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Electron Paramagnetic Resonance Signals from the S 3 State of the Oxygen-Evolving Complex. A Broadened Radical Signal Induced by Low-Temperature Near-Infrared Light Illumination †
Abstract
The tetranuclear manganese cluster responsible for the oxidation of water in photosystem II cycles through five redox states denoted S(i)() (i = 0, 1, 2, 3, 4). Progress has been made recently in the detection of weak low-field EPR absorptions in both the perpendicular and parallel modes, associated with the integer spin state S(3) [Matsukawa, T., Mino, H., Yoneda, D., and Kawamori, A. (1999) Biochemistry 38, 4072-4077]. We confirm observation of these signals and have obtained them in high yield by illumination of photosystem II membranes, in which the non-heme iron was chemically preoxidized. It is shown that a split g = 4 signal accompanies the S(3) state signals. The signals diminish in the presence of ethanol and vanish in the presence of methanol. This effect is similar to that exerted by these alcohols to the high-spin component (g = 4.1) of the S(2) state and suggests that the latter spin configuration is the precursor of the S(3) state low-field signals. The S(3) state shows similar sensitivity to infrared illumination as has been observed previously in the S(2) state [Boussac, A., Un, S., Horner, O., and Rutherford, A. W. (1998) Biochemistry 37, 4001-4007]. Illumination of the S(3) state with near-infrared light (700-900 nm), at temperatures around 50 K, results in the modification of the low-field signals and most notably to the appearance of a broad (DeltaH approximately 200 G) radical-type signal centered at g = 2. The signal is tentatively assigned to the interaction of the Mn cluster in a modified S(2) state with a radical.
... The observations of the S 2 -S 3 transition have received two The S 3 state has been particularly challenging to study by electron paramagnetic resonance (EPR) methods because of the even number of unpaired electrons in the cluster, which results in an integer value for the total spin. Early X-band EPR studies of the S 3 state documented weak low-field signals [64,65] that were considered consistent with a spin S = 1 assignment. Subsequent Q-band studies by Sanakis et al. [66] showed that only a total effective spin of S = 3 could fit both the X-and the Q-band data. ...
... Inorganics 2019, 7, x FOR PEER REVIEW 5 of 30 [64,65] that were considered consistent with a spin S = 1 assignment. Subsequent Q-band studies by Sanakis et al. [66] showed that only a total effective spin of S = 3 could fit both the X-and the Q-band data. ...
... In parallel to the above coherent picture, there are clear and incontrovertible indications that the S 3 state is heterogeneous, i.e., that it contains multiple forms that are spectroscopically distinct [65,[80][81][82][83][84]. A somewhat simplified account of the observations is that the S 3 state contains a population (perhaps up to 30-40%) that can absorb near-infrared (NIR) light and "regress" to an S 2 Y Z · state. ...
The catalytic cycle of the oxygen-evolving complex (OEC) of photosystem II (PSII) comprises five intermediate states Si (i = 0–4), from the most reduced S0 state to the most oxidized S4, which spontaneously evolves dioxygen. The precise geometric and electronic structure of the Si states, and hence the mechanism of O–O bond formation in the OEC, remain under investigation, particularly for the final steps of the catalytic cycle. Recent advances in protein crystallography based on X-ray free-electron lasers (XFELs) have produced new structural models for the S3 state, which indicate that two of the oxygen atoms of the inorganic Mn4CaO6 core of the OEC are in very close proximity. This has been interpreted as possible evidence for “early-onset” O–O bond formation in the S3 state, as opposed to the more widely accepted view that the O–O bond is formed in the final state of the cycle, S4. Peroxo or superoxo formation in S3 has received partial support from computational studies. Here, a brief overview is provided of spectroscopic information, recent crystallographic results, and computational models for the S3 state. Emphasis is placed on computational S3 models that involve O–O formation, which are discussed with respect to their agreement with structural information, experimental evidence from various spectroscopic studies, and substrate exchange kinetics. Despite seemingly better agreement with some of the available crystallographic interpretations for the S3 state, models that implicate early-onset O–O bond formation are hard to reconcile with the complete line of experimental evidence, especially with X-ray absorption, X-ray emission, and magnetic resonance spectroscopic observations. Specifically with respect to quantum chemical studies, the inconclusive energetics for the possible isoforms of S3 is an acute problem that is probably beyond the capabilities of standard density functional theory.
... Owing to the interaction between Mn 4 Ca and TyrZ to the water oxidation process, the study of the Mn-TyrZ • metalloradical intermediates has gained interest in recent years. EPR spectroscopy has been used extensively for the study of these intermediates (Nugent et al. 2002;Ioannidis and Petrouleas 2000;Nugent et al. 2003;Zhang and Styring 2003;Ioannidis et al. 2006;Sioros et al. 2007;Havelius et al. 2010;Zahariou et al. 2014). The magnetic interaction between the Mn-cluster and TyrZ • at temperatures lower than 100 K causes the fine features in the EPR spectra of all the metalloradical intermediates (Zahariou et al. 2007, Szalai et al. 1998. ...
... Å expected values. An alternative explanation for the above deviation is the following: while the other metalloradical states are formed by illumination at cryogenic temperatures (Nugent et al. 2002;Ioannidis and Petrouleas 2000;Nugent et al. 2003;Zhang and Styring 2003), the S 3 TyrZ • in the presence of MeOH is trapped at temperatures near the onset of the S 3 to S 0 transition (*250 K). It is therefore likely that during the oxidation of TyrZ • its proton moves closer to Asn298, via His190 (Chrysina et al. 2011). ...
... This results in the shifting of the center of the effective spin density of the two fragments of the cluster and therefore in the modification of the effective distances of TyrZ from both of Mn(IV) 3 Ca and Mn(IV) species. Furthermore, contrary to the other metalloradical states which are formed by illumination at temperatures \77 K (Nugent et al. 2002;Ioannidis and Petrouleas 2000;Nugent et al. 2003;Zhang and Styring 2003), S 3 TyrZ • in the presence of MeOH is trapped at much higher temperature (*250 K) (Zahariou et al. 2014). Consequently, a secondary proton transfer from TyrZ to Asn298, via His190, is possible. ...
The intermediates trapped during the transitions between the consecutive S-states of the oxygen-evolving complex (OEC) of photosystem II (PSII) contain the free radical TyrZ• interacting magnetically with the Mn-cluster (Mn4Ca). In this paper, we present a theoretical study of the EPR spectrum of the S3TyrZ• metalloradical intermediate state, which has been recently detected in MeOH-containing PSII preparations. For this analysis, we use two different approximations: the first, simpler one, is the point-dipole approach, where the two interacting spins are the S = 1/2 of TyrZ• and the ground spin state of S = 3 of the OEC being in the S3 state. The second approximation is based on previous proposals indicating that the ground spin state (S
G = 3) of the S3 state arises from an antiferromagnetic exchange coupling between the S = 9/2 of the Mn(IV)3CaO4 and the S = 3/2 of the external Mn(IV) of the OEC. Under the above assumption, the second approximation involves three interacting spins, denoted S
A(Mn(IV)3Ca) = 9/2, S
B(Mn(IV)) = 3/2 and S
C(TyrZ•) = 1/2. Accordingly, the tyrosine radical is exposed to dipolar interactions with both fragments of the OEC, while an antiferromagnetic exchange coupling within the “3 + 1” structural motif of the OEC is also considered. By application of the first-point-dipole approach, the inter-spin distance that simulates the experimental spectrum is not consistent with the theoretical models that were recently reported for the OEC in the S3 state. Instead, the recent models are consistent with the results of the analysis that is performed by using the second, more detailed, approach.
... This population responds to NIR illumination at cryogenic temperatures by forming a split S 0 2 Y Z c EPR signal, where the inorganic cluster is reduced by the nearby Y Z residue to an "S 2 -like" electronic conguration. [24][25][26][27][28][29][30][31][32] As such, this species is expected to represent a precursor of the alloctahedral S 3 state form. The structural identity of this species is unknown and its properties are puzzling because Mn NIR photochemistry, observed also in lower S-states, is typically associated with the oxidation state 3+, whereas an all-Mn(IV) cluster is expected for the S 3 state. ...
... To study the S 3 state by EPR, dark-adapted (S 1 -state) samples are illuminated by two ashes at room temperature and rapidly frozen to cryogenic temperatures. 24,25,93 As described in the introduction, this procedure captures an EPR signal consistent with a structure that contains four octahedrally coordinated Mn(IV) ions, which is considered the end point of the S 2 -S 3 transition. 21 Compared to the structural forms present in the S 2 state, this "nal" S 3 species is characterized by an open-cubane topology, but with the crucial difference that the coordination number of all Mn ions is six, due to insertion of a water-derived ligand (see the S 3 state model of Fig. 1). ...
... Specically, the state formed aer NIR excitation at 77 K of the NIR-active form of S 3 contains a tyrosyl radical (with an EPR signal at g z 2) interacting with a high-spin form of the inorganic cluster (attributed to S ¼ 7/2, with an EPR signal observed around g z 5). [24][25][26][27][28][29]32 Since this state results from the one-electron oxidation of tyrosine Y Z by the inorganic cofactor, it is designated as S 0 2 Y Z c, where S 0 2 denotes an electronic conguration of the Mn cluster that is singly reduced compared to S 3 , but at a geometry similar to its parent S 3 state. A comparable EPR signal measured in samples advanced to S 3 and stored for extended periods at liquid nitrogen temperature, points towards a similar conguration of the OEC formed in the absence of the Y Z c radical, where the reduced state of the cluster is generated via charge recombination with the plastoquinone Q A À . ...
Among the four photo-driven transitions of the water-oxidizing tetramanganese-calcium cofactor of biological photosynthesis, the second-last step of the catalytic cycle, that is the S2 to S3 state transition, is the crucial step that poises the catalyst for the final O-O bond formation. This transition, whose intermediates are not yet fully understood, is a multi-step process that involves the redox-active tyrosine residue and includes oxidation and deprotonation of the catalytic cluster, as well as the binding of a water molecule. Spectroscopic data has the potential to shed light on the sequence of events that comprise this catalytic step, which still lacks a structural interpretation. In this work the S2-S3 state transition is studied and a key intermediate species is characterized: it contains a Mn3O4Ca cubane subunit linked to a five-coordinate Mn(iv) ion that adopts an approximately trigonal bipyramidal ligand field. It is shown using high-level density functional and multireference wave function calculations that this species accounts for the near-infrared absorption and electron paramagnetic resonance observations on metastable S2-S3 intermediates. The results confirm that deprotonation and Mn oxidation of the cofactor must precede the coordination of a water molecule, and lead to identification of a novel low-energy water binding mode that has important implications for the identity of the substrates in the mechanism of biological water oxidation.
... Trapping of the Mn-TyrZ Å metalloradical intermediates has been at the focus of considerable interest in recent years. Various intermediates have been identified and characterized by EPR spectroscopy, including the S 0 Y Å Z , S 1 Y Å Z , S 2 Y Å Z and variants of them obtained after treatments that alter the electronic configuration of the OEC but preserve the oxygen evolution (addition of a few percent (v/v) methanol, replacement of Ca with Sr)[5][6][7][8][9][10][11]. These signals are produced by direct visible-light illumination at liquid helium temperatures[5][6][7][8][9][10][11]with the exception of the S 2 Y Å Z in the untreated sample, which is trapped by visible-light illumination at 77–190 K[9]. ...
... Various intermediates have been identified and characterized by EPR spectroscopy, including the S 0 Y Å Z , S 1 Y Å Z , S 2 Y Å Z and variants of them obtained after treatments that alter the electronic configuration of the OEC but preserve the oxygen evolution (addition of a few percent (v/v) methanol, replacement of Ca with Sr)[5][6][7][8][9][10][11]. These signals are produced by direct visible-light illumination at liquid helium temperatures[5][6][7][8][9][10][11]with the exception of the S 2 Y Å Z in the untreated sample, which is trapped by visible-light illumination at 77–190 K[9]. The notable missing metalloradical intermediate is that of the S 3 to S 0 transition. ...
... intermediates in PSII preparations containing methanol, at temperatures close to the half inhibition temperature of the S state transitions. The trapping of both metalloradicals at such high temperatures is an important result, since until recently it was thought that the metalloradical intermediates could only be trapped at temperatures well below the onset of the S-state transitions[5][6][7][8][9][10][11]. Moreover, the trapping of intermediates during the S-state transitions, likely indicates that they represent structures relevant to mechanistic events which occur at ambient temperatures. ...
We report the trapping of two metalloradical intermediates corresponding to the transitions S2 to S3 and S3 to S0 of the oxygen evolving complex of photosystem II, in preparations containing methanol, at temperatures near that of half inhibition of the respective S-state transitions. The first intermediate, with an EPR width of 160 G, is assigned to S2Yz(•), based on its similarity to the one previously characterized after trapping at 10 K. The second with a splitting of ∼100 G is tentatively assigned to S3Yz(•). The S3Yz(•) EPR signal is weaker than the S2Yz(•) one, and both are stable at cryogenic temperatures.
... However, in the S 2 and S 3 states split EPR signals can also be induced by near-infrared (NIR) light [17,18]. The wavelength dependence has been described for the S 3 state and stretches to 900 nm [16,19], clearly out of reach for photosynthetic charge separation, which is known to be inactive above 730 nm at the very low temperatures used [20]. ...
... The wavelength dependence has been described for the S 3 state and stretches to 900 nm [16,19], clearly out of reach for photosynthetic charge separation, which is known to be inactive above 730 nm at the very low temperatures used [20]. It was previously thought that only NIR illumination and not visible light illumination could induce the split EPR signal in the S 3 state [17,21,22]. However, recently [16] we demonstrated that the Split S 3 EPR signal can be induced by monochromatic light in the spectral range 415-900 nm. ...
