The Origin of the Multiline and g = 4.1 Electron Paramagnetic Resonance Signals from the Oxygen-Evolving System of Photosystem II

Continuous illumination at 200 K of photosystem (PS) II-enriched membranes generates two electron paramagnetic resonance (EPR) signals that both are connected with the S₂ state: a multiline signal at g 2 and a single line at g = 4.1. From measurements at three different X-band frequencies and at 34 GHz, the g tensor of the multiline species was found to be isotropic with g = 1.982. It has an excited spin multiplet at ~30 cm^-1, inferred from the temperature-dependence of the linewidth. The intensity ratio of the g = 4.1 signal to the multiline signal was found to be almost constant from 5 to 23 K. Based on these findings and on spin quantitation of the two signals in samples with and without 4% ethanol, it is concluded that they arise from the ground doublets of paramagnetic species in different PS II centers. It is suggested that the two signals originate from separate PS II electron donors that are in a redox equilibrium with each other in the S₂ state and that the g = 4.1 signal arises from monomeric Mn(IV).     

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

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

Studies of the slowly exchanging chloride in Photosystem II of higher plants

³⁶Cl^- was used to study the slow exchange of chloride at a binding site associated with Photosystem II (PS II). When PS II membranes were labeled with different concentrations of ³⁶Cl^-, saturation of binding at about I chloride/PS II was observed. The rate of binding showed a clear dependence on the concentration of chloride approaching a limiting value of about 3·10^-4 s^-1 at high concentrations, similar to the rate of release of chloride from labeled membranes. These rates were close to that found earlier for the release of chloride from PS II membranes isolated from spinach grown on ³⁶Cl^-, which suggests that we are observing the same site for chloride binding. The similarity between the limiting rate of binding and the rate of release of chloride suggests that the exchange of chloride with the surrounding medium is controlled by an intramolecular process. The binding of chloride showed a pH-dependence with an apparent pK_a of 7.5 and was very sensitive to the presence of the extrinsic polypeptides at the PS II donor side. The binding of chloride was competitively inhibited by a few other anions, notably Br^- and NO₃ ^-. The slowly exchanging Cl^- did not show any significant correlation with oxygen evolution rate or yield of EPR signals from the S₂ state. Our studies indicate that removal of the slowly exchanging chloride lowers the stability of PS II as indicated by the loss of oxygen evolution activity and S₂ state EPR signals.Abbreviations Chl chlorophyll - EPR electron paramagnetic resonance - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - Mes 4-morpholineethanesulfonic acid - MWCO molecular weight cut off - PPBQ phenyl-p-benzoquinone - PS II Photosystem II     

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.     

The origin of split EPR signals in the Ca2+-depleted Photosystem II

A light-driven reaction model for the Ca²⁺-depleted Photosystem (PS) II is proposed to explain the split signal observed in electron paramagnetic resonance (EPR) spectra based on a comparison of EPR assignments with recent x-ray structural data. The split signal has a splitting linewidth of 160 G at around g = 2 and is seen upon illumination of the Ca²⁺-depleted PS II in the S₂ state associated with complete or partial disappearance of the S₂ state multiline signal. Another g=2 broad ESR signal with a 110 G linewidth was produced by 245 K illumination for a short period in the Ca²⁺-depleted PS II in S₁ state. At the same time a normal Y_Z^· radical signal was also efficiently trapped. The g=2 broad signal is attributed to an intermediate S₁X^· state in equilibrium with the trapped Y_Z^· radical. Comparison with x-ray structural data suggests that one of the split signals (doublet signal) is attributable to interaction between His 190 and the Y_Z^· radical, and other signals is attributable to interaction between His 337 and the manganese cluster, providing further clues as to the mechanism of water oxidation in photosynthetic oxygen evolution.     

