Over-reduced states of the Mn-cluster in cucumber leaves induced by dark-chilling treatment

Oxygen evolution is inhibited when leaves of chilling-sensitive plants like cucumber are treated at 0 degrees C in the dark. The activity is restored by moderate illumination at room temperature. We examined the changes in the redox state of the Mn-cluster in cucumber leaves in the processes of dark-chilling inhibition and subsequent light-induced reactivation by means of thermoluminescence (TL). A TL B-band arising from S(2)Q(B)(-) charge recombination in PSII was observed upon single-flash illumination of untreated leaves, whereas four flashes were required to yield the B-band after dark-chilling treatment for 24 h. This three-step delay indicates that over-reduced states of the Mn-cluster such as the S(-2) state were formed during the treatment. Fitting analysis of the flash-number dependence of the TL intensities showed that the Mn-cluster was more reduced with a longer period of the treatment and that S(-3) was the lowest S-state detectable in the dark-chilled leaves. Measurements of the Mn content by atomic absorption spectroscopy showed that Mn atoms were gradually released from PSII during the dark-chilling treatment but re-bound to PSII by illumination at 30 degrees C. Thus, dark-chilling inhibition of oxygen evolution can be ascribed to the disintegration of the Mn-cluster due to its over-reduction. The observation of the S(-3) state in the present in vivo system strongly suggests that S(-3), which has been observed only by addition of exogenous reductants into in vitro preparations, is indeed a redox intermediate of the Mn-cluster in the processes of its disintegration and photoactivation.     

Interaction of N,N,N′,N′-tetramethyl-p-phenylenediamine with photosystem II as revealed by thermoluminescence: Reduction of the higher oxidation states of the Mn cluster and displacement of plastoquinone from the QB niche

The flash-induced thermoluminescence (TL) technique was used to investigate the action of N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) on charge recombination in photosystem II (PSII). Addition of low concentrations (μM range) of TMPD to thylakoid samples strongly decreased the yield of TL emanating from S₂Q_B⁻ and S₃Q_B⁻ (B-band), S₂Q_A⁻ (Q-band), and Y_D⁺Q_A⁻ (C-band) charge pairs. Further, the temperature-dependent decline in the amplitude of chlorophyll fluorescence after a flash of white light was strongly retarded by TMPD when measured in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Though the period-four oscillation of the B-band emission was conserved in samples treated with TMPD, the flash-dependent yields (Y_n) were strongly declined. This coincided with an upshift in the maximum yield of the B-band in the period-four oscillation to the next flash. The above characteristics were similar to the action of the ADRY agent, carbonylcyanide m-chlorophenylhydrazone (CCCP). Simulation of the B-band oscillation pattern using the integrated Joliot-Kok model of the S-state transitions and binary oscillations of Q_B confirmed that TMPD decreased the initial population of PSII centers with an oxidized plastoquinone molecule in the Q_B niche. It was deduced that the action of TMPD was similar to CCCP, TMPD being able to compete with plastoquinone for binding at the Q_B-site and to reduce the higher S-states of the Mn cluster.     

Afterglow thermoluminescence band as a possible early indicator of changes in the photosynthetic electron transport in leaves

The afterglow (AG) band of thermoluminescence (TL) has been investigated in leaves of Arabidopsis thaliana. Excitation of dark-adapted leaves with two saturating single turn-over flashes induced the appearance of a complex TL glow curve that could be well simulated by three components: the two components, B₁ and B₂, of the usually called B-band, peaking at 18 and 26 °C, respectively, and a band with t_max at 41 °C, which we attributed to an AG emission. Illumination of dark-adapted leaves with 720 nm monochromatic and FR lights generated the emission of a sharp single band peaking also around at 41 °C, that it is usually assigned to an AG emission band. Dark-incubation of whole plants increased the intensity of AG-band in TL curves induced by two flashes and, in parallel, decreased B-bands. Selective illumination of leaves with light mostly absorbed by PS II (650 nm light) completely abolished the AG-band induced by two flashes, B-band being the only TL band observed. The single AG-band induced by 720 nm light was abolished if leaves were also illuminated with 650 nm light. On the other hand, AG-band could be restored if 650 nm illuminated leaves were afterwards illuminated with 720 nm light. The changes in the intensity of B and AG bands induced by selective illuminations seem to be related to alterations in the redox state of Q_B and plastoquinone pool.     

