No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Q-band EPR of the S2 state of photosystem II confirms an S = 5/2 origin of the X-band g = 4.1 signal
Abstract
Disagreement has remained about the spin state origin of the g = 4.1 EPR signal observed at X-band (9 GHz) from the S2 oxidation state of the Mn cluster of Photosystem II. In this study, the S2 state of PSII-enriched membrane fragments was examined at Q-band (34 GHz), with special interest in low-field signals. Light-induced signals at g = 3.1 and g = 4.6 were observed. The intensity of the signal at g = 3.1 was enhanced by the presence of F- and suppressed by the presence of 5% ethanol, indicating that it was from the same spin system as the X-band signal at g = 4.1. The Q-band signal at g = 4.6 was also enhanced by F-, but not suppressed by 5% ethanol, making its identity less clear. Although it can be accounted for by the same spin system, other sources for the signal are considered. The observation of the signal at g = 3.1 agrees well with a previous study at 15.5 GHz, in which the X-band g = 4.1 signal was proposed to arise from the middle Kramers doublet of a near rhombic S = 5/2 system. Zero-field splitting values of D = 0.455 cm(-1) and E/D = 0.25 are used to simulate the spectra.
... The EPR spectrum of dark-adapted D1-D170E PSII core complexes measured with continuous wave X-band (9.40 GHz) at 5.0 K shows a signal at g = 4.3 and multiple signals between g = 5.9 and 6.7 (Fig. 2, blue trace). These signals correspond to free and protein-bound ferric ions normally observed in PS II (Miller and Brudvig 1991;Haddy et al. 2004). The signal at g = 4.3 corresponds to rhombic iron whereas the features between g = 5.9 and g = 6.7 arise in part from the oxidation of the non-heme iron in the Q A Fe complex from Fe 2+ to Fe 3+ induced by the presence of DCMU in a fraction of PSII reaction centers (Boussac et al. 2011). ...
... The light-minus-dark D1-D170E spectrum could be simulated with the parameters S = 5 / 2 , |D|= 0.41 cm −1 , E/D = 0.18 (Fig. 2, red line). These values are comparable to, although slightly lower than, those of the S 2 state g = 4.1 signal that is observed in spinach PSII: S = 5 / 2 , D = 0.455 cm −1 , E/D = 0.25 (Haddy et al. 2004). It should be noted that the value of D cannot be determined accurately from X-band measurements alone. ...
The residue D1-D170 bridges Mn4 with the Ca ion in the O2-evolving Mn4CaO5 cluster of Photosystem II. Recently, the D1-D170E mutation was shown to substantially alter the Sn+1-minus-Sn FTIR difference spectra [Debus RJ (2021) Biochemistry 60:3841-3855]. The mutation was proposed to alter the equilibrium between different Jahn–Teller conformers of the S1 state such that (i) a different S1 state conformer is stabilized in D1-D170E than in wild-type and (ii) the S1 to S2 transition in D1-D170E produces a high-spin form of the S2 state rather than the low-spin form that is produced in wild-type. In this study, we employed EPR spectroscopy to test if a high-spin form of the S2 state is formed preferentially in D1-D170E PSII. Our data show that illumination of dark-adapted D1-D170E PSII core complexes does indeed produce a high-spin form of the S2 state rather than the low-spin multiline form that is produced in wild-type. This observation provides further experimental support for a change in the equilibrium between S state conformers in both the S1 and S2 states in a site-directed mutant that retains substantial O2 evolving activity.
... [5,[10][11][12][13][14][15] In the parallel efforts of spectroscopic and quantum chemical studies to provide experimentally consistent atomistic models for other catalytic states and intermediates, a most intriguing outcome has been the role of polymorphism in the OEC. Specifically, the S 2 state of the Mn 4 CaO 5 cluster with oxidation states Mn III Mn IV 3 is known to exhibit two types of EPR signal, [16][17][18][19] at g = 2 (multiline signal, spin S = 1/2) and g ! 4.1 (S ! ...