... wide spectral range between 415 and 900 nm, and not only by light in the near-infrared region as first proposed [17]. A question remaining from the previous study is whether a single mechanism can explain the induction of the Split S 3 signal across the entire wavelength range, or whether wavelength-dependent mechanisms are required. ...
Metalloradical EPR signals have been found in intact Photosystem II at cryogenic temperatures. They reflect the light-driven formation of the tyrosine Z radical (Y(Z)) in magnetic interaction with the CaMn(4) cluster in a particular S state. These so-called split EPR signals, induced at cryogenic temperatures, provide means to study the otherwise transient Y(Z) and to probe the S states with EPR spectroscopy. In the S(0) and S(1) states, the respective split signals are induced by illumination of the sample in the visible light range only. In the S(3) state the split EPR signal is induced irrespective of illumination wavelength within the entire 415-900nm range (visible and near-IR region) [Su, J. H., Havelius, K. G. V., Ho, F. M., Han, G., Mamedov, F., and Styring, S. (2007) Biochemistry 46, 10703-10712]. An important question is whether a single mechanism can explain the induction of the Split S(3) signal across the entire wavelength range or whether wavelength-dependent mechanisms are required. In this paper we confirm that the Y(Z) radical formation in the S(1) state, reflected in the Split S(1) signal, is driven by P680-centered charge separation. The situation in the S(3) state is different. In Photosystem II centers with pre-reduced quinone A (Q(A)), where the P680-centered charge separation is blocked, the Split S(3) EPR signal could still be induced in the majority of the Photosystem II centers using both visible and NIR (830nm) light. This shows that P680-centered charge separation is not involved. The amount of oxidized electron donors and reduced electron acceptors (Q(A)(-)) was well correlated after visible light illumination at cryogenic temperatures in the S(1) state. This was not the case in the S(3) state, where the Split S(3) EPR signal was formed in the majority of the centers in a pathway other than P680-centered charge separation. Instead, we propose that one mechanism exists over the entire wavelength interval to drive the formation of the Split S(3) signal. The origin for this, probably involving excitation of one of the Mn ions in the CaMn(4) cluster in Photosystem II, is discussed.
... Cyanobacteria (untreated preparations) don't show the g = 4.1 form [32]. The S 3 state being an integer spin state is more difficult to probe, but it is characterized by broad bimodal signals at around g = 10 and ∼ 4 in perpendicular mode [33,34], and respective Q-band resonances [35], and several other smaller signals detected recently [36] all attributed to an S = 3 spin configuration [35,36]. The signals are similar in plant and cyanobacterial PSII preparations, implying that they result from oxidation of the multiline configuration of S 2 . ...
... No evidence for heterogeneity in S 3 has been reported so far. A number of transient isoelectronic configurations can be however obtained by NIR and visible excitation at liquid helium temperatures [34,373839 a property that parallels the NIR-induced conversion of the S 2 multiline form to higher spin-state forms [40]. The question, which arises then is, what is the fate of the g = 4.1 conformation of S 2 upon advancement to S 3 . ...
... The S 3 state was approximately quantified by comparison of the size of the characteristic signal at g = 10 with earlier spectra (after appropriate normalization of their S 2 state signals) obtained from samples in which the S 3 to S 0 transition was inhibited by the presence of atrazine [34]. ...
The oxygen evolving complex of Photosystem II undergoes four light-induced oxidation transitions, S(0)-S(1),...,S(3)-(S(4))S(0) during its catalytic cycle. The oxidizing equivalents are stored at a (Mn)(4)Ca cluster, the site of water oxidation. EPR spectroscopy has yielded valuable information on the S states. S(2) shows a notable heterogeneity with two spectral forms; a g=2 (S=1/2) multiline, and a g=4.1 (S=5/2) signal. These oscillate in parallel during the period-four cycle. Cyanobacteria show only the multiline signal, but upon advancement to S(3) they exhibit the same characteristic g=10 (S=3) absorption with plant preparations, implying that this latter signal results from the multiline configuration. The fate of the g=4.1 conformation during advancement to S(3) is accordingly unknown. We searched for light-induced transient changes in the EPR spectra at temperatures below and above the half-inhibition temperature for the S(2) to S(3) transition (ca 230K). We observed that, above about 220K the g=4.1 signal converts to a multiline form prior to advancement to S(3). We cannot exclude that the conversion results from visible-light excitation of the Mn cluster itself. The fact however, that the conversion coincides with the onset of the S(2) to S(3) transition, suggests that it is triggered by the charge-separation process, possibly the oxidation of tyr Z and the accompanying proton relocations. It therefore appears that a configuration of (Mn)(4)Ca with a low-spin ground state advances to S(3).
... At the start of the new millennium, split EPR signals of a metalloradical nature induced in intact PSII were discovered (127)(128)(129)(130)(131). They can all be induced by illumination at cryogenic temperatures and found to oscillate with the S-states (summarized in Table 1, Figure 9). ...
... The split EPR signals in this category were first discovered to be induced by near-infrared illumination (131,153) and later confirmed to also be inducible with (pure) visible light (Paper I, IV, VI, VIII). ...
... The Split S 3 signal is formed in PSII samples exposed to 2 flashes (dominated by the S 3 -state) by illumination both with visible light [104,107] and near infrared light [104,113] at cryogenic temperatures. In contrast to the signal induced by visible light, the near infrared induced signal is very stable at 5 K and shows no observable decay for at least 30 min [104]. ...
... Fig. 6C displays the Split S 3 signals induced by visible light (black line) and 830 nm light (grey line) at 5 K in Arabidopsis. Both signals are very similar to the corresponding signals reported from spinach [107,109,113] and cyanobacteria [114]. The signal has a double trough at g = 1.95 and 1.93 position and a peak (for the near infrared induced signal) at g = 2.06 (Table 2). ...
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 S₀- and S₂-states of CaMn₄O(x)-cluster in the water oxidation complex were characterized. Moreover, split EPR signals, the interaction signals from Y(Z) and CaMn₄O(x)-cluster in the S₀-, S₁-, S₂-, and the S₃-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 b₅₅₉ 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.
... In order to further investigate the S 2 → S 3 transition at different temperatures, we also measured the formation of the S 3 state. The S 3 state of the WOC is characterized by several EPR signals (Boussac et al. 2009;Havelius et al. 2010;Petrouleas et al. 2005;Matsukawa et al. 1999;Ioannidis and Petrouleas 2000;Ioannidis et al. 2002). We used the socalled Split S 3 signal, obtained by the infrared light illumination of the S 3 state at 5 K. ...
In Photosystem II, light-induced water splitting occurs via the S state cycle of the CaMn4O5-cluster. To understand the role of various possible conformations of the CaMn4O5-cluster in this process, the temperature dependence of the S1 → S2 and S2 → S3 state transitions, induced by saturating laser flashes, was studied in spinach photosystem II membrane preparations under different conditions. The S1 → S2 transition temperature dependence was shown to be much dependent on the type of the cryoprotectant and presence of 3.5% methanol, resulting in the variation of transition half-inhibition temperature by 50 K. No similar effect was observed for the S2 → S3 state transition, for which we also show that both the low spin g = 2.0 multiline and high spin g = 4.1 EPR configurations of the S2 state advance with similar efficiency to the S3 state, both showing a transition half-inhibition temperature of 240 K. This was further confirmed by following the appearance of the Split S3 EPR signal. The results are discussed in relevance to the functional and structural heterogeneity of the water oxidizing complex intermediates in photosystem II.
... Integer spin state EPR signals, initially attributed to S = 1 by X-band EPR spectroscopy [27,29] and later revised to S = 3 by Q-band experiments, [21] have long been associated with the S 3 state. A commonly accepted geometric conformation of the OEC cluster that satisfies the spectroscopic requirements of an S = 3 ground state and for all Mn centers being isotropic octahedrally coordinated Mn IV ions, as indicated by electronelectron double resonance (ELDOR) detected nuclear magnetic resonance experiments (EDNMR), [6] is an "oxo-hydroxo" conformation (Figure 3), where a water-derived OH ligand completes the coordination sphere of Mn1 compared to the dominant isomeric conformation of the preceding S 2 state. ...
Electron paramagnetic resonance spectroscopy reveals that the final metastable catalytic state (S3) in biological water oxidation in higher plants is a mixture of a high‐spin and an intermediate‐spin form. These two forms represent distinct structural components related to water binding and activation on the path towards oxygen evolution.
Abstract
Among the intermediate catalytic steps of the water‐oxidizing Mn4CaO5 cluster of photosystem II (PSII), the final metastable S3 state is critically important because it binds one substrate and precedes O2 evolution. Herein, we combine X‐ and Q‐band EPR experiments on native and methanol‐treated PSII of Spinacia oleracea and show that methanol‐treated PSII preparations of the S3 state correspond to a previously uncharacterized high‐spin (S=6) species. This is confirmed as a major component also in intact photosynthetic membranes, coexisting with the previously known intermediate‐spin conformation (S=3). The high‐spin intermediate is assigned to a water‐unbound form, with a MnIV3 subunit interacting ferromagnetically via anisotropic exchange with a coordinatively unsaturated MnIV ion. These results resolve and define the structural heterogeneity of the S3 state, providing constraints on the S3 to S4 transition, on substrate identity and delivery pathways, and on the mechanism of O−O bond formation.
... Integer spin state EPR signals,initially attributed to S = 1 by X-band EPR spectroscopy [27,29] and later revised to S = 3 by Q-band experiments, [21] have long been associated with the S 3 state.Acommonly accepted geometric conformation of the OEC cluster that satisfies the spectroscopic requirements of an S = 3g round state and for all Mn centers being isotropic octahedrally coordinated Mn IV ions,asindicated by electronelectron double resonance (ELDOR) detected nuclear magnetic resonance experiments (EDNMR), [6] is an "oxo-hydroxo" conformation ( Figure 3), where aw ater-derived OH ligand completes the coordination sphere of Mn1 compared to the dominant isomeric conformation of the preceding S 2 state.M odified spectral forms of the S = 3E PR signal have been reported in cation (Ca 2+ /Sr 2+ )o ra nion (Cl À /I À )s ub-stituted and MeOH-treated cyanobacterial PSII. [15,28] By contrast, the S = 6s tate described in the present work for spinach PSII is experimentally identified for the first time, despite representing apparently the majority species.I ts drastically different spectroscopic properties are indicative of fundamental differences in the electronic and geometric structure of the cluster compared to the S = 3f orm. ...
Among the intermediate catalytic steps of the water‐oxidizing Mn4CaO5 cluster of photosystem II (PSII), the final metastable S3 state is critically important because it binds one substrate and precedes O2 evolution. Herein, we combine X‐ and Q‐band EPR experiments on native and methanol‐treated PSII of Spinacia oleracea and show that methanol‐treated PSII preparations of the S3 state correspond to a previously uncharacterized high‐spin (S=6) species. This is confirmed as a major component also in intact photosynthetic membranes, coexisting with the previously known intermediate‐spin conformation (S=3). The high‐spin intermediate is assigned to a water‐unbound form, with a MnIV3 subunit interacting ferromagnetically via anisotropic exchange with a coordinatively unsaturated MnIV ion. These results resolve and define the structural heterogeneity of the S3 state, providing constraints on the S3 to S4 transition, on substrate identity and delivery pathways, and on the mechanism of O−O bond formation.
... The WT-PSII core preparations show the typical S 3 EPR split signal [76] signified by a main radical peak at g ~ 2 having a width of 19.4 G (IR-illuminated) and two troughs at g = 1.93 and g = 1.95 (Fig. 6B). Subtraction of the IR induced split signal from the visible light induced split EPR spectrum reveals the Q A − Fe 2+ signal at g = 1.96, evidencing the successful charge separation at 5 K under visible illumination. ...
In photosynthesis, the oxygen-evolving complex (OEC) of the pigment-protein complex photosystem II (PSII) orchestrates the oxidation of water. Introduction of the V185N mutation into the D1 protein was previously reported to drastically slow O2-release and strongly perturb the water network surrounding the Mn4Ca cluster. Employing time-resolved membrane inlet mass spectrometry, we measured here the H218O/H216O-exchange kinetics of the fast (Wf) and slow (Ws) exchanging substrate waters bound in the S1, S2 and S3 states to the Mn4Ca cluster of PSII core complexes isolated from wild type and D1-V185N strains of Synechocystis sp. PCC 6803. We found that the rate of exchange for Ws was increased in the S1 and S2 states, while both Wf and Ws exchange rates were decreased in the S3 state. Additionally, we used EPR spectroscopy to characterize the Mn4Ca cluster and its interaction with the redox active D1-Tyr161 (YZ). In the S2 state, we observed a greatly diminished multiline signal in the V185N-PSII that could be recovered by addition of ammonia. The split signal in the S1 state was not affected, while the split signal in the S3 state was absent in the D1-V185N mutant. These findings are rationalized by the proposal that the N185 residue stabilizes the binding of an additional water-derived ligand at the Mn1 site of the Mn4Ca cluster via hydrogen bonding. Implications for the sites of substrate water binding are discussed.
... 3,13,25 Importantly, EPR experiments additionally indicate the presence of an EPR-silent form of the S 3 state that under IR illumination converts into the EPR-detectable S 2 Y Z state. 36,39,51 This indicates that the Mn 4 Ca cluster and D1-Tyr161 (Y Z ), the electron donor to P680 + , are in a delicate redox equilibrium in the S 3 state. [52][53][54] The EPR silent S 3 state has been tentatively assigned to the S B 3 or S BW 3 structures. ...