EPR studies of the water oxidizing complex in the S1 and the higher S states: the manganese cluster and YZ radical

The parallel polarization electron paramagnetic resonance (EPR) method has been applied to investigate manganese EPR signals of native S₁ and S₃ states of the water oxidizing complex (WOC) in photosystem (PS) II. The EPR signals in both states were assigned to thermally excited states with S=1, from which zero-field interaction parameters D and E were derived. Three kinds of signals, the doublet signal, the singlet-like signal and g=11–15 signal, were detected in Ca²⁺-depleted PS II. The g=11–15 signal was observed by parallel and perpendicular modes and assigned to a higher oxidation state beyond S₂ in Ca²⁺-depleted PS II. The singlet-like signal was associated with the g=11–15 signal but not with the Y_Z (the tyrosine residue 161 of the D1 polypeptide in PS II) radical. The doublet signal was associated with the Y_Z radical as proved by pulsed electron nuclear double resonance (ENDOR) and ENDOR-induced EPR. The electron transfer mechanism relevant to the role of Y_Z radical was discussed.     

EPR detection of a cryogenically photogenerated intermediate in photosynthetic oxygen evolution

In Photosystem II preparations at low temperature we were able to generate and trap an intermediate state between the S₁ and S₂ states of the Kok scheme for photosynthetic oxygen evolution. Illumination of dark-adapted, oxygen-evolving Photosystem II preparations at 140 K produces a 320-G-wide EPR signal centered near g = 4.1 when observed at 10 K. This signal is superimposed on a 5-fold larger and somewhat narrower background signal; hence, it is best observed in difference spectra. Warming of illuminated samples to 190 K in the dark results in the disappearance of the light-induced g = 4.1 feature and the appearance of the multiline EPR signal associated with the S₂ state. Low-temperature illumination of samples prepared in the S₂ state does not produce the g = 4.1 signal. Inhibition of oxygen evolution by incubation of PS II preparations in 0.8 M NaCl buffer or by the addition of 400 μM NH₂OH prevents the formation of the g = 4.1 signal. Samples in which oxygen evolution is inhibited by replacement of Cl^? with F^? exhibit the g = 4.1 signal when illuminated at 140 K, but subsequent warming to 190 K neither depletes the amplitude of this signal nor produces the multiline signal. The broad signal at g = 4.1 is typical for a $\text{S =}\text{5}\text{2}$ spin system in a rhombic environment, suggesting the involvement of non-heme Fe in photosynthetic oxygen evolution.     

Electron spin-lattice relaxation studies of different forms of the S2 state multiline EPR signal of the Photosystem II oxygen-evolving complex

The pulsed EPR inversion recovery sequence has been utilized to monitor the temperature dependence of the electron spin-lattice relaxation rate of the Mn cluster of the Photosystem II oxygen evolving complex poised in a variety of S ₂ state forms giving rise to g = 2 multiline EPR signals. A previous study (Lorigan and Britt (1994) Biochemistry 33: 12072–12076) showed that for PS II membranes treated with 5% ethanol, the S ₂ state Mn cluster relaxes via the Orbach spin-lattice relaxation mechanism, where the relaxation is enhanced via phonon scattering off an excited state spin manifold, in this case at an energy of Δ = 36.5 cm⁻¹ above the S = 1/2 ground state giving rise to the multiline EPR signal. Parallel experiments are reported for PS II membranes with 5% methanol, treated with ammonia, and following short and long term dark adaptation. In each case, the temperature dependence of the electron spin-lattice relaxation rate is consistent with Orbach relaxation, and the range of excited state energies is relatively narrow (33.8 cm⁻¹ ≤ Δ ≤ 39.7 cm⁻¹). In addition, short term dark adapted (6 min, ‘active state’) PS II membranes show biphasic recovery traces which indicate that a minority fraction of the oxygen evolving complexes are trapped in a form with greatly slowed spin-lattice relaxation. This revised version was published online in June 2006 with corrections to the Cover Date.     