... Several explanations for this phenomenon have been considered (see Supporting Information), but the most well-supported scenario suggests that the signals arise from two valence isomeric forms (referred to as S 2 A and S 2 B ) that differ in the position of the unique Mn III ion, Mn1 in S 2 A and Mn4 in S 2 B , respectively ( Figure 1). [20][21][22] The two valence isomers, which are close in energy and interconvertible, [20,[22][23] have distinct spin states and spectroscopic properties that correspond to the two known types of EPR signal [16][17][18][19] for the S 2 state: the S 2 A with spin S = 1/2 and g = 2, and the S 2 B with S ! 5/2 and g ! ...
The dark-stable resting state of the biological oxygen-evolving complex is shown to accommodate a rare type of functionally important MnIII orientational Jahn–Teller isomerism that is identified as the electronic origin of subsequent valence isomerism in the catalytic cycle of water oxidation.
Abstract
The tetramanganese–calcium cluster of the oxygen-evolving complex of photosystem II adopts electronically and magnetically distinct but interconvertible valence isomeric forms in its first light-driven oxidized catalytic state, S2. This bistability is implicated in gating the final catalytic states preceding O−O bond formation, but it is unknown how the biological system enables its emergence and controls its effect. Here we show that the Mn4CaO5 cluster in the resting (dark-stable) S1 state adopts orientational Jahn–Teller isomeric forms arising from a directional change in electronic configuration of the “dangler” MnIII ion. The isomers are consistent with available structural data and explain previously unresolved electron paramagnetic resonance spectroscopic observations on the S1 state. This unique isomerism in the resting state is shown to be the electronic origin of valence isomerism in the S2 state, establishing a functional role of orientational Jahn–Teller isomerism unprecedented in biological or artificial catalysis.
... [5,[10][11][12][13][14][15] In the parallel efforts of spectroscopic and quantum chemical studies to provide experimentally consistent atomistic models for other catalytic states and intermediates,amost intriguing outcome has been the role of polymorphism in the OEC. Specifically,t he S 2 state of the Mn 4 CaO 5 cluster with oxidation states Mn III Mn IV 3 is known to exhibit two types of EPR signal, [16][17][18][19] at g = 2( multiline signal, spin S = 1/2) and g ! 4.1 (S ! ...
... Several explanations for this phenomenon have been considered (see Supporting Information), but the most well-supported scenario suggests that the signals arise from two valence isomeric forms (referred to as S 2 A and S 2 B ) that differ in the position of the unique Mn III ion, Mn1 in S 2 A and Mn4 in S 2 B ,respectively ( Figure 1). [20][21][22] Thetwo valence isomers,which are close in energy and interconvertible, [20,[22][23] have distinct spin states and spectroscopic properties that correspond to the two known types of EPR signal [16][17][18][19] for the S 2 state:t he S 2 A with spin S = 1/2 and g = 2, and the S 2 B with S ! 5/2 and g ! ...
The tetramanganese–calcium cluster of the oxygen‐evolving complex of photosystem II adopts electronically and magnetically distinct but interconvertible valence isomeric forms in its first light‐driven oxidized catalytic state, S2. This bistability is implicated in gating the final catalytic states preceding O−O bond formation, but it is unknown how the biological system enables its emergence and controls its effect. Here we show that the Mn4CaO5 cluster in the resting (dark‐stable) S1 state adopts orientational Jahn–Teller isomeric forms arising from a directional change in electronic configuration of the “dangler” MnIII ion. The isomers are consistent with available structural data and explain previously unresolved electron paramagnetic resonance spectroscopic observations on the S1 state. This unique isomerism in the resting state is shown to be the electronic origin of valence isomerism in the S2 state, establishing a functional role of orientational Jahn–Teller isomerism unprecedented in biological or artificial catalysis.