Function of plastoquinone in heat stress reactions of plants

The effect of high temperature treatment (40 °C, 3 h, illumination at 100 μmol m^− 2 s^− 1) on the photosynthetic electron flow in barley seedlings of different age was investigated. Thermoinduced inhibition of the liner electron flow due to partial impairment of the water oxidizing complex (WOC) and the increase in the extent of Q_A⁻ reoxidation by Tyr_z^ox in thylakoids isolated from 4-day-old leaves was shown by measurements of oxygen evolution using benzoquinone or potassium ferricyanide as electron acceptors, as well as by following Q_A⁻ reoxidation kinetics in the absence and presence of exogenous electron acceptors, DCBQ and DMBQ. Using HPLC analysis, an increase in the oxidation of the photoactive plastoquinone pool in young leaves under heating was shown. In older, 11-day-old leaves, heat treatment limited both photosynthetic electron flow and oxygen evolution. The same effects of heat shock on oxygen evolution caused an inhibition of electron flow on the donor side of PSII only. However, a rise in the proportion of PSII with Q_A⁻ reoxidized through recombination with the S₂/S₃ state of the WOC was observed. The addition of exogenous electron acceptors (DCBQ and DMBQ) and a donor (DPC) showed that the thermoinduced decrease in the electron transport rate was caused by an impediment of electron flow from Q_A⁻ to acceptor pool. The decrease in size of the photoactive PQ-pool and a change in the proportions of oxidized and reduced PQ in older leaves under heat treatment were shown. It was suggested that a thermoinduced change of the redox state of the PQ-pool and a redistribution of plastoquinone molecules between photoactive and non-photoactive pools are the mechanisms which reflect and regulate the response of the photosynthetic apparatus under heat stress conditions.     

Light-dependent reversal of dark-chilling induced changes in chloroplast structure and arrangement of chlorophyll-protein complexes in bean thylakoid membranes

Changes in chloroplast structure and rearrangement of chlorophyll-protein (CP) complexes were investigated in detached leaves of bean (Phaseolus vulgaris L. cv. Eureka), a chilling-sensitive plant, during 5-day dark-chilling at 1 °C and subsequent 3-h photoactivation under white light (200 μmol photons m⁻² s⁻¹) at 22 °C. Although, no change in chlorophyll (Chl) content and Chl a/b ratio in all samples was observed, overall fluorescence intensity of fluorescence emission and excitation spectra of thylakoid membranes isolated from dark-chilled leaves decreased to about 50%, and remained after photoactivation at 70% of that of the control sample. Concomitantly, the ratio between fluorescence intensities of PSI and PSII (F736/F681) at 120 K increased 1.5-fold upon chilling, and was fully reversed after photoactivation. Moreover, chilling stress seems to induce a decrease of the relative contribution of LHCII fluorescence to the thylakoid emission spectra at 120 K, and an increase of that from LHCI and PSI, correlated with a decrease of stability of LHCI-PSI and LHCII trimers, shown by mild-denaturing electrophoresis. These effects were reversed to a large extent after photoactivation, with the exception of LHCII, which remained partly in the aggregated form. In view of these data, it is likely that dark-chilling stress induces partial disassembly of CP complexes, not completely restorable upon photoactivation. These data are further supported by confocal laser scanning fluorescence microscopy, which showed that regular grana arrangement observed in chloroplasts isolated from control leaves was destroyed by dark-chilling stress, and was partially reconstructed after photoactivation. In line with this, Chl a fluorescence spectra of leaf discs demonstrated that dark-chilling caused a decrease of the quantum yield PSII photochemistry (F_v/F_m) by almost 40% in 5 days. Complete restoration of the photochemical activity of PSII required 9 h post-chilling photoactivation, while only 3 h were needed to reconstruct thylakoid membrane organization and chloroplast structure. The latter demonstrated that the long-term dark-chilled bean leaves started to suffer from photoinhibition after transfer to moderate irradiance and temperature conditions, delaying the recovery of PSII photochemistry, independently of photo-induced reconstruction of PSII complexes.     