EPR and ENDOR studies of the water oxidizing complex of Photosystem II

A comparative study of X-band EPR and ENDOR of the S₂ state of photosystem II membrane fragments and core complexes in the frozen state is presented. The S₂ state was generated either by continuous illumination at T=200 K or by a single turn-over light flash at T=273 K yielding entirely the same S₂ state EPR signals at 10 K. In membrane fragments and core complex preparations both the multiline and the g=4.1 signals were detected with comparable relative intensity. The absence of the 17 and 23 kDa proteins in the core complex preparation has no effect on the appearance of the EPR signals. ¹H-ENDOR experiments performed at two different field positions of the S₂ state multiline signal of core complexes permitted the resolution of four hyperfine (hf) splittings. The hf coupling constants obtained are 4.0, 2.3, 1.1 and 0.6 MHz, in good agreement with results that were previously reported (Tang et al. (1993) J Am Chem Soc 115: 2382–2389). The intensities of all four line pairs belonging to these hf couplings are diminished in D₂O. A novel model is presented and on the basis of the two largest hfc's distances between the manganese ions and the exchangeable protons are deduced. The interpretation of the ENDOR data indicates that these hf couplings might arise from water which is directly ligated to the manganese of the water oxidizing complex in redox state S₂.Abbreviations cw continuous wave - ENDOR electron nuclear double resonance - EPR electron paramagnetic resonance - hf hyperfine - hfc hyperfine coupling - MLS multiline signal - PS II Photosystem II - rf radio frequency - WOC water oxidizing complex     

EPR studies of the oxygen-evolving enzyme of Photosystem II

The light-induced EPR multiline signal is studied in O₂-evolving PS II membranes. The following results are reported: (1) Its amplitude is shown to oscillate with a period of 4, with respect to the number of flashes given at room temperature (maxima on the first and fifth flashes). (2) Glycerol enhances the signal intensity. This effect is shown to come from changes in relaxation properties rather than an increase in spin concentration. (3) Deactivation experiments clearly indicate an association with the S₂ state of the water-oxidizing enzyme. A signal at g = 4.1 with a linewidth of 360 G is also reported and it is suggested that this arises from an intermediate donor between the S states and the reaction centre. This suggestion is based on the following observations: (1) The g = 4.1 signal is formed by illumination at 200 K and not by flash excitation at room temperature, suggesting that it arises from an intermediate unstable under physiological conditions. (2) The formation of the g = 4.1 signal at 200 K does not occur in the presence of DCMU, indicating that more than one turnover is required for its maximum formation. (3) The g = 4.1 signal decreases in the dark at 220 K probably by recombination with Q^?_AFe. This recombination occurs before the multiline signal decreases, indicating that the g = 4.1 species is less stable than S₂. (4) At short times, the decay of the g = 4.1 signal corresponds with a slight increase in the multiline S₂ signal, suggesting that the loss of the g = 4.1 signal results in the disappearance of a magnetic interaction which diminishes the multiline signal intensity. (5) Tris-washed PS II membranes illuminated at 200 K do not exhibit the signal.     

Acceptor and donor-side interactions of phenolic inhibitors in Photosystem II

Certain phenolic compounds represent a distinct class of Photosystem (PS) II Q_B site inhibitors. In this paper, we report a detailed study of the effects of 2,4,6-trinitrophenol (TNP) and other phenolic inhibitors, bromoxynil and dinoseb, on PS II energetics. In intact PS II, phenolic inhibitors bound to only 90-95% of Q_B sites even at saturating concentrations. The remaining PS II reaction centers (5-10%) showed modified Q_A to Q_B electron transfer but were sensitive to urea/triazine inhibitors. The binding of phenolic inhibitors was 30- to 300-fold slower than the urea/triazine class of Q_B site inhibitors, DCMU and atrazine. In the sensitive centers, the S₂Q_A⁻ state was 10-fold less stable in the presence of phenolic inhibitors than the urea/triazine herbicides. In addition, the binding affinity of phenolic herbicides was decreased 10-fold in the S₂Q_A⁻ state than the S₁Q_A state. However, removal of the oxygen-evolving complex (OEC) and associated extrinsic polypeptides by hydroxylamine (HA) washing abolished the slow binding kinetics as well as the destabilizing effects on the charge-separated state. The S₂-multiline electron paramagnetic resonance (EPR) signal and the ‘split’ EPR signal, originating from the S₂Y_Z state showed no significant changes upon binding of phenolic inhibitors at the Q_B site. We thus propose a working model where Q_A redox potential is lowered by short-range conformational changes induced by phenolic inhibitor binding at the Q_B niche. Long-range effects of HA-washing eliminate this interaction, possibly by allowing more flexibility in the Q_B site.     