In photosynthesis, dioxygen formation from water is catalyzed by the oxygen evolving complex (OEC) in Photosystem II (PSII) that harbours the Mn4Ca cluster. During catalysis, the OEC cycles through five redox states, S0 to S4. In the S2 state, the Mn4Ca cluster can exist in two conformations, which are signified by the low-spin (LS) g = 2 EPR multiline signal and the high-spin (HS) g = 4.1 EPR signal. Here, we employed time-resolved membrane inlet mass spectrometry to measure the kinetics of H218O/H216O exchange between bulk water and the two substrate waters bound at the Mn4Ca cluster in the S2LS, S2HS, and the S3 states in both Ca-PSII and Sr-PSII core complexes from T. elongatus. We found that the slowly exchanging substrate water exchanges 10-times faster in the S2HS than in the S2LS state, and that the S2LS S2HS conversion has at physiological temperature an activation barrier of 17 1 kcal/mol. Of the presently suggested S2HS models, our findings are best in agreement with a water exchange pathway involving a S2HS state that has an open cubane structure with a hydroxide bound between Ca and Mn1. We also show that water exchange in the S3 state is governed by different equilibrium than in S2, and that the exchange of the fast substrate water in the S2 state is unaffected by Ca/Sr substitution. These findings support that (i) O5 is the slowly exchanging substrate water, with W2 being the only other option, and (ii) either W2 or W3 is the fast exchanging substrate. The three remaining options for O-O bond formation in PSII are discussed.
... Elucidating the structure of the S 3 state is central for understanding the water splitting process, as this is the highest oxidation state intermediate that precedes the O-O bond formation process. The EPR properties of the S 3 state have received more attention recently [33][34][35][36][37][38][39]. Two forms of S 3 exist: a) an EPR detectable form, attributed to an S = 3 state, and b) an EPR silent form that can be monitored by its photosensitivity to near-IR light at low temperature, back-reacting to form Yz • and a high spin form of the Mn cluster in the S 2 valence state. ...
Photosystem II (PSII) catalyzes light-driven water splitting in nature and is the key enzyme for energy input into the biosphere. Important details of its mechanism are not well understood. In order to understand the mechanism of water splitting, we perform here large-scale density functional theory (DFT) calculations on the active site of PSII in different oxidation, spin and ligand states. Prior to formation of the O-O bond, we find that all manganese atoms are oxidized to Mn(IV) in the S3 state, consistent with earlier studies. We find here, however, that the formation of the S3 state is coupled to the movement of a calcium-bound hydroxide (W3) from the Ca to a Mn (Mn1 or Mn4) in a process that is triggered by the formation of a tyrosyl radical (Tyr-161) and its protonated base, His-190. We find that subsequent oxidation and deprotonation of this hydroxide on Mn1 result in formation of an oxyl-radical that can exergonically couple with one of the oxo-bridges (O5), forming an O-O bond. When O2 leaves the active site, a second Ca-bound water molecule reorients to bridge the gap between the manganese ions Mn1 and Mn4, forming a new oxo-bridge for the next reaction cycle. Our findings are consistent with experimental data, and suggest that the calcium ion may control substrate water access to the water oxidation sites.
... The two resulting schemes ( Fig. 1) are usually referred to in the literature 15,28 as the low and high oxidation state paradigms or the low-valent (LV) and high-valent (HV) schemes. Although EPR results by different groups have been interpreted in support of either the LV 29,30 or the HV schemes, [31][32][33][34] unambiguous assignments of the ground state spin multiplicity 35 of all S i states have been made: S ¼ 1/2 (S 0 ), 36-39 S ¼ 0 (S 1 ), 40,41 S ¼ 1/2 and 5/2 (S 2 , g ¼ 2 and g $ 4.1 signals), 16,42,43 and S ¼ 3 (S 3 ), [44][45][46] and these studies oen provide information on low-lying excited states. ...
A central question in biological water splitting concerns the oxidation states of the manganese ions that comprise the oxygen-evolving complex of photosystem II. Understanding the nature and order of oxidation events that occur during the catalytic cycle of five S
i
states (i = 0-4) is of fundamental importance both for the natural system and for artificial water oxidation catalysts. Despite the widespread adoption of the so-called "high-valent scheme"-where, for example, the Mn oxidation states in the S2 state are assigned as III, IV, IV, IV-the competing "low-valent scheme" that differs by a total of two metal unpaired electrons (i.e. III, III, III, IV in the S2 state) is favored by several recent studies for the biological catalyst. The question of the correct oxidation state assignment is addressed here by a detailed computational comparison of the two schemes using a common structural platform and theoretical approach. Models based on crystallographic constraints were constructed for all conceivable oxidation state assignments in the four (semi)stable S states of the oxygen evolving complex, sampling various protonation levels and patterns to ensure comprehensive coverage. The models are evaluated with respect to their geometric, energetic, electronic, and spectroscopic properties against available experimental EXAFS, XFEL-XRD, EPR, ENDOR and Mn K pre-edge XANES data. New 2.5 K 55Mn ENDOR data of the S2 state are also reported. Our results conclusively show that the entire S state phenomenology can only be accommodated within the high-valent scheme by adopting a single motif and protonation pattern that progresses smoothly from S0 (III, III, III, IV) to S3 (IV, IV, IV, IV), satisfying all experimental constraints and reproducing all observables. By contrast, it was impossible to construct a consistent cycle based on the low-valent scheme for all S states. Instead, the low-valent models developed here may provide new insight into the over-reduced S states and the states involved in the assembly of the catalytically active water oxidizing cluster.
... 31,[35][36][37]58,59 For a long time, the Cyt b 559 /Chl Z /Car D2 pathway was thought to be the only functional pathway at 5 K and comparable low temperatures (there were no indications of Y Z oxidation). However, the understanding of the lowtemperature electron donation to P 680 + changed with the discovery that split EPR signals, which originate from the Y Z • radical in magnetic interaction with the CaMn 4 cluster, could be induced at 5−10 K. 48,50,51,60 Thus, it became obvious that Y Z could donate an electron to P 680 + also at ultralow temperatures. 53,54 Both pathways also work and compete with each other at intermediate temperatures (77−273 K) where the temperature-dependent partition ratio between them was first studied. ...
We have earlier shown that all electron transfer reactions in Photosystem II are operational up to 800 nm at room temperature [Thapper et al. (2009), Plant Cell 21, 2391-2401]. This led us to suggest an alternative charge separation pathway for far-red excitation. Here we extend these studies to very low temperature (5 K). Illumination of photosystem II (PS II) with visible light at 5 K is known to result in oxidation of almost similar amounts of YZ and the Cyt b559/ChlZ/CarD2 pathway. This is reproduced here using laser flashes at 532 nm and we find the partition ratio between the two pathways to be 1:0.8 at 5 K (the partition ratio is here defined as (yield of YZ/CaMn4 oxidation):(yield of Cyt b559/ChlZ/CarD2 oxidation)). The result using far red laser flashes is very different. We find partition ratios of 1.8 at 730 nm; 2.7 at 740 nm and >2.7 at 750 nm. No photochemistry involving these pathways is observed above 750 nm at this temperature. Thus, far-red illumination preferentially oxidizes YZ while the Cyt b559/ChlZ/CarD2 pathway is hardly touched. We propose that the difference in the partition ratio between visible and far-red light at 5 K reflects the formation of different first stable charge pair. In visible light, the first stable charge pair is considered to be PD1+Qa-. In contrast, we propose that the electron hole is residing on the ChlD1 molecule after illumination by far red at light 5 K resulting in the first stable charge pair being ChlD1+QA-. ChlD1 is much closer to YZ (11.3 Å) than to any component in Cyt b559/ChlZ/CarD2 pathway (closest distance is ChlD1 - CarD2 is 28.8 Å). This would then explain that far-red illumination preferentially drives efficient electron transfer from YZ. We also discuss mechanisms to account for the absorption of the far-red light and the existence of a hitherto unobserved charge transfer states. The involvement of two or more of the porphyrin molecules in the core of the Photosystem II reaction center is proposed.
... In the S 3 state, NIR illumination at ϳ4 K results in the formation of a split EPR signal (48,49), attributed to a (S 2 Tyr Z ⅐ )Ј state formed by NIR-induced conversion of the manganese cluster into an "activated" state able to oxidize Tyr Z and thus leading to the formation of (S 2 Tyr Z ⅐ )Ј at the expense of the S 3 Tyr Z state (50). This split signal is attributed to the magnetic interaction between Tyr Z ⅐ , with a spin state S ϭ 1/2 and the Mn 4 CaO 5 cluster possibly in a S ϭ 7/2 spin state (51), and as such is very sensitive to the geometry of the Tyr Z ⅐ /Mn 4 CaO 5 ensemble. ...
The main cofactors that determine the photosystem II (PSII) oxygen evolution activity are borne by the D1 and D2 subunits. In the cyanobacterium Thermosynechococcus elongatus, there are three psbA genes coding for D1. Among the 344 residues constituting D1, there are 21 substitutions between PsbA1 and PsbA3, 31 between PsbA1 and PsbA2, and 27 between PsbA2 and PsbA3. Here, we present the first study of PsbA2-PSII. Using EPR and UV-visible time-resolved absorption spectroscopy, we show that: (i) the time-resolved EPR spectrum of Tyr(Z)(•) in the (S(3)Tyr(Z)(•))' is slightly modified; (ii) the split EPR signal arising from Tyr(Z)(•) in the (S(2)Tyr(Z)(•))' state induced by near-infrared illumination at 4.2 K of the S(3)Tyr(Z) state is significantly modified; and (iii) the slow phases of P(680)(+) reduction by Tyr(Z) are slowed down from the hundreds of μs time range to the ms time range, whereas both the S(1)Tyr(Z)(•) → S(2)Tyr(Z) and the S(3)Tyr(Z)(•) → S(0)Tyr(Z) + O(2) transition kinetics remained similar to those in PsbA(1/3)-PSII. These results show that the geometry of the Tyr(Z) phenol and its environment, likely the Tyr-O···H···Nε-His bonding, are modified in PsbA2-PSII when compared with PsbA(1/3)-PSII. They also point to the dynamics of the proton-coupled electron transfer processes associated with the oxidation of Tyr(Z) being affected. From sequence comparison, we propose that the C144P and P173M substitutions in PsbA2-PSII versus PsbA(1/3)-PSII, respectively located upstream of the α-helix bearing Tyr(Z) and between the two α-helices bearing Tyr(Z) and its hydrogen-bonded partner, His-190, are responsible for these changes.
... One consequence of the functionality of the S 1 → S 2 transition at 77 K (albeit small) is that Tyr-Z also must be operational at this temperature. This was long over looked but it is now clear that Tyr-Z, similar to Tyr-D, can be oxidized efficiently and to high degree by illumination of intact PSII even at ultralow temperatures (5-10 K). 2 Starting around the beginning of the new millennium a series of split EPR signals of metallo-radical character were discovered when PSII was illuminated at cryogenic temperature [93][94][95][96][97]. The signals were found to oscillate with the S states [93,98] and have with time been shown to originate (with high probability) from Tyr-Z• in magnetic interaction with the CaMn 4 cluster. ...
Photosystem II (PSII), the thylakoid membrane enzyme which uses sunlight to oxidize water to molecular oxygen, holds many organic and inorganic redox cofactors participating in the electron transfer reactions. Among them, two tyrosine residues, Tyr-Z and Tyr-D are found on the oxidizing side of PSII. Both tyrosines demonstrate similar spectroscopic features while their kinetic characteristics are quite different. Tyr-Z, which is bound to the D1 core protein, acts as an intermediate in electron transfer between the primary donor, P(680) and the CaMn₄ cluster. In contrast, Tyr-D, which is bound to the D2 core protein, does not participate in linear electron transfer in PSII and stays fully oxidized during PSII function. The phenolic oxygens on both tyrosines form well-defined hydrogen bonds to nearby histidine residues, His(Z) and His(D) respectively. These hydrogen bonds allow swift and almost activation less movement of the proton between respective tyrosine and histidine. This proton movement is critical and the phenolic proton from the tyrosine is thought to toggle between the tyrosine and the histidine in the hydrogen bond. It is found towards the tyrosine when this is reduced and towards the histidine when the tyrosine is oxidized. The proton movement occurs at both room temperature and ultra low temperature and is sensitive to the pH. Essentially it has been found that when the pH is below the pK(a) for respective histidine the function of the tyrosine is slowed down or, at ultra low temperature, halted. This has important consequences for the function also of the CaMn₄ complex and the protonation reactions as the critical Tyr-His hydrogen bond also steer a multitude of reactions at the CaMn₄ cluster. This review deals with the discovery and functional assignments of the two tyrosines. The pH dependent phenomena involved in oxidation and reduction of respective tyrosine is covered in detail. This article is part of a Special Issue entitled: Photosystem II.
Nitric oxide (NO) is an important signaling molecule in plants, which regulates many processes in the cell. The various studies have been tried to understand the role of NO in plant cells especially in the chloroplast by exogenous application of gaseous molecule or donor of NO. Here, we review the important target sites of NO in plastids which are directly affected by the exogenous application of NO gas or NO donor, e.g., sodium nitroprusside (SNP). The studies show the positive effect of NO at low concentration (nanomoles) and inhibitory effect of NO at higher concentrations (micro- or millimole) on photosynthetic machinery in chloroplast. NO, thus by interacting with plastids proteins/enzymes, influencing photophosphorylation, electron transport activity, and oxidoreduction state of the Mn clusters of the oxygen-evolving complex, RuBisCo activity, stomatal conductance, and thereby modulating the overall photosynthesis of the plant cells. The detailed modulation of NO-induced changes in the photosynthetic apparatus on its functions and sensitivity are discussed here in this chapter.