Spermine and spermidine inhibition of photosystem II: Disassembly of the oxygen evolving complex and consequent perturbation in electron donation from TyrZ to P680 and the quinone acceptors QA to QB

Polyamines are implicated in plant growth and stress response. However, the polyamines spermine and spermidine were shown to elicit strong inhibitory effects in photosystem II (PSII) submembrane fractions. We have studied the mechanism of this inhibitory action in detail. The inhibition of electron transport in PSII submembrane fractions treated with millimolar concentrations of spermine or spermidine led to the decline of plastoquinone reduction, which was reversed by the artificial electron donor diphenylcarbazide. The above inhibition was due to the loss of the extrinsic polypeptides associated with the oxygen evolving complex. Thermoluminescence measurements revealed that charge recombination between the quinone acceptors of PSII, Q_A and Q_B, and the S₂ state of the Mn-cluster was abolished. Also, the dark decay of chlorophyll fluorescence after a single turn-over white flash was greatly retarded indicating a slower rate of Q_A⁻ reoxidation.     

Heterogeneity and Photoinhibition of Photosystem II Studied with Thermoluminescence

Thermoluminescence (TL) signals were recorded from grana stacks, margins, and stroma lamellae from fractionated, dark-adapted thylakoid membranes of spinach (Spinacia oleracea L.) in the absence and in the presence of 2,6-dichlorphenylindophenol (DCMU). In the absence of DCMU, the TL signal from grana fractions consisted of a homogenous B-band, which originates from recombination of the semi-quinone Q_B⁻ with the S₂ state of the water-splitting complex and reflects active photosystem II (PSII). In the presence of DCMU, the B-band was replaced by the Q-band, which originates from an S₂Q_A⁻ recombination. Margin fractions mainly showed two TL-bands, the B- and C-bands, at approximately 50°C in the absence of DCMU, and Q- and C-bands in the presence of DCMU. The C-band is ascribed to a Tyr_D⁺-Q_A⁻ recombination. In the absence of DCMU, the fractions of stromal lamellae mainly gave rise to a TL emission at 42°C. The intensity of this band was independent of the number of excitation flashes and was shifted to higher temperatures (52°C) after the addition of DCMU. Based on these observations, this band was considered to be a C-band. After photoinhibitory light treatment of uncoupled thylakoid membranes, the TL intensities of the B- and Q-bands decreased, whereas the intensity at 45°C (C-band) slightly increased. It is proposed that the 42 to 52°C band that was observed in marginal and stromal lamellae and in photoinhibited thylakoid membranes reflects inactive PSII centers that are assumed to be equivalent to inactive PSII Q_B-nonreducing centers.     

PSII manganese cluster: Protonation of W2, O5, O4 and His337 in the S1 state explored by combined quantum chemical and electrostatic energy computations

Photosystem II (PSII) is a membrane-bound protein complex that oxidizes water to produce energized protons, which are used to built up a proton gradient across the thylakoidal membrane in the leafs of plants. This light-driven reaction is catalyzed by withdrawing electrons from the Mn₄CaO₅-cluster (Mn-cluster) in four discrete oxidation steps [S₁ − (S₄ / S₀)] characterized in the Kok-cycle. In order to understand in detail the proton release events and the subsequent translocation of such energized protons, the protonation pattern of the Mn-cluster need to be elucidated. The new high-resolution PSII crystal structure from Umena, Kawakami, Shen, and Kamiya is an excellent basis to make progress in solving this problem. Following our previous work on oxidation and protonation states of the Mn-cluster, in this work, quantum chemical/electrostatic calculations were performed in order to estimate the pKa of different protons of relevant groups and atoms of the Mn-cluster such as W2, O4, O5 and His337. In broad agreement with previous experimental and theoretical work, our data suggest that W2 and His337 are likely to be in hydroxyl and neutral form, respectively, O5 and O4 to be unprotonated. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Substitution of chloride by bromide modifies the low-temperature tyrosine Z oxidation in active photosystem II