Interactions of Chloride and Formate at the Donor and the Acceptor Side of Photosystem II

Chloride is required for the maximum activity of the oxygen evolving complex (OEC) while formate inhibits the function of OEC. On the basis of the measurements of oxygen evolution rates and the S₂ state multiline EPR signal, an interaction between the action of chloride and formate at the donor side of PS II has been suggested. Moreover, the Fe₂+Q–A EPR signals were measured to investigate a common binding site of both these anions at the PS II acceptor side. Other monovalent anions like bromide, nitrate etc. could influence the effects of formate to a small extent at the donor side of PS II, but not significantly at the acceptor side of PS II. The results presented in this paper clearly suggest a competitive binding of formate and chloride at the PS II acceptor side.     

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.     

Fluoride inhibition of photosystem II and the effect of removal of the PsbQ subunit

Photosystem II (PSII), the light-absorbing complex of photosynthesis that evolves oxygen, requires chloride for activation of the oxygen evolving complex (OEC). In this study, fluoride was characterized as an inhibitor of Cl⁻-activated oxygen evolution in higher plant PSII. It was confirmed to be primarily a competitive inhibitor in intact PSII, with Cl⁻-competitive inhibition constant K_i = 2 mM and uncompetitive inhibition constant \textK_\texti^￠ {\text{K}}_{\text{i}}^{\prime }  = 79 mM. A pH dependence study showed that fluoride inhibition was more pronounced at lower pH values. In order to determine the location of the fluoride effect, PSII preparations lacking various amounts of the PsbQ subunit were prepared. The competitive F⁻ inhibition constant and the Michaelis constant for Cl⁻ activation increased with loss of the PsbQ subunit, while the uncompetitive F⁻ inhibition constant was relatively insensitive to loss of PsbQ. The S₂ state EPR signals from PSII lacking PsbQ responded to Ca²⁺ and Cl⁻ removal and to F⁻ treatment similar to intact PSII, with enhancement of the g = 4.1 signal and suppression of the multiline signal, but the effects were more pronounced in PSII lacking PsbQ. Together, these results support the interpretation that the PsbQ subunit has a role in retaining anions within the OEC.     

NMR paramagnetic relaxation enhancements due to manganese in the S0 and S2 states of Photosystem II-enriched membrane fragments and in the detergent-solubilized Photosystem II complex