The S3 state is currently the last observable intermediate prior to O−O bond formation at the oxygen evolving complex (OEC) of Photosystem II, and its electronic structure has been assigned to a homovalent MnIV4 core with an S = 3 ground state. While structural interpretations based on the EPR spectroscopic features of the S3 state provide valuable mechanistic insight, corresponding synthetic and spectroscopic studies on tetranuclear complexes mirroring the Mn oxidation states of the S3 state remain rare. Herein, we report the synthesis and characterization by XAS and multifrequency EPR spectroscopy of a MnIV4O4 cuboidal complex as a spectroscopic model of the S3 state. Results show that this MnIV4O4 complex has an S = 3 ground state with isotropic 55Mn hyperfine coupling constants of −75, −88, −91, and 66 MHz. These parameters are consistent with an αααβ spin topology approaching the trimer-monomer magnetic coupling model of pseudo-octahedral MnIV centers. Importantly, the spin ground state changes from S = 1/2 to S = 3 as the OEC is oxidized from the S2 state to the S3 state. This same spin state change is observed following the oxidation of the previously reported MnIIIMnIV3O4 cuboidal complex to the MnIV4O4 complex described here. This sets a synthetic precedent for the observed low-spin to high-spin conversion in the OEC.
Heterogeneity in intermediate catalytic states of the oxygen-evolving complex (OEC) of Photosystem II is known from a wide range of experimental and theoretical data, but its potential implications for the mechanism of water oxidation remain unexplored. We delineate the consequences of structural heterogeneity for the final step of the catalytic cycle by tracing the evolution of three spectroscopically relevant and structurally distinct components of the last metastable S3 state to the transient O2-evolving S4 state of the OEC. Using quantum chemical calculations, we show that each S3 isomer leads to a different electronic structure formulation for the active S4 state. Crucially, in addition to previously hypothesized Mn(IV)-oxyl species, we establish for the first time, how a genuine Mn(V)-oxo can be obtained in the catalytically active S4 state: this takes the form of a five-coordinate and locally high-spin (SMn = 1) Mn(V) site. This formulation for the S4 state evolves naturally from a preceding S3-state structural intermediate that contains a quasi-trigonal-bipyramidal Mn(IV) ion. The results strongly suggest that water binding in the S3 state is not prerequisite for reaching the oxygen-evolving S4 state of the complex, supporting the notion that both substrates are preloaded at the beginning of the catalytic cycle. This scenario allows true four-electron metal-centered hole accumulation to precede OO bond formation and hence the latter can proceed via a genuine even-electron mechanism. This can occur as intramolecular nucleophilic coupling of two oxo units synchronously with the binding of a water substrate for the next catalytic cycle.
The sunlight-powered oxidation of water by photosystem II (PSII) of algae, plants, and cyanobacteria underpins the energy conversion processes that sustain most of life on our planet. Understanding the structure and function of the “engine of life”, the oxygen-evolving complex (OEC) in the active site of PSII, has been one of the great and persistent challenges of modern science. Immense progress has been achieved in recent years through combined contributions of diverse disciplines and research approaches, yet the challenge remains. The improved understanding of the tetramanganese-calcium cluster of the OEC for the experimentally accessible catalytic states often creates a more complex picture of the system than previously imagined, while the various strands of evidence cannot always be unified into a coherent model. This review focuses on selected current problems that relate to structural–electronic features of the OEC, emphasizing conceptual aspects and highlighting topics of structure and function that remain uncertain or controversial. The Mn4CaOx cluster of the OEC cycles through five redox states (S0–S4) to store the oxidizing equivalents required for the final step of dioxygen evolution in the spontaneously decaying S4 state. Remarkably, even the dark-stable state of the OEC, the S1 state, is still incompletely understood because the available structural models do not fully explain the complexity revealed by spectroscopic investigations. In addition to the nature of the dioxygen-evolving S4 state and the precise mechanism of O–O bond formation, major current open questions include the type and role of structural heterogeneity in various intermediate states of the OEC, the sequence of events in the highly complex S2–S3 transition, the heterogeneous nature of the S3 state, the accessibility of substrate or substrate analogues, the identification of substrate oxygen atoms, and the role of the protein matrix in mediating proton removal and substrate delivery. These open questions and their implications for understanding the principles of catalytic control in the OEC must be convincingly addressed before biological water oxidation can be understood in its full complexity on both the atomic and the systemic levels.
Light driven water oxidation is a fundamental reaction in the biosphere. The Mn4Ca cluster of Photosystem II cycles through five redox states termed S0-S4 after which, oxygen is evolved. Critically, the timing of O–O bond formation within the Kok cycle remains unknown. Combining recent crystallographic, spectroscopic and DFT results, we demonstrate an atomistic S3-state model with the possibility of a low barrier to O–O bond formation prior to the final oxidation step. Furthermore, the associated one electron oxidized S4-state does not provide more advantages in terms of spin alignment or the energy of O–O bond formation. We propose that a high energy peroxide isoform of the S3-state can preferentially be oxidized by Tyrzox in the course of final electron transfer leading to O2 evolution. Such a mechanism may explain the peculiar kinetic behavior as well as serve as an evolutionary adaptation which avoids release of the harmful peroxides.
Plants, algae and cyanobacteria capture sunlight, extracting electrons from H2O to reduce CO2 into sugars, while releasing O2 in the oxygenic photosynthetic phenomenon. Because of the important role of water oxidation for artificial photosynthesis and many solar fuel systems, understanding the structure and function of this unique biological catalyst forms a requisite research field. Herein the structure of the water-oxidizing complex and its ligand environment are described with reference to the 1.9 Å resolution X-ray-derived crystallographic model of the water-oxidizing complex from the cyanobacterium Thermosynechococcus vulcanus. Proposed mechanisms for water oxidation by Photosystem II and nanosized manganese oxides are also reviewed and discussed in the paper.
Methanol has long being used as a substrate analogue to probe access pathways and investigate water delivery at the oxygen-evolving complex (OEC) of photosystem-II. In this contribution we study the interaction of methanol with the OEC by assembling available spectroscopic data into a quantum mechanical treatment that takes into account the local channel architecture of the active site. The effect on the magnetic energy levels of the Mn4Ca cluster in the S2 state of the catalytic cycle can be explained equally well by two models that involve either methanol binding to the calcium ion of the cluster, or a second-sphere interaction in the vicinity of the "dangler" Mn4 ion. However, consideration of the latest ¹³C hyperfine interaction data shows that only one model is fully consistent with experiment. In contrast to previous hypotheses, methanol is not a direct ligand to the OEC, but is situated at the end-point of a water channel associated with the O4 bridge. Its effect on magnetic properties of plant PS-II results from disruption of hydrogen bonding between O4 and proximal channel water molecules, thus enhancing superexchange (antiferromagnetic coupling) between the Mn3 and Mn4 ions. The same interaction mode applies to the dark-stable S1 state and possibly to all other states of the complex. Comparison of protein sequences from cyanobacteria and plants reveals a channel-altering substitution (D1-Asn87 versus D1-Ala87) in the proximity of the methanol binding pocket, explaining the species-dependence of the methanol effect. The water channel established as the methanol access pathway is the same that delivers ammonia to the Mn4 ion, supporting the notion that this is the only directly solvent-accessible manganese site of the OEC. The results support the pivot mechanism for water binding at a component of the S3 state and would be consistent with partial inhibition of water delivery by methanol. Mechanistic implications for enzymatic regulation and catalytic progression are discussed.
The origin of photosynthesis research in Greece can be traced to the early 1960s, and the first dedicated laboratory was established by George Akoyunoglou in the Nuclear Reseach Center (now National Center for Scientific Research) Demokritos, in Athens. More photosynthesis groups subsequently emerged, in Demokritos and in the universities. Research in Greece benefited greatly from the links of Greek scientists with laboratories and personalities, primarily in the USA and western Europe. The local research output is a proportional part of global research and, more or less, in tune with the shifting priorities of the latter. The list of references provided includes only a sample of publications: it is not inclusive.
Syntheses and magnetic functionalities of exchange-coupled magnetic systemsin a controlled fashion of molecular basis have been the focus of the current topics in chemistry and materials science; particularly extremely large spins in molecular frames and molecular high-spin clusters have attracted much attention among the diverse topics of molecule-based magnetics and high spin chemistry. Magnetic characterizations of molecule-based exchange-coupled high-spin clusters are described in terms of conventional as well as highfield/high-frequency ESR spectroscopy. Off-principal-axis extra lines as a salient feature of fine structure ESR spectroscopy in non-oriented media are emphasized in the spectral analyses. Pulse-ESR-based two-dimensional electron spin transient nutation spectroscopy applied to molecular high-spin clusters is also dealt with, briefly. Solution-phase fine-structure ESR spectroscopy is reviewed in terms of molecular magnetics. In addition to finite molecular high-spin clusters, salient features of molecule-based low-dimensional magnetic materials are dealt with. Throughout the chapter, electron spin resonance for high-spin systems is treated in a general manner in terms of theory. Hybrid eigenfield method is formulated in terms of direct products, and is described as a powerful and facile approach to the exact numerical calculation of resonance fields and transition probabilities for molecular high spin systems. Exact analytical expressions for resonance fields of high spin systems in their principal orientations are for the first time given.
This chapter provides an overview of oxygenic photosynthesis, with primary focus on the oxygen-evolving complex (OEC) of photosystem II (PSII). The introduction includes a general discussion of photosynthesis and a brief overview of the metals involved. This is followed by an in-depth description of the metal centers within PSII, in which the structural and electronic characterizations of the OEC are reviewed in detail. The role of chloride in PSII and the proton exit pathway are discussed. Several mechanisms proposed for the O. O bond formation in the OEC are presented, along with a few detailed mechanisms for the complete catalytic cycle obtained from quantum mechanics/molecular mechanics and density functional theory calculations. Model complexes that are functional mimics of the OEC are also briefly discussed. General discussion on plastocyanin, iron-sulfur centers, [FeFe] and [NiFe] hydrogenases, and ribulose-1,5-bisphosphate carboxylase-oxygenase is included. The chapter concludes with the implications of natural photosynthesis on the development of an artificial photosynthetic framework.
A frequent challenge when dealing with multinuclear transition metal clusters in biology is to determine the absolute oxidation states of the individual metal ions and to identify how they evolve during catalytic turnover. The oxygen-evolving complex of biological photosynthesis, an active site that harbors an oxo-bridged Mn4Ca cluster as the water-oxidizing species, offers a prime example of such a challenge that withstood satisfactory resolution for decades. A multitude of experimental studies have approached this question and have offered insights from different angles, but they were also accompanied by incomplete or inconclusive interpretations. Only very recently, through a combination of experiment and theory, has a definitive assignment of the individual Mn oxidation states been achieved for all observable catalytic states of the complex. Here we review the information obtained by structural and spectroscopic methods, describe the interpretation and synthesis achieved through quantum chemistry, and summarize our current understanding of the electronic structure of nature’s water splitting catalyst.
Water splitting is considered as a method to storage of renewable energies to hydrogen. The water-oxidation reaction in water splitting is an efficiency-limiting process for water splitting and, thus, there has been notable progress to find highly efficient water-oxidizing catalysts made from cost-effective and earth-abundant elements. In addition to efficiency, the stability of the water-oxidizing compounds is very important. Herein we focus on self-healing in manganese-based water-oxidizing catalysts in artificial photosynthetic systems.
Nature relies on a unique and intricate biochemical setup to achieve sunlight-driven water splitting. Combined experimental and computational efforts have produced significant insights into the structural and functional principles governing the operation of the water-oxidizing enzyme Photosystem II in general, and of the oxygen-evolving manganese-calcium cluster at its active site in particular. Here we review the most important aspects of biological water oxidation, emphasizing current knowledge on the organization of the enzyme, the geometric and electronic structure of the catalyst, and the role of calcium and chloride cofactors. The combination of recent experimental work on the identification of possible substrate sites with computational modeling have considerably limited the possible mechanistic pathways for the critical O-O bond formation step. Taken together, the key features and principles of natural photosynthesis may serve as inspiration for the design, development, and implementation of artificial systems.
Syntheses and magnetic functionalities of exchange-coupled magnetic systems in a controlled fashion of molecular basis have been the focus of the current topics in chemistry and materials science; particularly extremely large spins in molecular frames and molecular high-spin clusters have attracted much attention among the diverse topics of molecule-based magnetics and high spin chemistry. Magnetic characterizations of molecule-based exchange-coupled high-spin clusters are described in terms of conventional as well as high-field/high-frequency ESR spectroscopy. Off-principal-axis extra lines as a salient feature of fine-structure ESR spectroscopy in non-oriented media are emphasized in the spectral analyses. Pulse-ESR-based two-dimensional electron spin transient nutation spectroscopy applied to molecular high-spin clusters is also dealt with, briefly. Solution-phase fine-structure ESR spectroscopy is reviewed in terms of molecular magnetics. In addition to finite molecular high-spin clusters, salient features of molecule-based low-dimensional magnetic materials are dealt with. Throughout the chapter, electron spin resonance for high-spin systems is treated in a general manner in terms of theory. Hybrid eigenfield method is formulated in terms of direct products, and is described as a powerful and facile approach to the exact numerical calculation of resonance fields and transition probabilities for molecular high spin systems. Exact analytical expressions for resonance fields of high spin systems in their principal orientations are for the first time given.
The EPR "split signals" represent key intermediates of the S-state cycle where the redox active D1-Tyr161 (YZ) has been oxidized by the reaction center of the photosystem II enzyme to its tyrosyl radical form, but the successive oxidation of the Mn4CaO5 cluster has not yet occurred (SiYZ˙). Here we focus on the S2YZ˙ state, which is formed en route to the final metastable state of the catalyst, the S3 state, the state which immediately precedes O-O bond formation. Quantum chemical calculations demonstrate that both isomeric forms of the S2 state, the open and closed cubane isomers, can form states with an oxidized YZ˙ residue without prior deprotonation of the Mn4CaO5 cluster. The two forms are expected to lie close in energy and retain the electronic structure and magnetic topology of the corresponding S2 state of the inorganic core. As expected, tyrosine oxidation results in a proton shift towards His190. Analysis of the electronic rearrangements that occur upon formation of the tyrosyl radical suggests that a likely next step in the catalytic cycle is the deprotonation of a terminal water ligand (W1) of the Mn4CaO5 cluster. Diamagnetic metal ion substitution is used in our calculations to obtain the molecular g-tensor of YZ˙. It is known that the gx value is a sensitive probe not only of the extent of the proton shift between the tyrosine-histidine pair, but also of the polarization environment of the tyrosine, especially about the phenolic oxygen. It is shown for PSII that this environment is determined by the Ca(2+) ion, which locates two water molecules about the phenoxyl oxygen, indirectly modulating the oxidation potential of YZ.
