S-state dependence of the miss probability in Photosystem II

The oxygen production of dark-adapted Photosystem II upon illumination by a series of single-turnover flashes shows a damped period four oscillation with flash number. The damping is attributed to `misses' resulting from a statistical probability that a reaction center fails to produce a stable charge separation after a saturating flash. The origin of misses is of interest because its probable dependence on flash number, in principle, affects the quantitative interpretation of all measurements on phenomena associated with the period four oscillation. We show that the kinetics of chlorophyll fluorescence yield transients induced by a flash series can be used to estimate the relative amplitudes of the miss probability on each flash. It is concluded that a major part of the misses must be caused by failure of the reduction of the oxidized primary electron donor chlorophyll P680⁺ by the secondary donor tyrosine Y_Z before the charge separation is lost by recombination. The probability of this failure is found to increase with the oxidation state of the oxygen-evolving complex: more than half of it occurs upon charge separation in the S₃ state, which is attributed to the presence of Y_Z ^ox S₂ in Boltzmann equilibrium with Y_ZS₃. This revised version was published online in June 2006 with corrections to the Cover Date.     

The S-state dependence of Cl binding to plant Photosystem II

A combined single-turnover flash and ³⁵Cl NMR technique has been used to monitor S-state dependence of Cl⁻ binding to PS-II particles derived from mangrove (Avicennia marina). No detectable high-affinity binding was found to particles in the S₀ and S₁ states, but binding with an affinity comparable to that which activates O₂ evolution was found in the S₂ and S₃ states.     

Absorbance difference spectra of the S-state transitions in Photosystem II core particles

Redox changes of the oxygen evolving complex in PS II core particles were investigated by absorbance difference spectroscopy in the UV-region. The oscillation of the absorbance changes induced by a series of saturating flashes could not be explained by the minimal Kok model (Kok et al. 1970) consisting of a 4-step redox cycle, S₀ S₁ S₂ S₃ S₀, although the values of most of the relevant parameters had been determined experimentally. Additional assumptions which allow a consistent fit of all data are a slow equilibration of the S₃ state with an inactive state, perhaps related to Ca²⁺-release, and a low quantum efficiency for the first turnover after dark-adaptation. Difference spectra of the successive S-state transitions were determined. At wavelengths above 370 nm, they were very different due to the different contribution of a Chl bandshift in each spectrum. At shorter wavelengths, the S₁ S₂ transition showed a difference spectrum similar to that reported by Dekker et al. 1984b and attributed to an Mn(III) to Mn(IV) oxidation. The spectrum of absorbance changes associated with the S₂ S₃ transition was similar to that reported by Lavergne 1991 for PS II membranes. The S₀ S₁ transition was associated with a smaller but still substantial absorbance increase in the UV. Differences with the spectra reported by Lavergne 1991 are attributed to electrostatic effects on electron transfer at the acceptor side associated with the S-state dependence of proton release in PS II membranes.Abbreviations Bis-Tris (bis[2-hydroxyethyl]imino-tris[hydroxymethyl]methane) - DCBQ 2,5-dichloro-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PS II Photosystem II - Q_A secondary electron acceptor of PS II - S₀ to S₄ redox state of the oxygen evolving complex - Z secondary electron donor of PS II     

Improved 5-step modeling of the Photosystem II S-state mechanism in cyanobacteria

We present a model of the S-state mechanism, as well as an improved eigenvalue analysis, that integrate into a coherent ensemble several features found since the S-state model was initially developed. These features include the presence of S_–1, deactivations in the dark interval between flashes, and the change in the number of active PS II centers by photoinhibition or photoactivation. A new feature is the capacity to predict the steady-state distribution of S-states under conditions of steady photoinhibition or photoactivation. The improved eigenvalue analysis allowed the calculation of the initial S-state distribution. In addition, the model resolved true photochemical misses from apparent misses due to deactivations in the dark interval between flashes. The model suggested that most of the misses that are commonly reported are due to deactivations, and not to an intrinsic inefficiency of the photochemical mechanism of PS II. Because models that allow double-hits encompassing the S₂ to S₃ transition often predict negative initial quantities of S₂ in cyanobacteria, our proposed model specifically prohibited them. The model accounts for inhomogeneous misses and a steady-state distribution of the type (S₂)(S₁)>(S₃)(S₀). This 5-step model uses only 4 probabilities, and is therefore easy to handle. The use of this model is critical for the analysis of several cyanobacterial strains, as well as for any species that show non-negligible deactivations in the dark interval between flashes.     