The NMR paramagnetic relaxation enhancement (NMR-PRE) produced in the solvent proton resonance by manganese in the S₀ and S₂ states of the oxygen evolving center (OEC) has been recorded for three Photosystem II (PS II)-enriched preparations: (1) PS II-enriched thylakoid membrane fragments (TMF-2 particles); (2) salt-washed (2M NaCl) TMF-2 particles; and (3) the octylglucopyranoside (OGP)-solubilized PS II complex. The second and third preparations, but not the first, are depleted of the peripheral 17 and 23 kD polypeptides associated with the OEC. It has been proposed that depletion of these polypeptides increases the exposure of OEC manganese to the aqueous phase. The NMR-PRE response measures the quantity (T_1m+_m)^-1, where T_1m is the spin relaxation time and _m is the mean residence time with respect to chemical exchange reactions of solvent protons in the manganese coordination sphere, and, thus, the NMR-PRE provides a direct measure of the solvent proton chemical exchange rate constant _m ^-1. This study tested whether the 17 and 23 kD polypeptides shield the OEC from the solvent phase and whether their depletion enhances the S₂ and S₀ NMR-PRE signals by removing a kinetic barrier to the solvent proton chemical exchange reaction. The amplitude of the S₂ NMR-PRE signal, measured in its chemical exchange-limited regime (_m>T_1m), is slightly decreased, rather than increased, in preparations (2) and (3) relative to (1), indicating that removal of the 17 and 23 kD polypeptides slightly slows, rather than accelerates, the rate-limiting steps of the solvent proton chemical exchange reactions. In addition, the lifetime of the S₂ state was shortened several-fold in the solubilized PS II complex and in salt-washed TMF-2 membranes relative to untreated TMF-2 control samples. The S₀ NMR-PRE signal, which is present in TMF-2 suspensions, was not detected in suspensions of the solubilized PS II complex, even though these samples contained high concentrations of active manganese centers (approximately double those of the TMF-2 control) and exhibited an S₂ NMR-PRE signal of comparable amplitude to that of the TMF-2 preparation. These results suggest that the 17 and 23 kD extrinsic polypeptides do not shield the NMR-visible water binding site in the OEC from the aqueous phase, although their removal substantially alters the proton relaxation efficiency by shortening T_1m.Abbreviations ADRY acceleration of the deactivation reactions of the water splitting enzyme Y - BBY Photosystem II-enriched membrane fragments prepared by the method of Berthold et al. (1981) - CCCP carbonyl cyanide m-chlorophenyl hydrazone - Chl chlorophyll - DCBQ 2,5-dichlorobenzoquinone - MES morpholinoethanesulfonate - NMR nuclear magnetic resonance - OEC oxygen evolving complex - OGP octylglucopyranoside - PRE paramagnetic relaxation enhancement - PS II Photosystem II - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TMF-2 Photosystem II-enriched thylakoid membrane fragments prepared by the method of Radmer et al. (1986) - T₁, T₂ longitudinal and transverse nuclear spin relaxation times     

Influence of protein phosphorylation on the electron-transport properties of Photosystem II

Many of the core proteins in Photosystem II (PS II) undergo reversible phosphorylation. It is known that protein phosphorylation controls the repair cycle of Photosystem II. However, it is not known how protein phosphorylation affects the partial electron transport reactions in PS II. Here we have applied variable fluorescence measurements and EPR spectroscopy to probe the status of the quinone acceptors, the Mn cluster and other electron transfer components in PS II with controlled levels of protein phosphorylation. Protein phosphorylation was induced in vivo by varying illumination regimes. The phosphorylation level of the D1 protein varied from 10 to 58% in PS II membranes isolated from pre-illuminated spinach leaves. The oxygen evolution and Q_A ⁻ to Q_B(Q_B ⁻) electron transfer measured by flash-induced fluorescence decay remained similar in all samples studied. Similar measurements in the presence of DCMU, which reports on the status of the donor side in PS II, also indicated that the integrity of the oxygen-evolving complex was preserved in PS II with different levels of D1 protein phosphorylation. With EPR spectroscopy we examined individual redox cofactors in PS II. Both the maximal amplitude of the charge separation reaction (measured as photo-accumulated pheophytin⁻) and the EPR signal from the Q_A ⁻ Fe²⁺ complex were unaffected by the phosphorylation of the D1 protein, indicating that the acceptor side of PS II was not modified. Also the shape of the S₂ state multiline signal was similar, suggesting that the structure of the Mn-cluster in Photosystem II did not change. However, the amplitude of the S₂ multiline signal was reduced by 35% in PS II, where 58% of the D1 protein was phosphorylated, as compared to the S₂ multiline in PS II, where only 10% of the D1 protein was phosphorylated. In addition, the fraction of low potential Cyt b ₅₅₉ was twice as high in phosphorylated PS II. Implications from these findings, were precise quantification of D1 protein phosphorylation is, for the first time, combined with high-resolution biophysical measurements, are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Inhomogeneity of the EPR multiline signal from the S2-state of the photosystem II oxygen-evolving enzyme