Full geometry optimizations followed by the vibrational analysis were performed for eight spin configurations of the CaMn4O4X(H2O)3Y (X = O, OH; Y = H2O, OH) cluster in the S1 and S3 states of the oxygen evolution complex (OEC) of photosystem II (PSII). The energy gaps among these configurations obtained by vertical, adiabatic and adiabatic plus zero-point-energy (ZPE) correction procedures have been used for computation of the effective exchange integrals (J) in the spin Hamiltonian model. The J values are calculated by the (1) analytical method and the (2) generalized approximate spin projection (AP) method that eliminates the spin contamination errors of UB3LYP solutions. Using J values derived from these methods, exact diagonalization of the spin Hamiltonian matrix was carried out, yielding excitation energies and spin densities of the ground and lower-excited states of the cluster. The obtained results for the right (R)- and left (L)-opened structures in the S1 and S3 states are found to be consistent with available optical and magnetic experimental results. Implications of the computational results are discussed in relation to (a) the necessity of the exact diagonalization for computations of reliable energy levels, (b) magneto-structural correlations in the CaMn4O5 cluster of the OEC of PSII, (c) structural symmetry breaking in the S1 and S3 states, and (d) the right- and left-handed scenarios for the O-O bond formation for water oxidation.
We report the detection of a “split” electron paramagnetic resonance (EPR) signal during illumination of dark-adapted (S1 state) oxygen-evolving photosystem II (PSII) membranes at <20 K. The characteristics of this signal indicate that it arises from an interaction between an organic radical and the Mn cluster of PSII. The broad radical signal decays in the dark following illumination either by back-reaction with Qa•- or by forward electron transfer from the Mn cluster. The forward electron transfer (either from illumination at 11 K followed by incubation in the dark at 77 K or by illumination at 77 K) results in the formation of a multiline signal similar to, but distinct from, other well-characterized multiline forms found in the S0 and S2 states. The relative yield of the “S1 split signal”, which we provisionally assign to S1X•, where X could be YZ• or Car•+, and that of the 77 K multiline signal indicate a relationship between the two states. An approximate quantitation of the yield of these signals indicates that up to 40−50% of PSII centers can form the S1 split signal. Ethanol addition removes the ability to observe the S1 split signal, but the multiline signal is still formed at 77 K. The multiline forms with <700 nm light and is not affected by near-infrared (IR) light, showing that we are detecting electron transfer in centers not responsive to IR illumination. The results provide important new information about the mechanism of electron abstraction from the water oxidizing complex (WOC).
Illuminating a photosystem II sample at low temperatures (here 5-10 K) yields so called split signals detectable with CW-EPR. These signals reflect the oxidized, deprotonated radical of D1-Tyr161 (YZ(•)) in a magnetic interaction with the CaMn4 cluster in a particular S state. The intensity of the split EPR signals are affected by the addition of the water substrate analogue methanol. This was previously shown by the induction of split EPR signals from the S1, S3 and S0 states [Su, J-H. et al. (2006) Biochemistry 45, 7617-7627.]. Here, we use two split EPR signals induced from photosystem II trapped in the S2 state to further probe the binding of methanol in an S state dependent manner. The signals are induced with either visible or near-infrared light illumination provided at 5-10 K where methanol cannot bind or un-bind from its site. The results imply that the binding of methanol not only changes the magnetic properties of the CaMn4 cluster but also the hydrogen bond network in the OEC, thereby affecting the relative charge of the S2 state. The induction mechanisms for the two split signals are different resulting in two different redox states, S2YZ(•) and S1YZ(•) respectively. The two states show different methanol dependence for their induction. This indicates the existence of two binding sites for methanol in the CaMn4 cluster. It is proposed that methanol binds to MnA with high affinity and to MnD with lower affinity. The molecular nature and S-state dependence of the methanol binding to each respective site is discussed.
We have performed continuous-wave electron paramagnetic resonance (CW-EPR) and electron spin echo electron nuclear double resonance (ESE-ENDOR) experiments on the multiline form of the S2-state of untreated, MeOH-treated, and ammonia-treated spinach photosystem II (PS II) centers. Through simultaneously constrained simulations of the CW-EPR and ESE-ENDOR data, we conclude that four effective 55Mn hyperfine tensors (AX, AY, AZ) are required to properly simulate the experimental data [untreated and MeOH-treated PS II centers (MHz): −232, −232, −270; 200, 200, 250; −311, −311, −270; 180, 180, 240; ammonia-treated PS II centers (MHz): 208, 208, 158; −150, −150, −112; 222, 222, 172; −295, −315, −390]. We further show that these effective hyperfine tensors are best supported by a trimer/monomer arrangement of three Mn(IV) ions and one Mn(III) ion. In this topology, MnA, MnB, and MnC form a strongly exchange coupled core (JAB and JBC < −100 cm-1) while MnD is weakly exchange coupled (JCD) to one end of the trinuclear core. For untreated and MeOH-treated PS II centers, the Mn(III) ion is either MnA or MnC, with a zero-field-splitting of D = −1.25 to −2.25 cm-1. For ammonia-treated PS II centers, the Mn(III) ion is MnD, with a zero-field-splitting of D = +0.75 to +1.75 cm-1. The binding of the ammonia ligand results in a shift of the Mn(III) ion from the trinuclear core to the monomer Mn ion. This structural model can also account for the higher spin of the g = 4.1 signal and the magnetic properties of the S0-state.
Photosystem II (PS II), found in oxygenic photosynthetic organisms, catalyses the most energetically demanding reaction in nature, the oxidation of water to molecular oxygen and protons. The water oxidase in PS II contains a Mn(4)Ca cluster (oxygen evolving complex, OEC), whose catalytic mechanism has been extensively investigated but is still unresolved. In particular the precise Mn oxidation levels through which the cluster cycles during functional turnover are still contentious. In this, the first of several planned parts, we examine a broad range of published data relating to this question, while considering the recent atomic resolution PS II crystal structure of Umena et al. (Nature, 2011, 473, 55). Results from X-ray, UV-Vis and NIR spectroscopies are considered, using an approach that is mainly empirical, by comparison with published data from known model systems, but with some reliance on computational or other theoretical considerations. The intention is to survey the extent to which these data yield a consistent picture of the Mn oxidation states in functional PS II - in particular, to test their consistency with two current proposals for the mean redox levels of the OEC during turnover; the so called 'high' and 'low' oxidation state paradigms. These systematically differ by two oxidation equivalents throughout the redox accumulating catalytic S state cycle (states S(0)···S(3)). In summary, we find that the data, in total, substantially favor the low oxidation proposal, particularly as a result of the new analyses we present. The low oxidation state scheme is able to resolve a number of previously 'anomalous' results in the observed UV-Visible S state turnover spectral differences and in the resonant inelastic X-ray spectroscopy (RIXS) of the Mn pre-edge region of the S(1) and S(2) states. Further, the low oxidation paradigm is able to provide a 'natural' explanation for the known sensitivity of the OEC Mn cluster to cryogenic near infra-red (NIR) induced turnover to alternative spin/redox states in S(2) and S(3).
Inspired by the Photosystem II reaction center and the water oxidation chemistry that it performs, we aim to develop artificial
photosynthesis for fuel production. Besides the original work we do in this direction, we also acquire knowledge feedback
from our novel compounds. Our man-made systems create new perspectives on electron and proton transfer, bioinorganic chemistry,
excitation energy transfer and other issues that are central to photosynthesis research. In this chapter we describe some
of the highlights in our research and the conclusions they have generated.
We have applied Q-band EPR spectroscopy to study the S3 state of the Mn cluster in untreated and in methanol treated PSII membranes, extending earlier bimodal studies at X band
EPR. Prominent EPR signals are observed in both sets of samples. A simultaneous analysis of the spectra at both frequency
bands indicates that the spin associated with the signals of S3 is S = 3 rather than S = 1. An S = 3 spin state is also indicated in the presence of methanol, but with significantly different zero field splitting parameters,
which explain the absence of X-band signals in this case.
Recent spectroscopic studies on the water oxidizing complex (WOC) of Photosystem II are discussed in terms of the possible
nature and structures for the Mn containing catalytic site. Emphasis is given to the various electron paramagnetic resonance
techniques which have been increasingly employed, as well as examination of complementary data from optical and X-ray absorption
spectroscopies. All of the quasi-stable intermediate states of the catalytic turnover cycle of the WOC (S0 to S3) are now accessible spectroscopically. We show that the available data may be rationalized by a scheme in which the ‘active,’
catalytically cycling component of the water oxidizing site contains a pair of coupled, oxo bridged Mn ions, closely associated
with a Ca ion. The structure of the active Mn site resembles the structure of the dinuclear Mn catalase. During functional
turnover, oxidizing equivalents are stored both in Mn ions and ligand groups and one Mn ion has a unique, low symmetry ligand
environment.
The redox-active tyrosine residue (YZ) plays a crucial role in the mechanism of the water oxidation. Metalloradical electron paramagnetic resonance (EPR) signals
reflecting the light-induced YZ· in magnetic interaction with the CaMn4-cluster in the particular S-state, YZ·SX intermediates, have been found in intact photosystem II. These so-called split EPR signals are induced by illumination at
cryogenic temperatures and provide means to both study the otherwise transient YZ· and to probe the S-states with EPR spectroscopy. The illumination used for signal induction grouped the observed split EPR signals in two categories:
(i) YZ in the lower S-states was oxidized by P680+ formed via charge separation, while (ii) YZ in the higher S-states was oxidized by an excited, highly oxidizing Mn species. Applied mechanistic studies of the YZ·SX intermediates in the different S-states are reviewed and compared to investigations in photosystem II at physiological temperature. Addition of methanol induced
S-state characteristic changes in the split signals’ formation which reflect changes in the magnetic coupling within the CaMn4-cluster due to methanol binding. The pH titration of the split EPR signals, on the other hand, could probe the proton-coupled
electron transfer properties of the YZ oxidation. The apparent pK
as found for decreased split signal induction were interpreted in the fate of the phenol proton.
A split EPR signal can be induced at 5 K in Photosystem II in the S3-state by light in the range of 400–900 nm. To investigate if the same mechanism is involved in the signal induction in the
full spectral range we compared the properties of the S3 signal induced by 830 nm light or white light. Our results indicate that the same mechanism is responsible for the formation
of the “Split S3” signal in the whole spectral range. The mechanism of the “Split S3” signal is not P680 driven but is instead driven by manganese excitation.
Cryogenic illumination of Photosystem II (PSII) can lead to the trapping of the metastable radical Y(Z)(•), the radical form of the redox-active tyrosine residue D1-Tyr161 (known as Y(Z)). Magnetic interaction between this radical and the CaMn(4) cluster of PSII gives rise to so-called split electron paramagnetic resonance (EPR) signals with characteristics that are dependent on the S state. We report here the observation and characterization of a split EPR signal that can be directly induced from PSII centers in the S(2) state through visible light illumination at 10 K. We further show that the induction of this split signal takes place via a Mn-centered mechanism, in the same way as when using near-infrared light illumination [Koulougliotis, D., et al. (2003) Biochemistry 42, 3045-3053]. On the basis of interpretations of these results, and in combination with literature data for other split signals induced under a variety of conditions (temperature and light quality), we propose a unified model for the mechanisms of split signal induction across the four S states (S(0), S(1), S(2), and S(3)). At the heart of this model is the stability or instability of the Y(Z)(•)(D1-His190)(+) pair that would be formed during cryogenic oxidation of Y(Z). Furthermore, the model is closely related to the sequence of transfers of protons and electrons from the CaMn(4) cluster during the S cycle and further demonstrates the utility of the split signals in probing the immediate environment of the oxygen-evolving center in PSII.
The stability of the S(3) and S(2) states of the oxygen evolving complex in photosystem II (PSII) was directly probed by EPR spectroscopy in PSII membrane preparations from spinach in the presence of the exogenous electron acceptor PpBQ at 1, 10, and 20 °C. The decay of the S(3) state was followed in samples exposed to two flashes by measuring the split S(3) EPR signal induced by near-infrared illumination at 5 K. The decay of the S(2) state was followed in samples exposed to one flash by measuring the S(2) state multiline EPR signal. During the decay of the S(3) state, the S(2) state multiline EPR signal first increased and then decreased in amplitude. This shows that the decay of the S(3) state to the S(1) state occurs via the S(2) state. The decay of the S(3) state was biexponential with a fast kinetic phase with a few seconds decay half-time. This occurred in 10-20% of the PSII centers. The slow kinetic phase ranged from a decay half-time of 700 s (at 1 °C) to ~100 s (at 20 °C) in the remaining 80-90% of the centers. The decay of the S(2) state was also biphasic and showed quite similar kinetics to the decay of the S(3) state. Our experiments show that the auxiliary electron donor Y(D) was oxidized during the entire experiment. Thus, the reduced form of Y(D) does not participate to the fast decay of the S(2) and S(3) states we describe here. Instead, we suggest that the decay of the S(3) and S(2) states reflects electron transfer from the acceptor side of PSII to the donor side of PSII starting in the corresponding S state. It is proposed that this exists in equilibrium with Y(Z) according to S(3)Y(Z) ⇔ S(2)Y(Z)(•) in the case of the S(3) state decay and S(2)Y(Z) ⇔ S(1)Y(Z)(•) in the case of the S(2) state decay. Two kinetic models are discussed, both developed with the assumption that the slow decay of the S(3) and S(2) states occurs in PSII centers where Y(Z) is also a fast donor to P(680)(+) working in the nanosecond time regime and that the fast decay of the S(3) and S(2) states occurs in centers where Y(Z) reduces P(680)(+) with slower microsecond kinetics. Our measurements also demonstrate that the split S(3) EPR signal can be used as a direct probe to the S(3) state and that it can provide important information about the redox properties of the S(3) state.
