Electron paramagnetic resonance study of the electron transfer reactions in photosystem II membrane preparations from Arabidopsis thaliana

Arabidopsis thaliana is widely used as a model organism in plant biology as its genome has been sequenced and transformation is known to be efficient. A large number of mutant lines and genomic resources are available for Arabidopsis. All this makes Arabidopsis a useful tool for studies of photosynthetic reactions in higher plants. In this study, photosystem II (PSII) enriched membranes were successfully isolated from thylakoids of Arabidopsis plants and for the first time the electron transfer cofactors in PSII were systematically studied using electron paramagnetic resonance (EPR) spectroscopy. EPR signals from both of the donor and acceptor sides of PSII, as well as from auxiliary electron donors were recorded. From the acceptor side of PSII, EPR signals from Q(A)- Fe2(+) and Phe- Q(A)- Fe2(+) as well as from the free Phe- radical were observed. The multiline EPR signals from the S?- and S?-states of CaMn?O(x)-cluster in the water oxidation complex were characterized. Moreover, split EPR signals, the interaction signals from Y(Z) and CaMn?O(x)-cluster in the S?-, S?-, S?-, and the S?-state were induced by illumination of the PSII membranes at 5K and characterized. In addition, EPR signals from auxiliary donors Y(D), Chl(+) and cytochrome b??? were observed. In total, we were able to detect about 20 different EPR signals covering all electron transfer components in PSII. Use of this spectroscopic platform opens a possibility to study PSII reactions in the library of mutants available in Arabidopsis.     

Spectroscopic studies of photosystem II in chlorophyll d-containing Acaryochloris marina

Photosystem II (PSII) electron transfer (ET) in the chlorophyll d-containing cyanobacterium Acaryochloris marina (A. marina) was studied by time-resolved electron paramagnetic resonance (EPR) spectroscopy at room temperature, chlorophyll fluorescence, and low-temperature optical spectroscopy. To maximize the ability to measure PSII ET in the intact cells of this organism, growth conditions were optimized to provide the highest specific O(2) activity and the instrumental parameters for the EPR measurements of tyrosine Z (Y(Z)) reduction were adjusted to give the best signal-to-noise over spectral resolution. Analysis of the Y(Z)(*) reduction kinetics revealed that ET to the oxygen-evolving complex on the donor side of PSII in A. marina is indistinguishable from that in higher plants and other cyanobacteria. Likewise, the charge recombination kinetics between the first plastoquinone acceptor Q(A) and the donor side of PSII monitored by the chlorophyll fluorescence decay on the seconds time scale are not significantly different between A. marina and non-chlorophyll d organisms, while low-temperature optical absorption spectroscopy identified the primary electron acceptor in A. marina as pheophytin a. The results indicate that, if the PSII primary electron donor in A. marina is made up of chlorophyll d instead of chlorophyll a, then there must be very different interactions with the protein environment to account for the ET properties, which are similar to higher plants and other cyanobacteria. Nevertheless, the water oxidation mechanism in A. marina is kinetically unaltered.     

Inhibition of tyrosine Z photooxidation after formation of the S3 state in Ca(2+)-depleted and Cl(-)-depleted photosystem II.

Ca2+ and Cl- are obligatory cofactors in photosystem II (PS-II), the oxygen-evolving enzyme of plants. The sites of inhibition in both Ca(2+)- and Cl(-)-depleted PS-II were compared using EPR and flash absorption spectroscopies to follow the extent of the photooxidation of the redox-active tyrosine (TyrZ) and of the primary electron donor chlorophyll (P680) and their subsequent reduction in the dark. The inhibition occurred after formation of the S3 state in Ca(2+)-depleted PS-II. In Cl(-)-depleted photosystem II, the inhibition occurred after formation of the S3 state in about half of the centers and probably after S2TyrZ+ formation in the remaining centers. After the S3 state was formed in Ca(2+)- and Cl(-)-depleted photosystem II, electron transfer from TyrZ to P680 was inhibited. This inhibition is discussed in terms of electrostatic constraints resulting from S3 formation in the absence of Ca2+ and Cl-.     

Photoinhibition affects the non-heme iron center in photosystem II.