... During the second oxidation of the OEC it was observed that no proton is released from the cluster and positive charge is accumulated in the OEC during the transition of S1 → S2 + [44,45]. The S2 state is paramagnetic and has been extensively studied using EPR spectroscopy and two different EPR signal at approximately g = 4.1 is observed and dramatic multiline EPR signal at g = 2 is observed based on the conditions used for the EPR measurement [46][47][48][49][50][51]. The g = 4.1 and g = 2 EPR signals represents two spin isomers of the S2 state with a ground state of S = 5/2 and S = 1/2 respectively. ...
... Adapted with permission from [34]. Copyright 2011, Nature publishing group) distances and promotes antiferromagnetic coupling of the Mn center leading to a low spin state [45,47,48,51]. During the third oxidation of the OEC by the nearby tyrosine radical, the Mn oxidation is coupled with the proton transfer and followed by water coordination and the resulting S3 state's contain four Mn(IV) centers with six-coordination [35,52,53]. ...
Manganese plays multiple role in many biological redox reactions in which it exists in different oxidation states from Mn(II) to Mn(IV). Among them the high-valent manganese-oxo intermediate plays important role in the activity of certain enzymes and lessons from the natural system provide inspiration for new developments of artificial systems for a sustainable energy supply and various organic conversions. This review describes recent advances and key lessons learned from the nature on high-valent Mn-oxo intermediates. Also we focus on the elemental science developed from the natural system, how the novel strategies are realised in nano particles and molecular sites at heterogeneous and homogeneous reaction conditions respectively. Finally, perspectives on the utilisation of the high-valent manganese-oxo species towards other organic reactions are proposed.
... The g = 2 signal arises from the S = 1/2 ground state, and the g = 4.1 signal arises from the S = 5/2 ground state. 19 Although the generation of the g = 4.1 EPR signal in native PSII is possible by other techniques, 19 in this work we will only discuss the g = 4.1 EPR signal induced by chloride depletion. Computational studies have further shown that the identity of the manganese ion oxidized in the S 1 to S 2 transition leads to two different S 2 states. ...
... The g = 2 signal arises from the S = 1/2 ground state, and the g = 4.1 signal arises from the S = 5/2 ground state. 19 Although the generation of the g = 4.1 EPR signal in native PSII is possible by other techniques, 19 in this work we will only discuss the g = 4.1 EPR signal induced by chloride depletion. Computational studies have further shown that the identity of the manganese ion oxidized in the S 1 to S 2 transition leads to two different S 2 states. ...
Chloride is an essential cofactor in the oxygen-evolution reaction that takes place in photosystem II (PSII). The oxygen-evolving complex (OEC) is oxidized in a linear four-step photocatalytic cycle in which chloride is required for the OEC to advance beyond the S2 state. Here, using Density Functional Theory, we compare the energetics and spin configuration of two different states of the Mn4CaO5 cluster in the S2 state; state A with Mn13+ and B with Mn43+ with and without chloride. The calculations suggest that model B with an S=5/2 ground state occurs in the chloride depleted PSII, which may explain the presence of the EPR signal at g=4.1. Moreover, we use Multi Conformer Continuum Electrostatics to study the effect of chloride depletion on the redox potential associated with the S1/S2 and S2/S3 transitions.
... Here, Mn1, Mn2, and Mn3 are Mn(IV) (S 1 = S 2 = S 3 = 3/2) and Mn4 is Mn(III) (S 4 = 2) with D = -0.445 and E/D = 0.25(52). ...
In photosystem II (PSII), one-electron oxidation of the most stable state of the oxygen-evolving Mn4CaO5 cluster (S1) leads to the S2 state formation, Mn1(III)Mn2(IV)Mn3(IV)Mn4(IV) (open-cubane S2) or Mn1(IV)Mn2(IV)Mn3(IV)Mn4(III) (closed-cubane S2). In electron paramagnetic resonance (EPR) spectroscopy, the g = 4.1 signal is not observed in cyanobacterial PSII but in plant PSII, whereas the g = 4.8 signal is observed in cyanobacterial PSII and extrinsic-subunit-depleted plant PSII. Here we investigated the closed-cubane S2 conformation, a candidate for a higher spin configuration that accounts for g > 4.1 EPR signal, considering all pairwise exchange couplings in the PSII protein environment (i.e. instead of considering only a single exchange coupling between the [Mn3(CaO4)] cubane region and the dangling Mn4 site). Only when a ligand water molecule that forms an H-bond with D1-Asp61 (W1) is deprotonated at dangling Mn4(IV), the g = 4.1 EPR spectra can be reproduced using the cyanobacterial PSII crystal structure. The closed-cubane S2 is less stable than the open-cubane S2 in cyanobacterial PSII, which may explain why the g = 4.1 EPR signal is absent in cyanobacterial PSII.