The photosystem II core complex (PSIIcc) is the key enzyme of oxygenic photosynthesis, as it catalyzes the light-induced oxidation of water to form dioxgyen and protons. It is located in the thylakoid membrane of cyanobacteria, algae, and plants and consists of 20 protein subunits binding about 100 cofactors. In this review, we discuss what is presently known about the "donor side" of PSIIcc, covering the photosynthetic reaction center and the water oxidase part. The focus is on the catalytic Mn4Ca cluster and its protein environment. An attempt is made to connect recent crystallographic data (up to 2.9 Å resolution) with the wealth of information about Nature's water oxidation device from spectroscopic, biochemical and theoretical work.
The electrons extracted from the CaMn(4) cluster during water oxidation in photosystem II are transferred to P(680)(+) via the redox-active tyrosine D1-Tyr161 (Y(Z)). Upon Y(Z) oxidation a proton moves in a hydrogen bond toward D1-His190 (His(Z)). The deprotonation and reprotonation mechanism of Y(Z)-OH/Y(Z)-O is of key importance for the catalytic turnover of photosystem II. By light illumination at liquid helium temperatures (∼5 K) Y(Z) can be oxidized to its neutral radical, Y(Z)(•). This can be followed by the induction of a split EPR signal from Y(Z)(•) in a magnetic interaction with the CaMn(4) cluster, offering a way to probe for Y(Z) oxidation in active photosystem II. In the S(3) state, light in the near-infrared region induces the split S(3) EPR signal, S(2)'Y(Z)(•). Here we report on the pH dependence for the induction of S(2)'Y(Z)(•) between pH 4.0 and pH 8.7. At acidic pH the split S(3) EPR signal decreases with the apparent pK(a) (pK(app)) ∼ 4.1. This can be correlated to a titration event that disrupts the essential H-bond in the Y(Z)-His(Z) motif. At alkaline pH, the split S(3) EPR signal decreases with the pK(app) ∼ 7.5. The analysis of this pH dependence is complicated by the presence of an alkaline-induced split EPR signal (pK(app) ∼ 8.3) promoted by a change in the redox potential of Y(Z). Our results allow dissection of the proton-coupled electron transfer reactions in the S(3) state and provide further evidence that the radical involved in the split EPR signals is indeed Y(Z)(•).
Electron spin echo electron-nuclear double resonance (ESE-ENDOR) experiments performed on a broad radical electron paramagnetic resonance (EPR) signal observed in photosystem II particles depleted of Ca2+ indicate that this signal arises from the redox-active tyrosine YZ. The tyrosine EPR signal width is increased relative to that observed in a manganese-depleted preparation due to a magnetic interaction between the photosystem II manganese cluster and the tyrosine radical. The manganese cluster is located asymmetrically with respect to the symmetry-related tyrosines YZ and YD. The distance between the YZ tyrosine and the manganese cluster is estimated to be approximately 4.5 A. Due to this close proximity of the Mn cluster and the redox-active tyrosine YZ, we propose that this tyrosine abstracts protons from substrate water bound to the Mn cluster.
Following illumination at 200 K, the charge recombination reactions and the origin of the thermoluminescence (TL) bands appearing at about 0 degree C (Q band) and +50 degrees C (C band) in the glow curve were investigated by comparative TL and EPR measurements in DCMU-treated photosystem II particles. Decay half-time measurements carried out at -25 degrees C and +25 degrees C, respectively, suggest that the S2 state (multi-line signal) undergoes charge recombination with the g = 1.9 form of the semiquinone-iron complex, QA-.Fe2+, resulting in the appearance of the Q band, and that the g = 1.82 form of QA-.Fe2+ back-reacts with the oxidized tyrosine, YD+ (Signal IIs), accounting for the generation of the C band.
Electron paramagnetic resonance of spinach chloroplasts given a series of laser flashes, n = 0, 1,..., 6, at room temperature and rapidly cooled to -140 degrees C reveals a signal possessing at least 16 and possibly 21 or more hyperfine lines when observed below 35 K. The spectrum is consistent with a pair of antiferromagnetically coupled Mn ions, or possibly a tetramer of Mn ions, in which Mn(III) and Mn(IV) oxidation states are present. The intensity of this signal peaks on the first and fifth flashes, suggesting a cyclic change in oxidation state of period 4. The multiline signal produced on the first flash is not affected by the electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea but is abolished by agents that influence the state of bound manganese, such as incubation with alkaline Tris, or dithionite, and by extraction with cholate detergent in the presence of ammonium sulfate. These results indicate that the paramagnetic signal is monitoring oxidation state changes in the enzyme involved in oxidation of water.
Redox events that occur in photosynthetic O2-evolving centers in NaCl/EDTA-washed PS II membranes were investigated by means of low temperature EPR and thermoluminescence. The following results have been obtained: (i) In the washed membranes, O2 centers could maintain the S2 state more than 3 h in darkness at room temperature. This dark-stable S2 was modified as seen by a multiline EPR signal with reduced hyperfine line spacing and also by a thermoluminescence band with upshifted peak temperature. (ii) This modified S2 state had an abnormally long life of at 20°C, and its appearance required the presence of EDTA in the medium. On addition of exogenous Ca2+, the modified S2 was converted in darkness to normal S2, and then decayed rapidly to be undetectable. (iii) On illuminating this modified S2, an EPR signal centering at aroung g = 2 was newly induced at no expense of the dark-stable EPR multiline signal. This EPR signal was accompanied by a new thermoluminescence band peaking at around 5°C, suggesting the presence of a new redox component whose oxidized form is capable of providing a positive charge for thermoluminescence in place of Mn. (iv) This new component was efficiently oxidized by illumination at −5°C but much less at −60°C, showing a half-inhibition temperature at around −40°C. (v) Addition of various divalent cations in place of Ca2+ variously affected both thermoluminescence glow peaks arising from the dark-stable S2 or from the new redox component, suggesting a cation-species-dependent modulation of the redox properties of both components. (vi) Both of these two thermoluminescence bands showed no dependency on flash number, suggesting interruption of further oxidation beyond their respective abnormal states. On addition of Ca2+, all these abnormal properties were abolished and normal period-four flash pattern was restored. These abnormal properties of the redox events in NaCl/EDTA-washed PS II membranes were discussed in relation to the demand for exogenous Ca2+ in recovery of normal properties.
The authors report the detection of a new electron paramagnetic resonance (EPR) signal that demonstrates the presence of a paramagnetic intermediate in the resting (S{sub 1}) state of the photosynthetic oxygen-evolving complex. The signal was detected using the method of parallel polarization EPR, which is sensitive to {Delta}m = 0 transitions in high spin systems. The properties of the parallel polarization EPR signal in the S{sub 1} state are consistent with an S=1 spin state of and exchange-coupled manganese center that corresponds to the reduced form of the species giving rise to the multiline EPR signal in the light-induced S{sub 2} state. The implications for the electronic structure of the oxygen-evolving complex are discussed. 36 refs., 2 fig., 1 tab.
The decay kinetics for the S2 and S3 states of the oxygen-evolving complex in Photosystem II have been measured in the presence of an external electron acceptor. The S2- and S3-states decay monophasically with half-decay times at 18°C of 3–3.5 min and 3.5–4 min, respectively. The results also show that S3 decays via S2 under these circumstances. The temperature dependence of the individual S-state transitions has been measured in single flash experiments in which the multiline EPR signal originating from the S2 state has been used as spectroscopic probe. The half-inhibition temperatures are for S0 to S1 220–225 K, for S1 to S2 135–140 K, for S2 to S3 230 K and for the S3-to-S0 transition 235 K.
Microwave power saturation studies have been performed over the range 4–20 K on EPR signals photogenerated in PS II particles by low-temperature illumination (180–240 K). In the presence of 3% methanol (+ MeOH), with no g 4.1 signal present, the multiline signal intensity (extrapolated to zero power) shows strict Curie law behaviour over the 4–20 K range. With no MeOH present in the suspension buffer (−MeOH), both the multiline and 4.1 signals show complementary deviations from Curie law behaviour. These are consistent with the signals arising respectively from the ground, S = 1/2, and first excited S = 3/2, states of a total spin = 7/2 multiplet, such as could occur in an MnIV-MnIII antiferromagnetically coupled pair. The deduced height of the 3/2 state above the ½ state is 9.0 K. An inferential estimate, from relaxation data, of this height for the +MeOH case is about 40 K. A broad, featureless component around g = 2 appears to underlie the multiline pattern in the presence of the 4.1 signal, and has a similar temperature behaviour to the latter. A possible exchange coupling model, involving four Mn centres, is presented to accommodate these and other findings on the S2 state signals.
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 spin system in a rhombic environment, suggesting the involvement of non-heme Fe in photosynthetic oxygen evolution.
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.
New evidence on the chloride requirement for photosynthetic O 2 evolution has indicated that Cl - facilitates oxidation of the manganese cluster by the photosystem II (PSII) Tyr-Z + radical. Illumination above 250K of spinach PSII centers which are inhibited in O 2 evolution bu either Cl - depletion of F - substitution produces a new EPR signal which has magnetic characteristics similar to one recently discovered in samples inhibited by depletion of Ca 2+ only.
We have investigated the effects of temperature on the formation and decay of the light-induced multiline EPR signal species associated with photosynthetic oxygen evolution (Dismukes, G.C. and Siderer, Y. (1980) FEBS Lett. 121, 78–80). (1) The decay rate following illumination is temperature dependent: at 295 K the half-time of decay is about 40 s, at 253 K the half-time is approx. 40 min. (2) A single intense flash of light becomes progressively less effective in generating the multiline signal below about 240 K. (3) Continuous illumination is capable of generating the signal down to almost 160 K. (4) Continuous illumination after a preilluminating flash generates less signal above 200 K than at lower temperatures. Our results support the conclusion of Dismukes and Siderer that the S2 state gives rise to this multiline signal; we find that the S1 state can be fully advanced to the S2 state at temperatures as low as 160 K. The S2 state is capable of further advancement at temperatures above about 210 K, but not below that temperature.
The light-driven oxidation of water to dioxygen is catalyzed by the enzyme photosystem II. A four-manganese ion cluster and a tyrosine, YZ, are present in the catalytic site. In preparations inhibited by addition of acetate or removal of the calcium cofactor, it is possible to trap the tyrosyl radical in interaction with the metal cluster. The coupled species is characterized by a broad split EPR signal at 9 GHz. In this work, high-field EPR has been used for further characterization of the coupling. The 285, 190 and 95 GHz EPR spectra of the interacting system are reported. Analysis of these spectra yielded exchange and dipolar couplings of the same magnitude as those found with 9 GHz EPR. However, the high-field spectra show that the coupling between the radical and the manganese cluster has opposite sign in acetate-treated compared to calcium-depleted samples. The sign difference indicates differences in the electronic structure of the radical−metal center pair. Comparisons are made between photosystem II and other enzymes containing radicals interacting with metal centers. Possible explanations for the difference in sign are proposed. The difficulty in obtaining reliable structural information for the spin coupled system is addressed.
Upon room-temperature illumination, acetate-inhibited photosystem II membranes are known to exhibit a 240 G wide X-band (9.5 GHz) electron paramagnetic resonance (EPR) signal at 10 K. This EPR signal arises from an interaction between the S = 1/2 multiline S2 state of the tetranuclear manganese cluster and an oxidized tyrosine residue, YZ•. In the present study, the exchange and dipolar interactions between the two paramagnetic species are simulated at X- and Q-band (33 GHz) frequencies utilizing second-order perturbation theory. The positions and relative intensities of the hyperfine lines in the S = 1/2 S2 state multiline EPR signal of the noninteracting Mn4 cluster are accurately simulated by including g anisotropy and four sets of axially symmetric 55Mn hyperfine tensors. These parameters are then used to simulate the dipolar and exchange interactions giving rise to the interacting S2YZ• (formerly referred to as S3) EPR signal. Relative intensities of components of the S2YZ• EPR spectrum, at both X- and Q-band frequencies, are best reproduced with a dipolar coupling corresponding to an interspin distance of 7.7 Å and an exchange coupling (J) of −280 × 10-4 cm-1.
The photosynthetic oxidation of water to oxygen occurs in photosystem II (PSII) at an active site composed of a tetranuclear cluster of manganese ions, a redox active tyrosine, YZ, and two essential cofactors, calcium and chloride. Recently, several experimental observations have led to the proposal of a metalloradical catalytic cycle in which water oxidation occurs via hydrogen-atom abstraction by the tyrosyl radical from water bound to the manganese cluster. This model predicts a close proximity between the radical tyrosine, Yz•, and the Mn cluster and the involvement of the radical in a bifurcated hydrogen bond. Magnetic resonance techniques have been used in this work to probe the interaction of the tyrosyl radical with its environment in PSII samples in which the catalytic cycle is blocked by acetate treatment and the enzyme is trapped in a paramagnetic S2Yz• state. Radical interaction with the metal cluster has been studied via simulations of the EPR spectra obtained for this state. The simulations were based on a radical-pair model and included terms for both electron−electron dipolar and exchange interactions. The results show a dominant exchange interaction between the radical and the manganese cluster in these preparations and led to an estimate of 8−9 Å for the spin−spin distance. ESEEM spectroscopy and 1H2O/2H2O exchange were used to study interactions of the S2Yz• state with exchangeable hydrogen nuclei in the site. Two-pulse ESEEM data show features expected for a radical-pair species, in support of our analysis of the continuous-wave EPR spectrum. An ESEEM analysis based on an electron spin 1/2, nuclear spin 1 model shows that both two- and three-pulse ESEEM data are consistent with four deuterons that exhibit an electron−nuclear dipole−dipole coupling of 0.42 MHz. The validity of this analysis and its implications for the oxygen-evolving apparatus are discussed.