Effects on the PS II acceptor side caused by exposure to strong white light (180 W/m2) of PS II membrane fragments (spinach) at pH 6.5 and 0 degrees C were analyzed by measuring low temperature EPR signals and flash-induced transient changes of the fluorescence quantum yield. The following results were obtained: (a) the extent of the light induced g = 1.9 EPR signal as a measure of photochemical Fe2+QA- formation declines with progressing photoinhibition. The half-life of this effect is independent of the absence or presence of an exogenous electron acceptor during the photoinhibitory treatment; (b) in samples photoinhibited in the absence of an electron acceptor and subsequently incubated with K3[Fe(CN)6] in the dark, the extent of the g = 8 EPR signal (reflecting the oxidized Fe3+ form of the endogenous non-heme iron center) and of the flash-induced change of the fluorescence yield (as a measure of fast electron transfer from QA- to Fe3+ after the first flash; [see (1992) Photosynth. Res. 31, 113-126] exhibits the same dependence on photoinhibition time as the g = 1.9 EPR signal; (c) in samples photoinhibited in the presence of an exogenous electron acceptor, the signals reflecting Fe(3+)-formation and fast electron transfer from QA- to Fe3+ decline faster than the g = 1.9 EPR signal. These results provide for the first time direct evidence that the endogenous non-heme iron center located between QA and QB is susceptible to modifications by light stress. The implications of this finding will be discussed.     

Analysis of photosystem II using particle II preparation. I. Experimental evidence supporting the idea of the involvement of two light reactions in photosystem II of green plant photosynthesis

(1) To analyze the photoelectron flow related to photosystemII, particle II preparation, i.e., the chloroplast fragmenthaving only photosystem II activity, proved to be far betterthan the generally used chloroplast preparations having activitiesof both PS-I and PS-II. (2) By simultaneous measurements ofthe activities of O₂ evolution and DPIP- and ferricyanide photoreductionusing variously-treated particle II preparations, it was foundthat a noticeable activity of ferricyanide photoreduction wasstill observed, though the former two activities were completelylost in the course of treatments such as Tris-treatment, pre-illuminationand aging. (3) Besides this, differences were found betweenferricyanide- and DPIP-photoreduction in respect to susceptibilityto CCCP, availability of artificial electron donor, and theeffect of chloride addition. However, both photo-reductionswere equally inactivated by heat-treatment and addition of DCMU.(4) To explain the observed distinctions between DPIP and ferricyanidein their mode of action as electron acceptor for PS-II, a schemesuggesting the involvement of two light reactions in PS-II isproposed and the electron flow near PS-II is discussed. ¹ This work has been supported by Grants from the Ministry ofEducation (Nos. 8425- 70-'71; 4970l4-'69-'71), which are gratefullyacknowledged here. (Received January 12, 1972; )     

Glutamate 189 of the D1 polypeptide modulates the magnetic and redox properties of the manganese cluster and tyrosine Y(Z) in photosystem II

Recent models for water oxidation in photosystem II postulate that the tyrosine Y(Z) radical, Y(Z)(*), abstracts both an electron and a proton from the Mn cluster during one or more steps in the catalytic cycle. This coupling of proton- and electron-transfer events is postulated to provide the necessary driving force for oxidizing the Mn cluster in its higher oxidation states. The formation of Y(Z)(*) requires the deprotonation of Y(Z) by His190 of the D1 polypeptide. For Y(Z)(*) to abstract both an electron and a proton from the Mn cluster, the proton abstracted from Y(Z) must be transferred rapidly from D1-His190 to the lumenal surface via one or more proton-transfer pathways. The proton acceptor for D1-His190 has been proposed to be either Glu189 of the D1 polypeptide or a group positioned by this residue. To further define the role of D1-Glu189, 17 D1-Glu189 mutations were constructed in the cyanobacterium Synechocystis sp. PCC 6803. Several of these mutants are of particular interest because they appear to assemble Mn clusters in 70-80% of reaction centers in vivo, but evolve no O(2). The EPR and electron-transfer properties of PSII particles isolated from the D1-E189Q, D1-E189L, D1-E189D, D1-E189N, D1-E189H, D1-E189G, and D1-E189S mutants were examined. Intact PSII particles isolated from mutants that evolved no O(2) also exhibited no S(1) or S(2) state multiline EPR signals and were unable to advance beyond an altered Y(Z)(*)S(2) state, as shown by the accumulation of narrow "split" EPR signals under multiple turnover conditions. In the D1-E189G and D1-E189S mutants, the quantum yield for oxidizing the S(1) state Mn cluster was very low, corresponding to a > or =1400-fold slowing of the rate of Mn oxidation by Y(Z)(*). In Mn-depleted D1-Glu189 mutant PSII particles, charge recombination between Q(A)(*)(-) and Y(Z)(*) in the mutants was accelerated, showing that the mutations alter the redox properties of Y(Z) in addition to those of the Mn cluster. These results are consistent with D1-Glu189 participating in a network of hydrogen bonds that modulates the properties of both Y(Z) and the Mn cluster and are consistent with proposals that D1-Glu189 positions a group that accepts a proton from D1-His190.     