... In the S 2 state, in addition to the low-spin S eff = ½ form showing the characteristic multiline signal around g ≈ 2, the cluster can also be found in a high-spin S eff = 5/2 state under certain conditions. This is evident from an EPR signal around g = 4.1 (see Fig. 5), which has been observed earlier by several research groups (Boussac et al. 1996;Casey and Sauer 1984;Haddy et al. 2004;Zimmermann and Rutherford 1984). Pantazis et al. could show computationally that the two electronic structures are a direct consequence of two different spatial conformations of the manganese cluster, namely a "closed cubane" (S eff = 5/2) and an "open cubane" (S eff = ½) form, which have almost the same energy (Bovi et al. 2013;Isobe et al. 2012;Pantazis et al. 2012). ...
Biological water oxidation, performed by a single enzyme, photosystem II, is a central research topic not only in understanding the photosynthetic apparatus but also for the development of water splitting catalysts for technological applications. Great progress has been made in this endeavor following the report of a high-resolution X-ray crystallographic structure in 2011 resolving the cofactor site (Umena et al. in Nature 473:55–60, 2011), a tetra-manganese calcium complex. The electronic properties of the protein-bound water oxidizing Mn4OxCa complex are crucial to understand its catalytic activity. These properties include: its redox state(s) which are tuned by the protein matrix, the distribution of the manganese valence and spin states and the complex interactions that exist between the four manganese ions. In this short review we describe how magnetic resonance techniques, particularly EPR, complemented by quantum chemical calculations, have played an important role in understanding the electronic structure of the cofactor. Together with isotope labeling, these techniques have also been instrumental in deciphering the binding of the two substrate water molecules to the cluster. These results are briefly described in the context of the history of biological water oxidation with special emphasis on recent work using time resolved X-ray diffraction with free electron lasers. It is shown that these data are instrumental for developing a model of the biological water oxidation cycle.
... Mn III /Mn IV and Mn IV /Mn IV are anti-ferromagnetically- coupled, respectively) ground state, LS S 2 state (Dismukes and Siderer 1981, Hansson and Andréasson 1982, Brudvig et al. 1983, de Paula and Brudvig 1985, Randall et al. 1995, Peloquin et al. 2000, Charlot et al. 2005, Haddy 2007, Kulik et al. 2007, Cox et al. 2011). Another broad featureless EPR signal at g ≥ 4.1, attributed to a higher spin multiplicity (S total = 5/2, i.e. ferromagnetically-coupled three Mn IV with anti- ferromagnetically-coupled one Mn III ) ground state, HS S 2 state, is also observed under different experimental conditions (Casey and Sauer 1984, Zimmermann and Rutherford 1984, Boussac et al. 1996, Boussac et al. 1998b, Boussac et al. 1998a, Peloquin and Britt 2001, Haddy et al. 2004). ...