Chloride is an essential cofactor for photosynthetic water oxidation. However, its location and functional roles in active photosystem II are still a matter of debate. We have investigated this issue by studying the effects of Cl⁻ replacement by Br⁻ in active PSII. In Br⁻ substituted samples, Cl⁻ is effectively replaced by Br⁻ in the presence of 1.2 M NaBr under room light with protection of anaerobic atmosphere followed by dialysis. The following results have been obtained. i) The oxygen-evolving activities of the Br⁻-PSII samples are significantly lower than that of the Cl⁻-PSII samples; ii) The same S₂ multiline EPR signals are observed in both Br⁻ and Cl⁻-PSII samples; iii) The amplitudes of the visible light induced S₁Tyr_Z^• and S₂Tyr_Z^• EPR signals are significantly decreased after Br⁻ substitution; the S₁Tyr_Z^• EPR signal is up-shifted about 8 G, whereas the S₂Tyr_Z^• signal is down-shifted about 12 G after Br⁻ substitution. These results imply that the redox properties of Tyr_Z and spin interactions between Tyr_Z^• and Mn-cluster could be significantly modified due to Br⁻ substitution. It is suggested that Cl⁻/Br⁻ probably coordinates to the Ca²⁺ ion of the Mn-cluster in active photosystem II.     

Reprint of PSII Manganese Cluster: Protonation of W2, O5, O4 and His337 in the S1 state explored by combined quantum chemical and electrostatic energy computations

Photosystem II (PSII) is a membrane-bound protein complex that oxidizes water to produce energized protons, which are used to built up a proton gradient across the thylakoidal membrane in the leafs of plants. This light-driven reaction is catalyzed by withdrawing electrons from the Mn₄CaO₅-cluster (Mn-cluster) in four discrete oxidation steps [S₁ − (S₄ / S₀)] characterized in the Kok-cycle. In order to understand in detail the proton release events and the subsequent translocation of such energized protons, the protonation pattern of the Mn-cluster need to be elucidated. The new high-resolution PSII crystal structure from Umena, Kawakami, Shen, and Kamiya is an excellent basis to make progress in solving this problem. Following our previous work on oxidation and protonation states of the Mn-cluster, in this work, quantum chemical/electrostatic calculations were performed in order to estimate the pKa of different protons of relevant groups and atoms of the Mn-cluster such as W2, O4, O5 and His337. In broad agreement with previous experimental and theoretical work, our data suggest that W2 and His337 are likely to be in hydroxyl and neutral form, respectively, O5 and O4 to be unprotonated. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Towards a spin coupling model for the Mn4 cluster in Photosystem II

The X-band EPR spectra of the IR sensitive untreated PSII and of MeOH- and NH₃-treated PSII from spinach in the S₂-state are simulated with collinear and rhombic g- and Mn-hyperfine tensors. The obtained principal values indicate a 1Mn(III)3Mn(IV) composition for the Mn₄ cluster. The four isotropic components of the Mn-hyperfine tensors are found in good agreement with the previously published values determined from EPR and ⁵⁵Mn-ENDOR data. Assuming intrinsic isotropic components of the Mn-hyperfine interactions identical to those of the Mn-catalase, spin density values are calculated. A Y-shape 4J-coupling scheme is explored to reproduce the spin densities for the untreated PSII. All the required criteria such as a S=1/2 ground state with a low lying excited spin state (30 cm⁻¹) and an easy conversion to a S=5/2 system responsible for the g=4.1 EPR signal are shown to be satisfied with four antiferromagnetic interactions lying between −290 and −130 cm⁻¹.     