Spin state considerations are proposed to sharply limit the possible O−O bond-forming steps in water oxidation by the oxygen evolving center of Photosystem II. A series of intermediates are proposed for the Kok S states on the basis of quantum chemical studies on simple model complexes; these are also consistent with the main biophysical data. Only one Mn atom in the active site cluster is thought to be redox-active and mediate O−O bond formation. A key concept is the formation of an unreactive MnO oxo at the S2 state, followed by its conversion to a reactive Mn−O• oxyl form at the S3 level, with radical character on the oxyl oxygen, at which point O−O bond formation can occur by a coupling between the oxyl and an outer-sphere water molecule. An MnOOH intermediate at S3 is proposed to lose a hydrogen atom to give O2. The role of the Ca cofactor is to bring about a 5- to 6-coordination change at S2, necessary for formation of a reactive oxo in S3. The chloride cofactor is assigned the role of charge neutralization.
The Mn4 complex which is involved in water oxidation in photosystem II is known to exhibit three types of EPR signals in the S2 state, one of the five redox states of the enzyme cycle: a multiline signal (spin 1/2), signals at g > 5 (spin 5/2), and a signal at g = 4.1 (spin value 3/2 or 5/2). The multiline and g = 4.1 signals are those the most readily observed. The relative proportions of the g = 4.1 signal and of the multiline signal are affected by many biochemical treatments including the substitution of Ca2+and Cl- which are two essential cofactors for O2 evolution. The state responsible for the multiline signal can also be converted, reversibly, to that responsible for the g = 4.1 signal upon the absorption of near-IR light at around 150 K. These infrared-induced effects are confined to the Mn4 cluster, and no other redox change occurs in the enzyme. Here, we have used the IR-induced photochemistry of the Mn4 cluster to measure the changes in magnetization occurring upon interconversion of the state responsible for the spin 1/2 state and the g = 4.1 state. Measurements were performed with a SQUID magnetometer below 20 K and at magnetic fields ≤5.5 T. Simulations of experimental data provide strong indication that the spin value of the state responsible for the g = 4.1 state is 5/2. Results are discussed in terms of a model implying an IR-triggered spin conversion of the MnIII (from the spin 2 to spin 1) of the Mn4 cluster.
The microwave power for half-saturation (P1/2) for the radical in photosystem II giving rise to signal IIslow (SIIs) has been measured by EPR in samples illuminated by a series of flashes. The charge storage state of the oxygen-evolving complex (S0-S4) was monitored by measuring the multiline EPR signal arising from the S2 state. The following results were obtained: (1) SIIs becomes easier to saturate after tris(hydroxymethyl)aminomethane (Tris) washing, a treatment that partially removes the Mn cluster. (2) P1/2 for SIIs oscillates with the flash number. P1/2 is lower in S1 (in dark-adapted material and after four flashes) than in S2, S3, or S0. (3) P1/2(S2) = P1/2(S3). (4) At 8 K P1/2(S2) > P1/2(S2), but at 20 K P1/2(S0) < P1/2(S2). (5) P1/2 for SIIs increases with temperature (8-70 K) in the S1 state. SHs is more difficult to saturate in S2, S3, and S0 than in S1 over the investigated temperature range. In addition, the increase in P1/2 is complex around 20-30 K in S2, S3, and S0. (6) In S0, P1/2 for SIIs decreases with time (decay half-time 30-60 s) to a stable level significantly above the dark level. The data are explained in terms of cross relaxation between the radical giving rise to SIIs and an efficient relaxer, which is suggested to be the Mn cluster. This relaxes more slowly in S1 than in the other S states. Since it is known that a mixed-valence Mn cluster is present in S2, and because P1/2 of SIIs in S3 and S0 is comparable to that in S2, it is suggested that mixed-valence Mn clusters are present in the S3 and S0 states also. Different models with these features can be proposed, the simplest of which is the following: S0 [Mn(H)-Mn(III)], S1 [Mn(III)-Mn(III)], S2 [Mn(III)-Mn(IV)], and S3 [Mn(III)-Mn(IV)].
Electron paramagnetic resonance (EPR) signals arising from components in photosystem II have been studied in membranes isolated from spinach chloroplasts. A broad EPR signal at g = 4.1 can be photoinduced by a single laser flash at room temperature. When a series of flashes is given, the amplitude of the g = 4.1 signal oscillates with a period of 4, showing maxima on the first and fifth flashes. Similar oscillations occur in the amplitude of a multiline signal centered at g ≃ 2. Such an oscillation pattern is characteristic of the S2 charge accumulation state in the oxygen-evolving complex. Accordingly, both EPR signals are attributed to the S2 state. Earlier data from which the g = 4.1 signal was attributed to a component different from the S2 state [Zimmermann, J.-L., & Rutherford, A. W. (1984) Biochim. Biophys. Acta 767, 160-167; Casey, J. L., & Sauer, K. (1984) Biochim. Biophys. Acta 767, 21-28] are explained by the effects of cryoprotectants and solvents, which are shown to inhibit the formation of the g = 4.1 signal under some conditions. The g = 4.1 signal is less stable than the multiline signal when both signals are generated together at low temperature. This indicates that the two signals arise from different populations of centers. The differences in structure responsible for the two different EPR signals are probably minor since both kinds of centers are functional in cyclic charge accumulation and seem to be interconvertible. The difference between the two EPR signals, which arise from the same redox state of the same component (a mixed-valence manganese cluster), is proposed to be due to a spin-state change, where the g = 4.1 signal reflects an S = 3/2 state and the multiline signal an S = 1/2 state within the framework of the model of de Paula and Brudvig [de Paula, J. C., & Brudvig, G. W. (1985) J. Am. Chem. Soc. 107, 2643-2648]. The spin-state change induced by cryoprotectants is compared to that seen in the iron protein of nitrogenase.
Q400, a high-potential electron acceptor associated with Photosystem II (PS II) of oxygenic photosynthesis, originally described by Ikegami and Katoh [3], has recently been identified by Petrouleas and Diner [8] as the non-heme iron of the iron-quinone complex of the PS II reaction center. This acceptor, which can function as the Fe(Ill)/Fe(II) redox couple with an Em.7, of 400 mV, demonstrates a pH-dependence of −60 mV/pH unit, indicative of a protonation reaction coupled to Fe(III) reduction.In this review, we describe the chemical and physical properties of the acceptor which led to its identification. Through a combination of optical, EPR and Mössbauer spectroscopy, we also show how the iron, unlike its bacterial reaction center homologue, is capable of redox chemistry involving the neighboring quinones, and how it serves as a sensitive spectroscopic probe, not only of its immediate coordination sphere, but of the sites at which quinones and inhibitors bind to the reaction center. A theoretical description of the Fe(III) EPR spectrum which accounts for the positions, amplitudes and energetics of the observed resonances is also presented.
EPR was used to study the binding of NH3 to the photosynthetic O2-evolving center, NH3-treated, Ca2+-depleted Photosystem II (PS II) membranes exposed to continuous light at 250 K showed a 10 mT-wide asymmetric EPR signal, centered around g = 2. When dark-adapted material was illuminated with a sequence of laser flashes the same signal appeared after the second flash, indicating that the g = 2 signal arises from a modified S3 state. The signal is different from the 15–16.5 mT-wide EPR signal at g = 2 ascribed to the S3′ state. Illumination of native NH3-treated PS II membranes with continuous light results in the appearance of an EPR signal at g = 2 with a width similar to that in Ca2+-depleted. NH3-treated membranes. The conditions for the formation of the signal and its properties suggest that it also arises from a perturbed S3 state with NH3 in close association with the manganese.
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.
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.
Detergent-treatment of higher plant thylakoids with Triton X-100 at pH 6.3 has been used to purify a PS2 fraction with very high rates of oxygen evolution (1000 μmol.mg chl−1.h−1). A photosynthetic unit size of about 300 chlorophyll (chl) molecules has been determined by optical methods, suggesting an average turnover time for PS2 of about 2 ms. The donor system for P680+ is particularly well preserved in the preparation, as judged by P680+ reduction kinetics, the detection by EPR of Signal IILT and the presence of the high potential form of cytochrome b-559 (at a ratio of 1:1 with the reaction centre).
1.1. The kinetics of prompt and delayed fluorescence of isolated chloroplasts or algae have been monitored after flash preillumination (in the time-range extending from 0.4 s after the flash). A rapid mixing with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) may take place after the last flash.2.2. 1 s after the mixing with DCMU, the prompt fluorescence displays binary oscillations with the number of preilluminating flashes, similarly to the observation of Velthuys, B.R. and Amesz, J. ((1974) Biochim. Biophys. Acta 333, 85–94), with chloroplasts to which an artificial Photosystem II donor was added. These oscillations are due to a back-transfer of electrons from the secondary acceptor, B, to the primary acceptor, Q, caused by DCMU. At longer times after the mixing, charge recombination takes place to a variable extent according to the charge storage state Si on the donor side, yielding the oscillatory pattern observed by Wollman, F.A. ((1978) Biochim. Biophys. Acta 503, 263–273).3.3. Shifting the pH from 6 to 8 causes an acceleration of the DCMU-induced back-transfer to Q and an about 2-fold increase in the amplitude of the fluorescence oscillations. The rate of the DCMU-induced rise of fluorescence is sensitive to the pH during the mixing, whereas the amplitude of the oscillations depends on the pH during the preillumination. Even under optimal conditions, the oscillations account only for a fraction of the total variable fluorescence.4.4. The delayed light emitted by isolated chloroplasts in the 100 ms—seconds range oscillates weakly (periodicity 4) with the number of preilluminating flashes. Mixing with DCMU after the preillumination causes a delayed light stimulation which varies with the flash number. The enhancement factor oscillates with periodicities of both 2 and 4. The amplitude of the period-2 contribution varies with the amount of B oxidized in the dark, while that of the period-4 contribution depends on the extent of this type of oscillation in the control experiment.5.5. The period-2 oscillations of the DCMU-stimulation of delayed light behave similarly to the fluorescence oscillations. It is shown that they are not due to a modulation of luminescence by the fluorescence yield, but rather to the variations of the amount of Q− as a substrate.6.6. It is concluded that in the absence of DCMU, the reduced secondary acceptor B− is not the main source of electrons involved in radiative recombination of functional centers in the time-range we have studied. Possible models are discussed.
An S1-state parallel polarization "multiline" EPR signal arising from the oxygen-evolving complex has been detected in spinach (PSII) membrane and core preparations depleted of the 23 and 17 kDa extrinsic polypeptides, but retaining the 33 kDa extrinsic protein. This S1-state multiline signal, with an effective g value of 12 and at least 18 hyperfine lines, has previously been detected only in PSII preparations from the cyanobacterium sp. Synechocystis sp. PCC6803 [Campbell, K. A., Peloquin, J. M., Pham, D. P., Debus, R. J., and Britt, R. D. (1998) J. Am. Chem. Soc. 120, 447-448]. It is absent in PSII spinach membrane and core preparations that either fully retain or completely lack the 33, 23, and 17 kDa extrinsic proteins. The S1-state multiline signal detected in spinach PSII cores and membranes has the same effective g value and hyperfine spacing as the signal detected in Synechocystis PSII particles. This signal provides direct evidence for the influence of the extrinsic PSII proteins on the magnetic properties of the Mn cluster.
We have compared the temperature-dependence characteristics of the EPR signals of Qa and Qb iron-semiquinones from both purple bacterial and plant photosystems. The data obtained were analyzed and estimates of the splitting parameters of the non-heme Fe2+ spin sublevels obtained. The study confirms the similarities of the g = 1.8 Qa iron-semiquinone signal (D/k = 15.6 K, E/k = 3.3 K) formed in formate-treated plant photosystem 2 to the signal found in purple bacteria. However, the g = 1.9 Qa iron-semiquinone signal (D/k = 7.1 K, E/k = much less than 1 K), formed in photosystem 2 when bicarbonate remains bound, has a unique temperature behavior. A series of spectral features associated with the iron-semiquinone in bicarbonate-bound photosystem 2 appear as the temperature is lowered, and the analysis of these data requires that some of these features be assigned to the higher spin states. The results are discussed in terms of the requirement for bicarbonate to be a ligand of the non-heme iron.
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 studies have revealed that removal of calcium using citric acid from the site in spinach photosystem II which is coupled to the photosynthetic O2-evolving process produces a structural change in the manganese cluster responsible for water oxidation. If done in the dark, this yields a modified S1' oxidation state which can be photooxidized above 250 K to form a structurally altered S2' state, as seen by formation of a "modified" multiline EPR signal. Compared to the "normal" S2 state, this new S2'-state EPR signal has more lines (at least 25) and 25% narrower 55Mn hyperfine splittings, indicative of disruption of the ligands to manganese. The calcium-depleted S2' oxidation state is greatly stabilized compared to the native S2 oxidation state, as seen by a large increase in the lifetime of the S2' EPR signal. Calcium reconstitution results in the reduction of the oxidized tyrosine residue 161YD+ (Em approximately 0.7-0.8 V, NHE) within the reaction center D1 protein in both the S1' and S2' states, as monitored by its EPR signal intensity. We attribute this to reduction by Mn. Thus a possible structural role which calcium plays is to bring YD+ into redox equilibrium with the Mn cluster. Photooxidation of S2' above 250 K produces a higher S state (S3 or S4) having a new EPR signal at g = 2.004 +/- 0.003 and a symmetric line width of 163 +/- 3 G, suggestive of oxidation of an organic donor, possibly an amino acid, in magnetic contact with the Mn cluster. This EPR signal forms in a stoichiometry of 1-2 relative to YD+.(ABSTRACT TRUNCATED AT 250 WORDS)
The light-driven water-splitting/oxygen-evolving enzyme remains one of the great enigmas of plant biology. However, due to the recent expansion of research efforts on this enzyme, it is grudgingly giving up some of its secrets.