Formate-induced inhibition of the water-oxidizing complex of photosystem II studied by EPR

The effects of various formate concentrations on both the donor and the acceptor sides in oxygen-evolving PS II membranes (BBY particles) were examined. EPR, oxygen evolution and variable chlorophyll fluorescence have been observed. It was found that formate inhibits the formation of the S(2) state multiline signal concomitant with stimulation of the Q(A)(-)Fe(2+) signal at g = 1.82. The decrease and the increase in intensities of the multiline and Q(A)(-)Fe(2+) signals, respectively, had a linear relation for formate concentrations between 5 and 500 mM. The g = 4.1 signal formation measured in the absence of methanol was not inhibited by formate up to 250 mM in the buffer. In the presence of 3% methanol the g = 4.1 signal evolved as formate concentration increased. The evolved signal could be ascribed to the inhibited centers. Oxygen evolution measured in the presence of an electron acceptor, phenyl-p-benzoquinone, was also inhibited by formate proportionally to the decrease in the multiline signal intensity. The inhibition seemed to be due to a retarded electron transfer from the water-oxidizing complex to Y(Z)(+), which was observed in the decay kinetics of the Y(Z)(+) signal induced by illumination above 250 K. These results show that formate induces inhibition of water oxidation reactions as well as electron transfer on the PS II acceptor side. The inhibition effects of formate in PS II were found to be reversible, indicating no destructive effect on the reaction center induced by formate.     

Factors influencing the formation of modified S2 EPR signal and the S3 EPR signal in Ca(2+)-depleted photosystem II

NaCl/EGTA-washing of photosystem II (PS-II) results in the removal of Ca2+ and the inhibition of oxygen evolution. Two new EPR signals were observed in such samples: a stable and modified S2 multiline signal and an S3 signal [(1989) Biochemistry 28, 8984-8989]. Here, we report what factors are responsible for the modifications of the S2 signal and the observation of the S3 signal. The following results were obtained. (i) The stable, modified, S2 multiline signal can be induced by the addition of high concentrations of EGTA or citrate to PS-II membranes which are already inhibited by Ca(2+)-depletion. (ii) The carboxylic acids act in the S3-state, are much less effective in S2 and have no effect in the S1-state. (iii) The extrinsic polypeptides (17- and 23-kDa) are not required to observe either the modified S2 signal or the S3 signal. However, they do influence the splitting and the lifetime of the S3 signal, and they seem to have a slight influence on the hyperfine pattern of the S2 signal. (iv) The S3 signal can be observed in Ca(2+)-depleted PS-II which does not exhibit the modified multiline signal. Then, it is proposed that formation of histidine radical during the S2 to S3 transition in Ca(2+)-depleted PS-II [(1990) Nature 347, 303-306] also occurs in functional PS-II.     

Low-temperature photochemistry in photosystem II from Thermosynechococcus elongatus induced by visible and near-infrared light