D1-S169A substitution of photosystem II reveals a novel S2-state structure

In photosystem II (PSII), photosynthetic water oxidation occurs at the O₂-evolving complex (OEC), a tetramanganese-calcium cluster that cycles through light-induced redox intermediates (S₀–S₄) to produce oxygen from two substrate water molecules. The OEC is surrounded by a hydrogen-bonded network of amino-acid residues that plays a crucial role in proton transfer and substrate water delivery. Previously, we found that D1-S169 was crucial for water oxidation and its mutation to alanine perturbed the hydrogen-bonding network. In this study, we demonstrate that the activation energy for the S₂ to S₁ transition of D1-S169A PSII is higher than wild-type PSII with a ~1.7–2.7× slower rate of charge recombination with Q_A⁻ relative to wild-type PSII. Arrhenius analysis of the decay kinetics shows an E_a of 5.87 ± 1.15 kcal mol⁻¹ for decay back to the S₁ state, compared to 0.80 ± 0.13 kcal mol⁻¹ for the wild-type S₂ state. In addition, we find that ammonia does not affect the S₂-state EPR signal, indicating that ammonia does not bind to the Mn cluster in D1-S169A PSII. Finally, a QM/MM analysis indicates that an additional water molecule binds to the Mn4 ion in place of an oxo ligand O5 in the S₂ state of D1-S169A PSII. The altered S₂ state of D1-S169A PSII provides insight into the S₂➔S₃ state transition.     

Energetics of primary and secondary electron transfer in Photosystem II membrane particles of spinach revisited on basis of recombination-fluorescence measurements

Photon absorption by one of the roughly 200 chlorophylls of the plant Photosystem II (PSII) results in formation of an equilibrated excited state (Chl200*) and is followed by chlorophyll oxidation (formation of P680+) coupled to reduction of a specific pheophytin (Phe), then electron transfer from Phe− to a firmly bound quinone (QA), and subsequently reduction of P680+ by a redox-active tyrosine residue denoted as Z. The involved free-energy differences (ΔG) and redox potentials are of prime interest. Oxygen-evolving PSII membrane particles of spinach were studied at 5 °C. By analyzing the delayed and prompt Chl fluorescence, we determined the equilibrium constant and thus free-energy difference between Chl200* and the [Z+,QA−] radical pair to be −0.43 ± 0.025 eV, at 10 μs after the photon absorption event for PSII in its S₃-state. On basis of this value and previously published results, the free-energy difference between P680* and [P680+,QA−] is calculated to be −0.50 ± 0.04 eV; the free-energy loss associated with electron transfer from Phe to QA is found to be 0.34 ± 0.04 eV. The given uncertainty ranges do not represent a standard deviation or likely error, but an estimate of the maximal error. Assuming a QA−/QA redox potential of −0.08 V [Krieger et al., 1995, Biochim. Biophys. Acta 1229, 193], the following redox-potential estimates are obtained: +1.25 V for P680/P680+; +1.21 V for Z/Z+ (at 10 μs); −0.42 V for Phe−/Phe; −0.58 V for P680*/P680+.     

Impairment of the photosynthetic apparatus by oxidative stress induced by photosensitization reaction of protoporphyrin IX

Treatment with the herbicide acifluorfen-sodium (AF-Na), an inhibitor of protoporphyrinogen oxidase, caused an accumulation of protoporphyrin IX (Proto IX) , light-induced necrotic spots on the cucumber cotyledon within 12-24 h, and photobleaching after 48-72 h of light exposure. Proto IX-sensitized and singlet oxygen (¹O₂)-mediated oxidative stress caused by AF-Na treatment impaired photosystem I (PSI), photosystem II (PSII) and whole chain electron transport reactions. As compared to controls, the F_v/F_m (variable to maximal chlorophyll a fluorescence) ratio of treated samples was reduced. The PSII electron donor NH₂OH failed to restore the F_v/F_m ratio suggesting that the reduction of F_v/F_m reflects the loss of reaction center functions. This explanation is further supported by the practically near-similar loss of PSI and PSII activities. As revealed from the light saturation curve (rate of oxygen evolution as a function of light intensity), the reduction of PSII activity was both due to the reduction in the quantum yield at limiting light intensities and impairment of light-saturated electron transport. In treated cotyledons both the Q (due to recombination of Q_A⁻ with S₂) and B (due to recombination of Q_B⁻ with S₂/S₃) band of thermoluminescence decreased by 50% suggesting a loss of active PSII reaction centers. In both the control and treated samples, the thermoluminescence yield of B band exhibited a periodicity of 4 suggesting normal functioning of the S states in centers that were still active. The low temperature (77 K) fluorescence emission spectra revealed that the F₆₉₅ band (that originates in CP-47) increased probably due to reduced energy transfer from the CP47 to the reaction center. These demonstrated an overall damage to the PSI and PSII reaction centers by ¹O₂ produced in response to photosensitization reaction of protoporphyrin IX in AF-Na-treated cucumber seedlings.     