EPR characteristics of chloride-depleted photosystem II membranes in the presence of other anions.

Chloride is an essential cofactor for the oxidation of water to oxygen. Anion substitution (Br(-), I(-), NO(2)(-), F(-)) in Cl(-)-depleted PS II membranes brings out significant changes in the EPR signals arising from the S(2) state and from the iron-quinone complex of PS II. On the basis of the changes observed in the S(2) state multiline signal and the Q(A)Fe(3+) EPR signal in Cl(-)-depleted PS II membranes after substituting with various anions, we report a possible binding site of anions such as chloride and bromide at the PS II donor side as well as at the acceptor side.     

Kinetics of EPR signal IIvf in chloroplast Photosystem II

The risetime of EPR signal II_vf (S II_vf) has been measured in oxygen-evolving Photosystem II particles from spinach chloroplasts at pH 6.0. The EPR signal shows an instrument-limited rise upon induction ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 3 μ}\text{s}$). These data are consistent with a model where the species Z responsible for S II_vf is the immediate electron donor to P-680⁺ in spinach chloroplasts. A new, faster decay component of S II_vf has also been detected in these experiments.     

Rise time of EPR signal IIvf in chloroplast Photosystem II

The rise time of the photoinduced, reversible EPR Signal II_vf in spinach chloroplasts is found using flash excitation to be 20 ± 10 μs. The results are interpreted as evidence that the Signal II_vf radical is an electron carrier on the donor side of Photosystem II, but probably does not result from the first donor to P680⁺.     

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.     

Comparative EPR and thermoluminescence study of anoxic photoinhibition in Photosystem II particles

Photosystem II particles were exposed to 800 W m^–2 white light at 20 °C under anoxic conditions. The F_o level of fluorescence was considerably enhanced indicating formation of stable-reduced forms of the primary quinone electron acceptor, Q_A. The F_m level of fluorescence declined only a little. The g=1.9 and g=1.82 EPR forms characteristic of the bicarbonate-bound and bicarbonate-depleted semiquinone-iron complex, Q_A ^–Fe²⁺, respectively, exhibited differential sensitivity against photoinhibition. The large g=1.9 signal was rapidly diminished but the small g=1.82 signal decreased more slowly. The S₂-state multiline signal, the oxygen evolution and photooxidation of the high potential form of cytochrome b-559 were inhibited approximately with the same kinetics as the g=1.9 signal. The low potential form of oxidized cytochrome b-559 and Signal II_slow arising from Tyr_D ⁺ decreased considerably slower than the g=1.9 semiquinone-iron signal. The high potential form of oxidized cytochrome b-559 was diminished faster than the low potential form. Photoinhibition of the g=1.9 and g=1.82 forms of Q_A was accompanied with the appearance and gradual saturation of the spin-polarized triplet signal of P 680. The amplitude of the radical signal from photoreducible pheophytin remained constant during the 3 hour illumination period. In the thermoluminescence glow curves of particles the Q band (S₂Q_A ^– charge recombination) was almost completely abolished. To the contrary, the C band (Tyr_D ⁺Q_A ^– charge recombination) increased a little upon illumination. The EPR and thermoluminescence observations suggest that the Photosystem II reaction centers can be classified into two groups with different susceptibility against photoinhibition.Abbreviations C band thermoluminescence band associated with Tyr-D⁺Q _a ^– charge recombination - Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - EPR electron paramagnetic resonance - F_o initial fluorescence - F_m maximum fluorescence - Q band thermoluminescence band originating from S₂Q _a -charge recombination - Q _a the primary quinone electron acceptor of PS II - P 680 the primary electron donor chlorophyll of PS II - S₂ oxidation state of the water-splitting system - Phe pheophytin - TL thermoluminescence - Tyr _d redox active tyrosine-160 of the D₂ protein     

A new EPR signal attributed to the primary plastosemiquinone acceptor in Photosystem II