The active site for water oxidation in photosystem II (PSII) consists of a Mn4Ca cluster close to a redox-active tyrosine residue (TyrZ). The enzyme cycles through five sequential oxidation states (S0 to S4) in the water oxidation process. Earlier electron paramagnetic resonance (EPR) work showed that metalloradical states, probably arising from the Mn4 cluster interacting with TyrZ., can be trapped by illumination of the S0, S1 and S2 states at cryogenic temperatures. The EPR signals reported were attributed to S0TyrZ., S1TyrZ. and S2TyrZ., respectively. The equivalent states were examined here by EPR in PSII isolated from Thermosynechococcus elongatus with either Sr or Ca associated with the Mn4 cluster. In order to avoid spectral contributions from the second tyrosyl radical, TyrD., PSII was used in which Tyr160 of D2 was replaced by phenylalanine. We report that the metalloradical signals attributed to TyrZ. interacting with the Mn cluster in S0, S1, S2 and also probably the S3 states are all affected by the presence of Sr. Ca/Sr exchange also affects the non-haem iron which is situated approximately 44 A units away from the Ca site. This could relate to the earlier reported modulation of the potential of QA by the occupancy of the Ca site. It is also shown that in the S3 state both visible and near-infrared light are able to induce a similar Mn photochemistry.     

Low-temperature electron transfer in photosystem II: a tyrosyl radical and semiquinone charge pair

The states induced by illumination at 7 K in the oxygen-evolving enzyme (PSII) from Thermosynechococcus elongatus were studied by EPR. In the S(0) and S(1) redox states, two g approximately 2 EPR signals, a split signal and a g = 2.03 signal, respectively, were generated by illumination with visible light. These signals were comparable to those already reported in plant PSII in terms of their g value, shape, and stability at low temperatures. We report that the formation and decay of these signals correlate with EPR signals from the semiquinone of the first quinone electron acceptor, Q(A)(-). The light-induced EPR signals from oxidized side-path electron donors (Cyt b(559), Car, and Chl(Z)) were also measured, and from these and the signals from Q(A)(-), estimates were made of the proportion of centers involved in the formation of the g approximately 2 signals (approximately 50% in S(0) and 40% in S(1)). Comparisons with the signals generated in plant PSII indicated approximately similar yields for the S(0) split signal. A single laser flash at 7 K induced more than 75% of the maximum split and g = 2.03 EPR signal observed by continuous illumination, with no detectable oxidation of side-path donors. The matching electron acceptor side reactions, the high quantum yield, and the relatively large proportion of centers involved support earlier suggestions that the state being monitored is Tyr(Z)(*)Q(A)(-), with the g approximately 2 EPR signals arising from Tyr(Z)(*) interacting magnetically with the Mn complex. The current picture of the photochemical reactions occurring in PSII at low temperatures is reassessed.     

The YF161D1 mutant of Synechocystis 6803 exhibits an EPR signal from a light-induced photosystem II radical.

The currently accepted model for the location of the redox-active tyrosines, D and Z, in photosystem II suggests that they are symmetrically located on the D1 and D2 polypeptides, which are believed to form the heterodimer core of the reaction center. Z, the electron conduit from the manganese catalytic site to the primary chlorophyll donor, has been identified with tyrosine-161 of D1. The YF161D1 mutant of Synechocystis 6803 [Debus, R. J., Barry, B. A., Sithole, I., Babcock, G. T., & McIntosh, L. (1988b) Biochemistry 27, 9071-9074; Metz, J. G., Nixon, P. J., Rogner, M., Brudvig, G. W., & Diner, B. A. (1989) Biochemistry 28, 6960-6969], in which this tyrosine has been changed to a phenylalanine, should have no light-induced EPR (electron paramagnetic resonance) signal from a tyrosine radical. This negative result has indeed been obtained in analysis of one of two independently constructed mutants through the use of a non-oxygen-evolving core preparation (Metz et al., 1989). Here, we present an analysis of a YF161D1 mutant through the use of a photosystem II purification procedure that gives oxygen-evolving particles from wild-type Synechocystis cultures. In our mutant preparation, a light-induced EPR signal from a photosystem II radical is observed under conditions in which, in a wild-type preparation, we can accumulate an EPR signal from Z+. This EPR signal has a different lineshape from that of the Z+ tyrosine radical, and spin quantitation shows that this radical can be produced in up to 60% of the mutant reaction centers. The EPR lineshape of this radical suggests that photosystem II reaction centers of the YF161D1 mutant contain a redox-active amino acid.     