The manganese complex (Mn₄) which is responsible for water oxidation in photosystem II is EPR detectable in the S₂-state, one of the five redox states of the enzyme cycle. The S₂-state is observable at 10?K either as an EPR multiline signal (spin S?=?1/2) or as a signal at g?=?4.1 (spin S?=?3/2 or 5/2). It has recently been shown that the state responsible for the multiline signal is converted to that responsible for the g?=?4.1 signal upon the absorption of near-infrared light [Boussac A, Girerd J-J, Rutherford AW (1996) Biochemistry 35?:?6984–6989]. It is shown here that the yield of the spin interconversion may be variable and depends on the photosystem II (PSII) preparations. The EPR multiline signal detected after near-infrared illumination, and which originates from PSII centers not susceptible to the near-infrared light, is shown to be different from that which originates from infrared-susceptible PSII centers. The total S₂-multiline signal results from the superposition of the two multiline signals which originate from these two PSII populations. One S₂ population gives rise to a "narrow" multiline signal characterized by strong central lines and weak outer lines. The second population gives rise to a "broad" multiline signal in which the intensity of the outer lines, at low and high field, are proportionally larger than those in the narrow multiline signal. The larger the relative amplitude of the outer lines at low and high field, the higher is the proportion of the near-infrared-susceptible PSII centers and the yield of the multiline to g?=?4.1 signal conversion. This inhomogeneity of the EPR multiline signal is briefly discussed in terms of the structural properties of the Mn₄ complex.     

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.     

Thermoluminescence studies on the function of Photosystem II in the desiccation tolerant lichen Cladonia convoluta

The effect of desiccation and rehydration on the function of Photosystem II has been studied in the desiccation tolerant lichen Cladonia convoluta by thermoluminescence. We have shown that in functional fully hydrated thalli thermoluminescence signals can be observed from the recombination of the S₂₍₃₎Q_B ^– (B band), S₂Q_A ^– (Q band), Tyr-D⁺Q_A ^– (C band) and Tyr-Z⁺(His⁺)Q_A ^– (A band) charge stabilization states. These thermoluminescence signals are completely absent in desiccated thalli, but rapidly reappear on rehydration. Flash-induced oscillation in the amplitude of the thermoluminescence band from the S₂₍₃₎Q_B ^– recombination shows the usual pattern with maxima after 2 and 6 flashes when rehydration takes place in light. However, after rehydration in complete darkness, there is no thermoluminescence emission after the 1 st flash, and the maxima of the subsequent oscillation are shifted to the 3rd and 7th flashes. It is concluded that desiccation of Cladonia convoluta converts PS II into a nonfunctional state. This state is characterized by the lack of stable charge separation and recombination, as well as by a one-electron reduction of the water-oxidizing complex. Restoration of PS II function during rehydration can proceed both in the light and in darkness. After rehydration in the dark, the first charge separation act is utilized in restoring the usual oxidation state of the water-oxidizing comples.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DT desiccation tolerant - PS II Photosystem II - TL thermoluminescence - P₆₈₀ reaction center Chl of PS II - Q_A and Q_B puinone electron acceptors of PS II - S₀,...,S₄ the redox states of the water-oxidizing complex - Tyr-Z and Tyr-D redox-active tyrosine electron donors of PS II     

Direct quantification of the four individual S states in Photosystem II using EPR spectroscopy

EPR spectroscopy is very useful in studies of the oxygen evolving cycle in Photosystem II and EPR signals from the CaMn₄ cluster are known in all S states except S₄. Many signals are insufficiently understood and the S₀, S₁, and S₃ states have not yet been quantifiable through their EPR signals. Recently, split EPR signals, induced by illumination at liquid helium temperatures, have been reported in the S₀, S₁, and S₃ states. These split signals provide new spectral probes to the S state chemistry. We have studied the flash power dependence of the S state turnover in Photosystem II membranes by monitoring the split S₀, split S₁, split S₃ and S₂ state multiline EPR signals. We demonstrate that quantification of the S₁, S₃ and S₀ states, using the split EPR signals, is indeed possible in samples with mixed S state composition. The amplitudes of all three split EPR signals are linearly correlated to the concentration of the respective S state. We also show that the S₁ → S₂ transition proceeds without misses following a saturating flash at 1 °C, whilst substantial misses occur in the S₂ → S₃ transition following the second flash.