A thermoluminescence study of Photosystem II back electron transfer reactions in rice leaves – effects of salt stress

The recombination reactions of Photosystem II have been investigated in vivo in rice leaves by using the thermoluminescence (TL) emission technique. Excitation of dark-adapted leaf segments at 0 °C with different number of single turn-over flashes induced the appearance of complex TL glow curves. The mathematical analysis of these curves showed the existence of four TL components: B1-band (temperature maximum, t_max, at 24 °C, originating from S₃Q_B⁻ recombination), B2-band (t_max at 35 °C, from S₂Q_B⁻), AG-band (t_max at 46 °C) and C-band (t_max at 55 °C, from Tyr_D⁺Q_A⁻). Their contributions to the total TL signal were different depending on the number of flashes given. AG-band seems to reflect a special electron transfer from some unknown stroma donor to PS II. Q-band (t_max at 19 °C), originating from S₂Q_A⁻ recombination, was recorded after flashing samples incubated in the presence of DCMU. The recombination halftimes (t_1/2) at 20 °C of S₂Q_A⁻, S₃Q_B⁻, S₂Q_B⁻ and Tyr_D⁺Q_A⁻ were, respectively, 0.8 s, 48 s, 74 s and about 1 h. A sharp AG-band (t_max at 50 °C and t_1/2 of 210 s) could be also observed after illumination of leaves with far-red light and after a dark incubation period of whole plants. Incubation of leaf segments with 0.5 M NaCl abolished the inductions of AG-band by darkness and far-red illumination, significantly decreased Q-band intensity, whereas induced a strong increase in C-band intensity. The possible inhibition of S₂/S₃ formation and quinone oxidation by saline stress are discussed.     

Probing tyrosine Z oxidation in photosystem II core complex isolated from spinach by EPR at liquid helium temperatures

Tyrosine Z (Tyr_Z) oxidation observed at liquid helium temperatures provides new insights into the structure and function of Tyr_Z in active Photosystem II (PSII). However, it has not been reported in PSII core complex from higher plants. Here, we report Tyr_Z oxidation in the S₁ and S₂ states in PSII core complex from spinach for the first time. Moreover, we identified a 500 G-wide symmetric EPR signal (peak position g = 2.18, trough position g = 1.85) together with the g = 2.03 signal induced by visible light at 10 K in the S₁ state in the PSII core complex. These two signals decay with a similar rate in the dark and both disappear in the presence of 6% methanol. We tentatively assign this new feature to the hyperfine structure of the S₁Tyr_Z ^• EPR signal. Furthermore, EPR signals of the S₂ state of the Mn-cluster, the oxidation of the non-heme iron, and the S₁Tyr_Z ^• in PSII core complexes and PSII-enriched membranes from spinach are compared, which clearly indicate that both the donor and acceptor sides of the reaction center are undisturbed after the removal of LHCII. These results suggest that the new spinach PSII core complex is suitable for the electron transfer study of PSII at cryogenic temperatures.     