Hydroxyl radical generation by photosystem II

The photogeneration of hydroxyl radicals (OH(*)) in photosystem II (PSII) membranes was studied using EPR spin-trapping spectroscopy. Two kinetically distinguishable phases in the formation of the spin trap-hydroxyl (POBN-OH) adduct EPR signal were observed: the first phase (t(1/2) = 7.5 min) and the second phase (t(1/2) = 30 min). The generation of OH(*) was found to be suppressed in the absence of the Mn-complex, but it was restored after readdition of an artificial electron donor (DPC). Hydroxyl radical generation was also lost in the absence of oxygen, whereas it was stimulated when the oxygen concentration was increased. The production of OH(*) during the first kinetic phase was sensitive to the presence of SOD, whereas catalase and EDTA diminished the production of OH(*) during the second kinetic phase. The POBN-OH adduct EPR signal during the first phase exhibits a similar pH-dependence as the ability to oxidize the non-heme iron, as monitored by the Fe(3+) (g = 8) EPR signal: both EPR signals gradually decreased as the pH value was lowered below pH 6.5 and were absent at pH 5. Sodium formate decreases the production of OH(*) in intact and Mn-deleted PSII membranes. Upon illumination of PSII membranes, both superoxide, as measured by EPR signal from the spin trap-superoxide (EMPO-OOH) adduct, and H(2)O(2), measured colormetrically, were generated. These results indicated that OH(*) is produced on the electron acceptor side of PSII by two different routes, (1) O(2)(*)(-), which is generated by oxygen reduction on the acceptor side of PSII, interacts with a PSII metal center, probably the non-heme iron, to form an iron-peroxide species that is further reduced to OH(*) by an electron from PSII, presumably via Q(A)(-), and (2) O(2)(*)(-) dismutates to form free H(2)O(2) that is then reduced to OH(*) via the Fenton reaction in the presence of metal ions, the most likely being Mn(2+) and Fe(2+) released from photodamaged PSII. The two different routes of OH(*) generation are discussed in the context of photoinhibition.     

Detection of an electron paramagnetic resonance signal in the S0 state of the manganese complex of photosystem II from Synechococcus elongatus.

The Mn(4)-cluster of photosystem II (PSII) from Synechococcus elongatus was studied by electron paramagnetic resonance (EPR) spectroscopy after a series of saturating laser flashes given in the presence of either methanol or ethanol. Results were compared to those obtained in similar experiments done on PSII isolated from plants. The flash-dependent changes in amplitude of the EPR multiline signals were virtually identical in all samples. In agreement with earlier work [Messinger, J., Nugent, J. H. A., and Evans, M. C. W. (1997) Biochemistry 36, 11055-11060; Ahrling, K. A., Peterson, S., and Styring, S. (1997) Biochemistry 36, 13148-13152], detection of an EPR multiline signal from the S(0) state in PSII from plants was only possible with methanol present. In PSII from S. elongatus, it is shown that the S(0) state exhibits an EPR multiline signal in the absence of methanol (however, ethanol was present as a solvent for the artificial electron acceptor). The hyperfine lines are better resolved when methanol is present. The S(0) multiline signals detected in plant PSII and in S. elongatus were similar but not identical. Unlike the situation seen in plant PSII, the S(2) state in S. elongatus is not affected by the addition of methanol in that (i) the S(2) multiline EPR signal is not modified by methanol and (ii) the spin state of the S(2) state is affected by infrared light when methanol is present. It is also shown that the magnetic relaxation properties of an oxidized low-spin heme, attributed to cytochrome c(550), vary with the S states. This heme then is in the magnetic environment of the Mn(4) cluster.     

Characteristics of the Photosystem II reaction centre. II. Electron donors

The properties of Photosystem II electron donation were investigated by EPR spectrometry at cryogenic temperatures. Using preparations from mutants which lacked Photosystem I, the main electron donor through the Photosystem II reaction centre to the quinone-iron acceptor was shown to be the component termed Signal II. A radical of 10 G line width observed as an electron donor at cryogenic temperatures under some conditions probably arises through modification of the normal pathway of electron donation. High-potential cytochrome b-559 was not observed on the main pathway of electron donation. Two types of PS II centres with identical EPR components but different electron-transport kinetics were identified, together with anomalies between preparations in the amount of Signal II compared to the quinone-iron acceptor. Results of experiments using cells from mutants of Scenedesmus obliquus confirm the involvement of the Signal II component, manganese and high-potential cytochrome b-559 in the physiological process leading to oxygen evolution.     

