two fragments of about 23-24 kDa that contain either the C-terminal or N-terminal regions of the D1-protein. A 24 kDa C-terminal fragment appears when the water splitting reaction is not functional and an electron acceptor is present. This 'donor'-side inhibition also generates an N-terminal fragment of about 10 kDa and is suggested to be due to the cleavage of a peptide bond in the region connecting transmembrane segments I and II of the D1-protein. In contrast, an N-terminal 23 kDa D1-protein fragment is detected when the water splitting reactions of the isolated complex are active, and occurs in the absence of an added electron acceptor. This 'acceptor'-side photo-inhibition also generates a C-terminal fragment of about 10 kDa.     

Probing the turnover efficiency of photosystem II membrane fragments with different electron acceptors

In this study we employ isotope ratio membrane-inlet mass spectrometry to probe the turnover efficiency of photosystem II (PSII) membrane fragments isolated from spinach at flash frequencies between 1Hz and 50Hz in the presence of the commonly used exogenous electron acceptors potassium ferricyanide(III) (FeCy), 2,5-dichloro-p-benzoquinone (DCBQ), and 2-phenyl-p-benzoquinone (PPBQ). The data obtained clearly indicate that among the tested acceptors PPBQ is the best at high flash frequencies. If present at high enough concentration, the PSII turnover efficiency is unaffected by flash frequency of up to 30Hz, and at 40Hz and 50Hz only a slight decrease by about 5-7% is observed. In contrast, drastic reductions of the O(2) yields by about 40% and 65% were found at 50Hz for DCBQ and FeCy, respectively. Comparison with literature data reveals that PPBQ accepts electrons from Q(A)(-) in PSII membrane fragments with similar efficiency as plastoquinone in intact cells. Our data also confirm that at high flashing rates O(2) evolution is limited by the reactions on the electron-acceptor side of PSII. The relevance of these data to the evolutionary development of the water-splitting complex in PSII and with regard to the potential of artificial water-splitting catalysts is discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Studies on the protolytic reactions coupled with water cleavage in photosystem II membrane fragments from spinach

The protolytic reactions of PSII membrane fragments were analyzed by measurements of absorption changes of the water soluble indicator dye bromocresol purple induced by a train of 10 s flashes in dark-adapted samples. It was found that: a) in the first flash a rapid H⁺-release takes place followed by a slower H⁺-uptake. The deprotonation is insensitive to DCMU but is completely eliminated by linolenic acid treatment of the samples; b) the extent of the H⁺-uptake in the first flash depends on the redox potential of the suspension. In this time domain no H⁺-uptake is observed in the subsequent flashes; c) the extent of the H⁺-release as a function of the flash number in the sequence exhibits a characteristic oscillation pattern. Multiphasic release kinetics are observed. The oscillation pattern can be satisfactorily described by a 1, 0, 1, 2 stoichiometry for the redox transitions S_i S_i+1 (i=0, 1, 2, 3) in the water oxidizing enzyme system Y. The H⁺-uptake after the first flash is assumed to be a consequence of the very fast reduction of oxidized Q₄₀₀(Fe³⁺) formed due to dark incubation with K₃[Fe(CN)₆]. The possible participation of component Z in the deprotonation reactions at the PSII donor side is discussed.Abbreviations A protonizable group at the PSII acceptor side - BCP Bromocresol Purple - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - FWHM Full Width at Half Maximum - Q_A, Q_B primary and secondary plastoquinone at PSII acceptor side - Q₄₀₀ redox group at PSII-acceptor side (high spin Fe²⁺) - P680 Photoactive chlorophyll of PSII reaction center - S_i redox states of the catalytic site of water oxidation - Z redox component connecting the catalytic site of water oxidation with the reaction center