An S3 electron paramagnetic resonance (EPR) signal is observed in a variety of photosystem 2 (PS2) samples in which the oxygen-evolving complex (OEC) has been inhibited. These signals have been proposed to be due to an interaction, S2X+, between the manganese cluster in an oxidation state equivalent to S2 and an organic radical, either oxidized histidine [Boussac et al. (1990) Nature 347, 303-306] or the tyrosine radical Yz+ [Hallahan et al. (1992) Biochemistry 31, 4562-4573]. We report that treatment of PS2 with acetate at pH 5.5 leads to a slowing of the reduction of Yz+ and allows the trapping of an S3-type state on freezing to 77 K following illumination at 277 K. The S3 EPR signal in acetate-treated PS2 has a broader and more complex line shape but otherwise has similar properties to other S3 signals. The addition to acetate-treated samples in the S1 state of the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), which allows only a single turnover of the reaction center, causes a large reduction in the yield of the S3 signal. Various anion and cation treatments change the S3 signal line shape and are used to show that acetate probably acts by binding and displacing chloride. We propose that a variety of treatments which affect calcium and chloride cofactor binding cause a modification of the S2 state of the manganese cluster, slow the reduction of Yz+, and allow an S3 EPR signal to be observed following illumination.(ABSTRACT TRUNCATED AT 250 WORDS)
A 230-G-wide EPR signal is induced in acetate-treated photosystem II by 30 s of illumination at 277 K followed by freezing under illumination to 77 K [MacLachlan, D. J., & Nugent, J. H. A. (1993) Biochemistry 32, 9772-9780]. This signal, referred to as the S3 EPR signal, has been interpreted to arise from an S2X+ species where X+ is an amino acid radical. Investigation of the factors responsible for the formation and decay of the S3 EPR signal reveals that the yield of the S3 EPR signal is strongly temperature-dependent and depends on the rate of oxidation of QA-. Quantitation of the number of centers contributing to the S3 EPR signal produced by the optimal continuous illumination times of 3 min at 250 K, 30 s at 273 K, and 5 s at 294 K gave values of 13, 38, and 49 +/- 3%, respectively. By using 5 s of illumination at 294 K to induce the S3 EPR signal, and then illumination at 200 K to reduce QA, both the S3 and QA(-)Fe EPR signals were induced in high yield. This result indicates that the S3 EPR signal does not arise from an acceptor-side species. When saturating laser flashes were used to induce the S3 EPR signal in a dark-prepared, dark-adapted, acetate-treated sample, the yield was small after one flash and close to maximal after two flashes. An EPR signal at g = 4.1 was observed to be formed at intermediate times during the decay of the S3 EPR signal in the dark; the rates of decay of the S3 EPR signal at 273 and 294 K corresponded to the rates of formation of the g = 4.1 EPR signal. These results, together with the flash results, indicate that two steps are involved in both the generation and decay of the S3 EPR signal. The rates of formation and decay of both the S3 and QA(-)Fe EPR signals were measured at 250, 273, and 294 K. A kinetic model is presented that accounts for these kinetic data and the yield of the S3 EPR signal.
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 a multiline signal (spin 1/2) or as a signal at g = 4.1 (spin 3/2 or spin 5/2). It is shown here that at around 150 K the state responsible for the multiline signal is converted to that responsible for the g = 4.1 signal upon the absorption of infrared light. This conversion is fully reversible at 200 K. The action spectrum of this conversion has its maximum at 820 nm (12 200 cm-1) and is similar to the intervalence charge transfer band in di-mu-oxo-(MnIIIMnIV) model systems. It is suggested that the conversion of the multiline signal to the g = 4.1 signal results from absorption of infrared light by the Mn cluster itself, resulting in electron transfer from MnIII to MnIV. The g = 4.1 signal is thus proposed to arise from a state which differs from that which gives rise to the multiline signal only in terms of this change in its valence distribution. The near-infrared light effect was observed in the S2 state of Sr(2+)-reconstituted photosystem II and in Ca(2+)-depleted, EGTA (or citrate-)-treated photosystem II but not in ammonia-treated photosystem II. Earlier results in the literature which showed that the g = 4.1 state was preferentially formed by illumination at 130 K are reinterpreted as being the result of two photochemical events: the first being photosynthetic charge separation resulting in an S2 state which gives rise to the multiline signal and the second being the conversion of this state to the g = 4.1 state due to the simultaneous and inadvertent presence of 820 nm light in the broad-band illumination given. There is therefore no reason to consider the state responsible for the g = 4.1 signal as a precursor of that which gives rise to the multiline signal.
Magnetic properties of the S1-state manganese cluster in the oxygen-evolving photosystem II were studied by parallel polarization electron paramagnetic resonance spectroscopy. Dark minus light spectra gave rise to a broad S1-state signal with a g value of about 4.9 [Dexheimer, S. L., Klein, M. P. (1992) J. Am. Chem. Soc. 114, 2821-2826]. Temperature variation of the signal intensity between 1.9 and 10 K observed in PS II with a sucrose buffer indicates that the signal originates from an excited state with a spin S of 1 with separation from the ground state (S = 0) of about 2.5 K. The S1-state signal was also observed in the sucrose buffer supplemented by 50% glycerol. However, no S1-state signal was detected by addition of 3% methanol or 30% ethylene glycol in the sucrose buffer, although illumination at 200 K in the presence of these alcohols induced the normal multiline S2 signal. Furthermore, modification of the Mn cluster by Cl- or Ca2+ depletion from PS II membranes failed to produce a detectable S1-state signal. A possible magnetic structure of the Mn cluster responsible for the generation of the S1-state signal is discussed on the basis of these observations.
In plants and algae, photosystem II uses light energy to oxidize water to oxygen at a metalloradical site that comprises a
tetranuclear manganese cluster and a tyrosyl radical. A model is proposed whereby the tyrosyl radical functions by abstracting
hydrogen atoms from substrate water bound as terminal ligands to two of the four manganese ions. Molecular oxygen is produced
in the final step in which hydrogen atom transfer and oxygen-oxygen bond formation occur together in a concerted reaction.
This mechanism establishes clear analogies between photosynthetic water oxidation and amino acid radical function in other
enzymatic reactions.
The S0* state was generated by incubation of dark-adapted (S1 state) photosystem II membranes either with the exogenous two electron reductant hydrazine and subsequent 273 K illumination in the presence of DCMU or by dark incubation with low amounts of the one electron reductant hydroxylamine. In agreement with earlier reports, the S1 and S-1 states were found to be electron paramagnetic resonance (EPR) silent. However, in the presence of 0.5-1.5% methanol, a weak EPR multiline signal centered around g = 2.0 was observed at 7 K for the S0* states generated by both procedures. This signal has a similar average line splitting to the well-characterized S2 state multiline EPR signal, but can be clearly distinguished from that and other modified S2 multiline signals by differences in line position and intensities. In addition, at 4 K it can be seen that the S0* multiline has a greater spectral breadth than the S2 multilines and is composed of up to 26 peaks. The S0* signal is not seen in the absence of methanol and is not affected by 1 mM EDTA in the buffer medium. We assign the S0* multiline signal to the manganese cluster of the oxygen evolving complex in a mixed valence state of the form MnIIMnIIIMnIIIMnIII,MnIIMnIIIMnIVMnIV, or MnIIIMnIIIMnIIIMnIV. Addition of methanol may be helpful in future to find an EPR signal originating form the natural S0 state.
Photosynthesis produces the oxygen necessary for all aerobic life. During this process, the manganese-containing oxygen evolving complex (OEC) in photosystem II (PSII), cycles through five oxidation states, S0-S4. One of these, S2, is known to be paramagnetic and gives rise to electron paramagnetic resonance (EPR) signals used to probe the catalytic structure and function of the OEC. The S0 states has long been thought to be paramagnetic. We report here a Mn EPR signal from the previously EPR invisible S0 state. The new signal oscillates with a period of four, indicating that it originates from fully active PSII centers. Although similar to the S2 state multiline signal, the new signal is wider (2200 gauss compared with 1850 gauss in samples produced by flashing), with different peak intensity and separation (82 gauss compared with 89 gauss). These characteristics are consistent with the S0 state EPR signal arising from a coupled MnII-MnIII intermediate. The new signal is more stable than the S2 state signal and its decay in tens of minutes is indicative of it originating from the S0 state. The S0 state signal will provide invaluable information toward the understanding of oxygen evolution in plants.
The Mn4 complex which is involved in water oxidation in photosystem II (PSII) is known to exhibit two types of EPR signals in the S2 state, one of the five redox states of the enzyme cycle: either a multiline signal (S = 1/2) or a signal at g = 4.1 (S = 3/2 or S= 5/2). The S = 1/2 state can be converted to that responsible for the g = 4.1 signal upon the absorption of near-infrared (IR) light [Boussac, A., Girerd, J.-J., and Rutherford, A.W. (1996) Biochemistry 35, 6984-6989]. It is shown here that a third state gives rise to signals at g = 10 and 6. This state is formed by IR illumination of the S = 1/2 state at 65 K, a temperature where IR illumination leads to the loss of the S = 1/2 signal but to no formation of the g = 4.1 state. On the basis of the corresponding decrease of the S = 1/2 state, the new state can be trapped in approximately 40% of the PSII centers. Warming of the sample above 65 K, in the dark, leads to the loss of the g = 10 and 6 resonances with the corresponding appearance of the g = 4.1 signal. It is suggested that the IR-induced conversion of the S = 1/2 state into the g = 4.1 state at 150 K involves the transient formation of the new state. The new state is attributed to a S = 5/2 state of the Mn4 complex (although a S value > 5/2 is also a possibility). Spectral simulations indicate an E/D ratio of -0.05 with D </= 1 cm-1. The resonances at g = 10 and 6 correspond to the gz of the +/-5/2 and +/-3/2 transition, respectively. The temperature-dependent conversion of this S = 5/2 state into the g = 4.1 state is proposed to be due to relaxation of the ligand environment around the Mn4 cluster that leads to a change in the zero field splitting parameters, assuming an S = 5/2 value for the g = 4.1 state. The new form of the S2 state reported here may explain some earlier data where the S2 state was present and yet not detectable as either a S = 1/2 or a g = 4.1 EPR signal.
By application of microsecond light flashes the oxygen-evolving complex (OEC) was driven through its functional cycle, the S-state cycle. The S-state population distribution obtained by the application of n flashes (n = 0. 6) was determined by analysis of EPR spectra; Mn K-edge X-ray absorption spectra were collected. Taking into consideration the likely statistical error in the data and the variability stemming from the use of three different approaches for the determination of edge positions, we obtained an upshift of the edge position by 0.8-1.5, 0.5-0.9, and 0.6-1.3 eV for the S0-S1, S1-S2, and S2-S3 transitions, respectively, and a downshift by 2.3-3.1 eV for the S3-S0 transition. These results are highly suggestive of Mn oxidation state changes for all four S-state transitions. In the S0-state spectrum, a clearly resolved shoulder in the X-ray spectrum around 6555 eV points toward the presence of Mn(II). We propose that photosynthetic oxygen evolution involves cycling of the photosystem II manganese complex through four distinct oxidation states of this tetranuclear complex: Mn(II)-Mn(III)-Mn(IV)2 in the S0-state, Mn(III)2-Mn(IV)2 in the S1-state, Mn(III)1-Mn(IV)3 in the S2-state, and Mn(IV)4 in the S3-state.
The light-induced new EPR signals at g = 12 and 8 were observed in photosystem II (PS II) membranes by parallel polarization EPR. The signals were generated after two flashes of illumination at room temperature, and the signal intensity had four flashes period oscillation, indicating that the signal origin could be ascribed to the S3-state. Successful simulations were obtained assuming S = 1 spin for the values of the zero-field parameters, D = +/-0.435 +/- 0. 005 cm-1 and E/D = -0.317 +/- 0.002. Orientation dependence of the g =12 and 8 signal intensities shows that the axial direction of the zero-field interaction of the manganese cluster is nearly parallel to the membrane normal.
The essential involvement of manganese in photosynthetic water oxidation was implicit in the observation by Pirson in 1937 that plants and algae deprived of Mn in their growth medium lost the ability to evolve Oâ. Addition of this essential element to the growth medium resulted in the restoration of water oxidation within 30 min. There is increased interest in the study of Mn in biological chemistry and dioxygen metabolism in the last two decades with the discovery of several Mn redox enzymes. The list of enzymes where Mn is required for redox activity includes a Mn superoxide dismutase, a binuclear Mn-containing catalase, a binuclear Mn-containing ribonucleotide reductase, a proposed binuclear Mn site in thiosulfate oxidase, a Mn peroxidase that is capable of oxidative degradation of lignin, and perhaps the most complex and important, the tetranuclear Mn-containing oxygen-evolving complex in photosystem II (Mn-OEC). Mn is well suited for the redox role with accessible oxidation states of II, III, and IV, and possibly V: oxidation states that have all been proposed to explain the mechanisms of the Mn redox enzymes.
X-ray absorption near-edge structure spectra of the manganese (Mn) cluster in physiologically native intermediate states of photosynthetic water oxidation induced by short laser flash were measured with a compact heat-insulated chamber equipped with an x-ray detector near the sample surface. The half-height energy of the Mn Kedge showed a period-four oscillation dependent on cycling of the Joliot-Kok's oxygen clock. The flash number-dependent shift in the Mn K-edge suggests that the Mn cluster is oxidized by one electron upon the S(0)-to-S(1), S(1)-to-S(2), and S(2)-to-S(3) transitions and then reduced upon the S(3)-to-S(0) transition that releases molecular oxygen.