Spectroscopic characterization of triplet forming states in photosystem II.

Fluorescence and electron paramagnetic resonance (EPR) measurements have been applied to characterize chlorophyll triplet formation in the reaction center of photosystem II (PSII). A highly triplet forming state was generated in PSII membranes by chemical double reduction of the primary electron acceptor QA. In triplet forming PSII centers, the steady-state yield of chlorophyll fluorescence decreased to about 70% of the maximal fluorescence yield observed in closed PSII centers in which QA is singly reduced. The results are well interpreted in the framework of a model where the charge state of QA electrostatically controls the yield of primary charge separation [Schatz, G. H., Brock, H., & Holzwarth, A. R. (1988) Biophys. J. 54, 397-405]. Thus, high triplet yield and decreased, although still quite high, fluorescence indicate a charge-neutralized state of PSII in which QA is singly or doubly reduced and protonated or absent. The EPR signal of the triplet primary chlorophyll donor, 3P680, is suppressed by illumination at 77 K concomitant with the formation of a cationic radical (g = 2.0025-2.0027, and 0.92 mT wide) that is stable in the dark. This is attributed to the oxidation of an accessory chlorophyll (Chl) in the vicinity of P680. Electrostatic repulsion between Chl+ and P680+ is likely to prevent primary charge separation, and in turn triplet formation, providing a further example of electrostatic control of primary charge separation. The triplet P680 EPR signal is also suppressed in the presence of oxygen. This effect, which is almost completely reversible by removing the oxygen, is attributed to the interaction of triplet P680 with triplet O2.     

Light-adaptation of photosystem II is mediated by the plastoquinone pool

During the first few enzymatic turnovers after dark-adaptation of photosystem II (PSII), the relaxation rate of the EPR signals from the Mn cluster and Y(D)(*) are significantly enhanced. This light-adaptation process has been suggested to involve the appearance of a new paramagnet on the PSII donor side [Peterson, S., Ahrling, K., H?gblom, J., and Styring, S. (2003) Biochemistry 42, 2748-2758]. In the present study, a correlation is established between the observed relaxation enhancement and the redox state of the quinone pool. It is shown that the addition of quinol to dark-adapted PSII membrane fragments induces relaxation enhancement already after a single oxidation of the Mn, comparable to that observed after five oxidations in samples with quinones (PPBQ or DQ) added. The saturation behavior of Y(D)(*) revealed that with quinol added in the dark, a single flash was necessary for the relaxation enhancement to occur. The quinol-induced relaxation enhancement of PSII was also activated by illumination at 200 K. Whole thylakoids, with no artificial electron acceptor present but with an intact plastoquinone pool, displayed the same relaxation enhancement on the fifth flash as membrane fragments with exogenous quinones present. We conclude that (i) reduction of the quinone pool induces the relaxation enhancement of the PSII donor-side paramagnets, (ii) light is required for the quinol to effect the relaxation enhancement, and (iii) light-adaptation occurs in the intact thylakoid system, when the endogenous plastoquinone pool is gradually reduced by PSII turnover. It seems clear that a species on the PSII donor side is reduced by the quinol, to become a potent paramagnetic relaxer. On the basis of XANES reports, we suggest that this species may be the Mn ions not involved in the cyclic redox changes of the oxygen-evolving complex.     

Competitive binding of acetate and chloride in photosystem II.

The binding of chloride and acetate to photosystem II (PSII) was examined to elucidate the mechanism of acetate inhibition. The mode of inhibition was studied, and individual binding sites were assigned by steady-state O2 evolution measurements in correlation with electron paramagnetic resonance (EPR) results. Two binding sites were found for acetate, one chloride-sensitive on the electron donor side and one chloride-insensitive on the electron acceptor side. The respective binding constants were as follows: KCl = 0.5 +/- 0.2 mM (chloride binding to the donor side), KI = 16 +/- 5 mM (acetate binding to the donor side), and KI' = 130 +/- 40 mM (acetate binding to the acceptor side). When acetate was bound to the acceptor side of PSII, 200 K illumination induced a narrowed form of the QA-FeII EPR signal, the yield of which was independent of the chloride concentration. When acetate was bound to the donor side, room-temperature illumination produced the S2YZ* state. EPR measurements showed that both the yield and formation rate of this state increased with acetate concentration. Increasing chloride concentrations slowed the rate of formation of the S2YZ* state, but did not affect the steady-state yield of the S2YZ* state. These findings indicate that the light-induced reactions in acetate-inhibited PSII are modulated by both donor side and acceptor side binding of acetate, while the steady-state yield of the S2YZ* state at the high PSII concentrations used for EPR measurements depends primarily on acceptor side turnover. Our data further support a close proximity of chloride to YZ*, indicating a possible role for chloride in the electron-transfer mechanism at the O2-evolving complex.     