A study of signals, light-induced at 77 K in O₂-evolving Photosystem II (PS II) membranes showed that the EPR signal that has been attributed to the semiquinone-iron form of the primary quinone acceptor, Q^?_AFe, at g = 1.82 was usually accompanied by a broad signal at g = 1.90. In some preparations, the usual g = 1.82 signal was almost completely absent, while the intensity of the g = 1.90 signal was significantly increased. The g = 1.90 signal is attributed to a second EPR form of the primary semiquinone-iron acceptor of PS II on the basis of the following evidence. (1) The signal is chemically and photochemically induced under the same conditions as the usual g = 1.82 signal. (2) The extent of the signal induced by the addition of chemical reducing agents is the same as that photochemically induced by illumination at 77 K. (3) When the g = 1.82 signal is absent and instead the g = 1.90 signal is present, illumination at 200 K of a sample containing a reducing agent results in formation of the characteristic split pheophytin^? signal, which is thought to arise from an interaction between the photoreduced pheophytin acceptor and the semiquinone-iron complex. (4) Both the g = 1.82 and g = 1.90 signals disappear when illumination is given at room temperature in the presence of a reducing agent. This is thought to be due to a reduction of the semiquinone to the nonparamagnetic quinol form. (5) Both the g = 1.90 and g = 1.82 signals are affected by herbicides which block electron transfer between the primary and secondary quinone acceptors. It was found that increasing the pH results in an increase of the g = 1.90 form, while lowering the pH favours the g = 1.82 form. The change from the g = 1.82 form to the g = 1.90 form is accompanied by a splitting change in the split pheophytin^? signal from approx. 42 to approx. 50 G. Results using chloroplasts suggest that the g = 1.90 signal could represent the form present in vivo.     

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

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

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     

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.     

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.     

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 CaMn(4) cluster are known in all S states except S(4). Many signals are insufficiently understood and the S(0), S(1), and S(3) 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 S(0), S(1), and S(3) 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 S(0), split S(1), split S(3) and S(2) state multiline EPR signals. We demonstrate that quantification of the S(1), S(3) and S(0) 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 S(1) --> S(2) transition proceeds without misses following a saturating flash at 1 degrees C, whilst substantial misses occur in the S(2) --> S(3) transition following the second flash.     

EPR signal II in cyanobacterial Photosystem II reaction-center complexes with and without the 40 kDa chlorophyll-binding subunit

The steady-state amplitude and flash-induced kinetics of EPR signal II in two Photosystem II (PS II) reaction center protein complexes from Synechococcus were measured to probe the organization of species involved in the PS II electron-transfer chain. A PS II reaction center complex (E-1) which has 47, 40, 31, 28 and 9 kDa subunits shows both fast decaying (signal II_f) and slowly decaying (signal II_s+u) EPR components. The amplitude of signal II_f, which represents Z (the donor to P-680), is about 1 spin per 30 Chl. This corresponds to one spin per reaction center in this preparation. Signal II_s+u, the slowly decaying component of signal II, reflects D, a donor to PS II on a side chain from the path of water oxidation in higher plants and algae. Signal II_s+u is present in the E-1 preparation in a ratio of about 1 spin per 40 Chl. Flash-induced signal II_f in E-1 shows biexponential decay with half-times of 20 ms and 300 ms. In a PS II reaction center complex (CP2b) which has 47, 31, 28 and 9 kDa subunits, but no 40 kDa subunit, an appreciable amount of signal II_f is observed (about 1 per 50 Chl). Less than 1 spin per 400 Chl of signal II_s+u is visible in this sample. The kinetics of Z⁺ reduction (signal II_f) in CP2b is similar to that seen in E-1 preparations, indicating that CP2b contains all of the molecules necessary for primary charge separation and secondary electron donation from Z.     