Compared thermoluminescence characteristics of pea thylakoids studied in vitro and in situ (in leaves). The effect of photoinhibitory treatments

Characteristics of thermoluminescence (TL) glow curves were studied in thylakoids (isolated from pea leaves) or in intact pea leaves after an exposure to very high light for 2 min in the TL device. The inhibition of photosynthesis was detected as decreases of oxygen evolution rates and/or of variable fluorescence.In thylakoids exposed to high light, then dark adapted for 5 min, a flash regime induced TL glow curves which can be interpreted as corresponding to special B bands since: 1) they can be fitted by a single B band (leaving a residual band at –5°C) with a lower activation energy and a shift of the peak maximum by –5 to –6°C and, 2) the pattern of oscillation of their amplitudes was normal with a period of 4 and maxima on flashes 2 and 6. During a 1 h dark adaptation, no recovery of PS II activity occurred but the shift of the peak maximum was decreased to –1 to –2°C, while the activation energy of B bands increased. It is supposed that centers which remained active after the photoinhibitory treatment were subjected to reversible and probably conformational changes.Conversely, in intact leaves exposed to high light and kept only some minutes in the dark, TL bands induced by a flash regime were composite and could be deconvoluted into a special B band peaking near 30°C and a complex band with maximum at 2–5°C. In the case of charging bands by one flash, this low temperature band was largely decreased in size after a 10 min dark adaptation period; parallely, an increase of the B band type component appeared. Whatever was the flash number, bands at 2–5°C were suppressed by a short far red illumination given during the dark adaptation period and only remained a main band a 20°C; therefore, the origin of the low temperature band was tentatively ascribed to recombinations in centers blocked in state S₂Q_A ^–Q_B ^2–. In vivo, the recovery of a moderately reduced state in the PQ pool, after an illumination, would be slow and under the dependence of a poising mechanism, probably involving an electron transfer between cytosol and chloroplasts or the so-called chlororespiration process.Abbreviations E_a- activation energy - FR- far-red - MV- methylviologen - pBQ- p-benzoquinone - PQ- plastoquinone - PS II- Photosystem II - Q_A- primary quinone electron acceptor of PS II - Q_B- secondary quinone electron acceptor of PS II - TL- thermoluminescence     

An FTIR study on the structure of the oxygen-evolving Mn-cluster of Photosystem II in different spin forms of the S2 state

The S₂ state of the oxygen-evolving Mn-cluster of Photosystem II (PS II) is known to have different forms that exhibit the g =2 multiline and g = 4.1 EPR signals. These two spin forms are interconvertible at > 200 K and the relative amplitudes of the two signals are dependent on the species of cryoprotectant and alcohol contained in the medium. Also, it was recently found that the mutiline form can be converted to the g = 4.1 form by absorption of near-infrared light by the Mn-cluster itself at around 150 K [Boussac et al. (1996) Biochemistry 35: 6984–6989]. We have used light-induced Fourier transform infrared (FTIR) difference spectroscopy to study the structural difference in these two S₂ forms. FTIR difference spectra for S₂/S₁ as well as for S₂Q_A ^-/S₁Q_A measured at cryogenic temperatures using PS II membranes in the presence of various cryoprotectants, and monohydric alcohols did not show any specific differences except for intensities of amide I bands, which were larger when ethylene glycol or glycerol was present in addition to sucrose. This result was interpreted due to more flexible movement of the protein backbones upon S₂ formation with a higher cryoprotectant content. Light-induced difference spectra measured at 150 K using either blue light without near-infrared light or red plus near-infrared light also did not show any detectable difference. In addition, a different spectrum upon near-infrared illumination at 150 K of the PS II sample in which the S₂ state had been photogenerated at 200 K exhibited no meaningful signals. These results indicate that the two S₂ forms that give rise to the multiline and g = 4.1 signals have only minor differences, if any, in the structures of amino-acid ligands and polypeptide backbones. This conclusion suggests that conversion between the two spin states is caused by a spin-state transition in the Mn(III) ion rather than valence swapping within the Mn-cluster that would considerably affect the vibrations of ligands.This revised version was published online in October 2005 with corrections to the Cover Date.     