EPR signals from modified charge accumulation states of the oxygen evolving enzyme in Ca2+-deficient photosystem II

Photosystem II enriched membranes were depleted of Ca2+ and the 17- and 23-kDa polypeptides by treatment with NaCl and EGTA. The 17- and 23-kDa polypeptides were then reconstituted. This preparation was incapable of O2 evolution until Ca2+ was added. An EPR study revealed the presence of two new EPR signals. One of these is a modified S2 multiline signal with an isotropic g value of 1.96 with at least 26 hyperfine peaks (average spacing 55 G) distributed over approximately 1600 G. The other is a near-Gaussian signal with an isotropic g value of 2.004, which is attributed to a formal S3 state. Experiments involving the interconversion of these signals and the effect of Ca2+ and Sr2+ rebinding provide evidence for these assignments. From these results the following conclusions are drawn: (1) These results are consistent with our earlier demonstration that charge accumulation is blocked after formation of S3 when Ca2+ is deficient. (2) Binding of the 17- and 23-kDa polypeptides to photosystem II in the absence of Ca2+ results in the perturbation of the Mn cluster. This is taken as a further indication that the Ca2+-binding site is close to or even an integral part of the Mn cluster. (3) The S3 signal may arise from an organic free radical interacting magnetically with the Mn cluster. However, other possible origins for this signal, including the Mn cluster itself, must also be considered.     

EPR spectroscopy of the manganese cluster of photosystem II

Electron paramagnetic resonance (EPR) spectroscopy is a valuable tool for understanding the oxidation state and chemical environment of the Mn₄Ca cluster of photosystem II. Since the discovery of the multiline signal from the S₂ state, EPR spectroscopy has continued to reveal details about the catalytic center of oxygen evolution. At present EPR signals from nearly all of the S-states of the Mn₄Ca cluster, as well as from modified and intermediate states, have been observed. This review article describes the various EPR signals obtained from the Mn₄Ca cluster, including the metalloradical signals due to interaction of the cluster with a nearby organic radical.     

EPR characterization of photosystem II from different domains of the thylakoid membrane

We report electron paramagnetic resonance (EPR) studies on photosystem II (PSII) from higher plants in five different domains of the thylakoid membrane prepared by sonication and two-phase partitioning. The domains studied were the grana core, the entire grana stack, the grana margins, the stroma lamellae and the purified stromal fraction, Y100. The electron transport properties of both donor and acceptor sides of PSII such as oxygen evolution, cofactors Y D, Q A, the CaMn 4-cluster, and Cytb 559 were investigated. The PSII content was estimated on the basis of oxidized Y D and Q A (-) Fe (2+) signal from the acceptor side vs Chl content (100% in the grana core fraction). It was found to be about 82% in the grana, 59% in the margins, 35% in the stroma and 15% in the Y100 fraction. The most active PSII centers were found in the granal fractions as was estimated from the rates of electron transfer and the S 2 state multiline EPR signal. In the margin and stroma fractions the multiline signal was smaller (40 and 33%, respectively). The S 2 state multiline could not be induced in the Y100 fraction. In addition, the oxidized LP Cytb 559 prevailed in the stromal fractions while the HP form dominated in the grana core. The margins and entire grana fractions have Cytb 559 in both potential forms. These data together with previous analyses indicate that the sequence of activation of the PSII properties can be represented as: PSII content > oxygen evolution > reduced Cytb 559 > dimerization of PSII centers in all fractions of the thylakoid membrane with the gradual increase from stromal fractions via margin to the grana core fraction. The results further support the existence of a PSII activity gradient which reflects lateral movement and photoactivation of PSII centers in the thylakoid membrane. The possible role of the PSII redox components in this process is discussed.