Effects of azide on the S2 state EPR signals from Photosystem II

The anion azide, N₃ ^-, has been previously found to be an inhibitor of oxygen evolution by Photosystem II (PS II) of higher plants. With respect to chloride activation, azide acts primarily as a competitive inhibitor but uncompetitive inhibition also occurs [Haddy A, Hatchell JA, Kimel RA and Thomas R (1999) Biochemistry 38: 6104–6110]. In this study, the effects of azide on PS II-enriched thylakoid membranes were characterized by electron paramagnetic resonance (EPR) spectroscopy. Azide showed two distinguishable effects on the S₂ state EPR signals. In the presence of chloride, which prevented competitive binding, azide suppressed the formation of the multiline and g = 4.1 signals concurrently, indicating that the normal S₂ state was not reached. Signal suppression showed an azide concentration dependence that correlated with the fraction of PS II centers calculated to bind azide at the uncompetitive site, based on the previously determined inhibition constant. No evidence was found for an effect of azide on the Fe(II)Q_A ^- signals at the concentrations used. This result is consistent with placement of the uncompetitive site on the donor side of PS II as suggested in the previous study. In chloride-depleted PS II-enriched membranes azide and fluoride showed similar effects on the S₂ state EPR signals, including a notable increase and narrowing of the g = 4.1 signal. Comparable effects of other anions have been described previously and apparently take place through the chloride-competitive site. The two azide binding sites described here correlate with the results of other studies of Lewis base inhibitors.This revised version was published online in October 2005 with corrections to the Cover Date.     

Photosystem II single crystals studied by transient EPR: the light-induced triplet state

Transient electron paramagnetic resonance (TR EPR) at 9.8 GHz has been used to study the light-induced triplet state in single crystals of Photosystem II (PS II). The crystals were grown from a solution of PS II core complexes from the thermophilic cyanobacterium Synechococcus elongatus. The core complexes contain at least 17 subunits, including the water-oxidizing complex, and 32 chlorophyll a molecules per PS II complex. The PS II complexes are active in light-induced electron transfer and water oxidation. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with four dimers of PS II complexes per unit cell. Laser excitation was used to generate the recombination triplet state in PS II which was then studied by EPR at low temperatures (10 K). The crystal spectra show the same magnitude of the zero-field splitting (ZFS) values D, E as spectra obtained earlier for the triplet state of PS II in frozen solution. The orientation of the ZFS tensor D of the triplet state with respect to the crystallographic axes has been deduced from the analysis of angular-dependent EPR spectra. Knowledge of the orientation of the D tensor component perpendicular to the plane of the chlorophyll (D(Z)) allows an assignment on which chlorophyll of the reaction centre the triplet state is localized at low temperatures. Furthermore, the orientation of the D(X) and D(Y) components of the D tensor yielded the in-plane orientation of the respective chlorophyll in the reaction centre providing first experimental evidence for the orientation of this molecule in the PS II.     

Temperature-dependent changes in Photosystem II heterogeneity support a cycle of Photosystem II during photoinhibition

High light treatments were given to attached leaves of pumpkin (Cucurbita pepo L.) at room temperature and at 1°C where the diffusion- and enzyme-dependent repair processes of Photosystem II are at a minimum. After treatments, electron transfer activities and fluorescence induction were measured from thylakoids isolated from the treated leaves. When the photoinhibition treatment was given at 1°C, the Photosystem II electron transfer assays that were designed to require electron transfer to the plastoquinone pool showed greater inhibition than electron transfer from H₂O to paraphenyl-benzoquinone, which measures all PS II centers. When the light treatment was given at room temperature, electron transfer from H₂O to paraphenyl-benzoquinone was inhibited more than whole-chain electron transfer. Variable fluorescence measured in the presence of ferricyanide decreased only during room-temperature treatments. These results suggest that reaction centers of one pool of Photosystem II, non-Q_B-PS II, replace photoinhibited reaction centers at room temperature, while no replacement occurs at 1°C. A simulation of photoinhibition at 1°C supports this conclusion.Abbreviations BSA bovine serum albumin - Chl chlorophyll - DCMU 3-(3,4,-dichlorophenyl)-1,1,-dimethylurea - DCPIP dichlorophenol-indophenol (2,6-dichloro-4((4-hydroxyphenyl)imino)-2,5-cyclohexadien-1-one) - DPC diphenyl carbazide (2,2-diphenylcarbonic dihydrazide) - FeCN ferricyanide (hexacyanoferrate(III)) - _app apparent quantum yield of photosynthetic oxygen evolution - MV methyl viologen (1,1-dimethyl-4,4-bipyridinium dichloride) - PPBQ phenyl-p-benzoquinone - PPFD photosynthetic photon flux density - PQ pool plastoquinone - Q_B secondary quinone acceptor of PS II - RT room temperature - WC whole chain electron transfer