A carboxylic residue at the high-affinity, Mn-binding site participates in the binding of iron cations that block the site

The role of carboxylic residues at the high-affinity, Mn-binding site in the ligation of iron cations blocking the site [Biochemistry 41 (2000) 5854] was studied, using a method developed to extract the iron cations blocking the site. We found that specifically bound Fe(III) cations can be extracted with citrate buffer at pH 3.0. Furthermore, citrate can also prevent the photooxidation of Fe(II) cations by Y_Z. Participation of a COOH group(s) in the ligation of Fe(III) at the high-affinity site was investigated using 1-ethyl-3-[(3-dimethylamino)propyl] carbodiimide (EDC), a chemical modifier of carboxylic amino acid residues. Modification of the COOH groups inhibits the light-induced oxidation of exogenous Mn(II) cations by Mn-depleted photosystem II (PSII[−Mn]) membranes. The rate of Mn(II) oxidation saturates at ≥10 μM in PSII(−Mn) membranes and ≥500 μM in EDC-treated PSII (−Mn) samples. Intact PSII(−Mn) membranes have only one site for Mn(II) oxidation via Y_Z (dissociation constant, K_d = 0.64 μM), while EDC-treated PSII(−Mn) samples have two sites (K_d = 1.52 and 22 μM; the latter is the low-affinity site). When PSII(−Mn) membranes were incubated with Fe(II) before modifier treatment (to block the high-affinity site) and the blocking iron cations were extracted with citrate (pH 3.0) after modification, the membranes contained only one site (K_d = 2.3 μM) for exogenous Mn(II) oxidation by Y_Z radical. In this case, the rate of electron donation via Y_Z saturated at a Mn(II) concentration ≥15 μM. These results indicate that the carboxylic residue participating in Mn(II) coordination and the binding of oxidized manganese cations at the HA_Z site is protected from the action of the modifier by the iron cations blocking the HA_Z site. We concluded that the carboxylic residue (D1 Asp-170) participating in the coordination of the manganese cation at the HA_Z site (Mn4 in the tetranuclear manganese cluster [Science 303 (2004) 1831]) is also involved in the ligation of the Fe cation(s) blocking the high-affinity Mn-binding site.     

Low-barrier hydrogen bond plays key role in active photosystem II — A new model for photosynthetic water oxidation

The function and mechanism of Tyr_Z in active photosystem II (PSII) is one of the long-standing issues in the study of photosynthetic water oxidation. Based on recent investigations on active PSII and theoretical studies, a new model is proposed, in which D1-His190 acts as a bridge, to form a low-barrier hydrogen bond (LBHB) with Tyr_Z, and a coordination bond to Mn or Ca ion of the Mn-cluster. Accordingly, this new model differs from previous proposals concerning the mechanism of Tyr_Z function in two aspects. First, the LBHB plays a key role to decrease the activation energy for Tyr_Z oxidation and Tyr_Z^· reduction during photosynthetic water oxidation. Upon the oxidation of Tyr_Z, the hydrogen bond between Tyr_Z and His190 changes from a LBHB to a weak hydrogen bond, and vice versa upon Tyr_Z^· reduction. In both stages, the electron transfer and proton transfer are coupled. Second, the positive charge formed after Tyr_Z oxidation may play an important role for water oxidation. It can be delocalized on the Mn-cluster, thus helps to accelerate the proton release from substrate water on Mn-cluster. This model is well reconciled with observations of the S-state dependence of Tyr_Z oxidation and Tyr_Z^· reduction, proton release, isotopic effect and recent EPR experiments. Moreover, the difference between Tyr_Z and Tyr_D in active PSII can also be readily rationalized. The His190 binding to the Mn-cluster predicted in this model is contradictious to the recent structure data, however, it has been aware that the crystal structure of the Mn-cluster and its environment are significantly modified by X-ray due to radiation damage and are different from that in active PSII. It is suggested that the His190 may be protonated during the radiation damage, which leads to the loss of its binding to Mn-cluster and the strong hydrogen bond with Tyr_Z. This type of change arising from radiation damage has been confirmed in other enzyme systems.