In nature, an oxo‐bridged Mn4CaO5 cluster embedded in Photosystem II (PSII), a membrane‐bound multi‐subunit pigment protein complex, catalyzes the water oxidation reaction that is driven by light‐induced charge separations in the reaction center of PSII. The Mn4CaO5 cluster accumulates four oxidizing equivalents to enable the four‐electron four‐proton catalysis of two water molecules to one dioxygen molecule and cycles through five intermediate S‐states, S0 – S4 in the Kok cycle. One important question related to the catalytic mechanism of the oxygen‐evolving complex (OEC) that remains is, whether structural isomers are present in some of the intermediate S‐states and if such equilibria are essential for the mechanism of the O‐O bond formation. Here we compare results from electron paramagnetic resonance (EPR) and X‐ray absorption spectroscopy (XAS) obtained at cryogenic temperatures for the S2 state of PSII with structural data collected of the S1, S2 and S3 states by serial crystallography at neutral pH (~6.5) using an X‐ray free electron laser at room temperature. While the cryogenic data demonstrate the presence of at least two structural forms of the S2 state, the room temperature crystallography data can be well‐described by just one S2 structure. We discuss the deviating results and outline experimental strategies for clarifying this mechanistically important question.
This article is protected by copyright. All rights reserved.
... Structural determination and spectroscopic characterization of intermediates (and derivatives thereof) in the S-state catalytic cycle of the Oxygen Evolving Complex (OEC) of Photosystem II (PSII) heavily influence mechanistic proposals for O−O bond formation. [1][2][3][4][5][6][7][8][9][10][11][12][13][14] The dark-stable S1 state of the OEC consists of a CaMn4O5 cluster with Mn oxidation states Mn III 2 Mn IV 2. ...
Despite extensive biochemical, spectroscopic, and computational studies, the mechanism of biological water oxidation by the Oxygen Evolving Complex (OEC) of Photosystem II remains a subject of significant debate. Mechanistic proposals are guided by the characteri-zation of reaction intermediates such as the S2 state, which features two characteristic EPR signals at g = 2 and g = 4.1. Two nearly isoen-ergetic structural isomers have been proposed as the source of these distinct signals, but relevant structure-electronic structure studies remain rare. Herein, we report the synthesis, crystal structure, electrochemistry, XAS, magnetic susceptibility, variable temperature CW-EPR, and pulse EPR data for a series of [MnIIIMn3IVO4] cuboidal complexes as spectroscopic models of the S2 state of the OEC. Re-sembling the oxidation state and EPR spectra of the S2 state of the OEC, these model complexes show two EPR signals, a broad low field signal and a multiline signal, that are remarkably similar to the biological system. The effect of systematic changes in the nature of the bridging ligands on spectroscopy were studied. Results show that the electronic structure of tetranuclear Mn complexes is highly sensitive to even small geometric changes and the nature of the bridging ligands. Our model studies suggest that the spectroscopic properties of the OEC may also react very sensitively to small changes in structure; the effect of protonation state and other reorganization processes need to be carefully assessed.
... Due to the ease of generation and detection of the S 2 state during S-state cycling, this state has been studied extensively. EPR studies of the S 2 state in PSII from spinach reveal two distinct spin isomers corresponding to S = 1/2 (g = 2) and S = 5/2 (g = 4.1) states that exist in equilibrium with each other (6)(7)(8). In cyanobacterial PSII, however, the S 2 -state EPR spectrum exhibits only the S = 1/2 spin isomer (8). ...
Photoinduced water oxidation at the O2-evolving complex (OEC) of photosystem II (PSII) is a complex process involving a tetramanganese-calcium cluster that is surrounded by a hydrogen-bonded network of water molecules, chloride ions, and amino-acid residues. Although the structure of the OEC has remained conserved over eons of evolution, significant differences in the chloride-binding characteristics exist between cyanobacteria and higher plants. An analysis of amino-acid residues in and around the OEC has identified residue 87 in the D1 subunit as the only significant difference between PSII in cyanobacteria and higher plants. We substituted the D1-N87 residue in the cyanobacterium Synechocystis sp. PCC 6803 (wild-type) with alanine, present in higher plants, or with aspartic acid. We studied PSII core complexes purified from D1-N87A and D1-N87D variant strains to probe the function of the D1-N87 residue in the water-oxidation mechanism. EPR spectra of the S2 state and flash-induced FTIR spectra of both D1-N87A and D1-N87D PSII core complexes exhibited characteristics similar to those of wildtype Synechocystis PSII core complexes. However, flash-induced O2-evolution studies revealed a decreased cycling efficiency of the D1-N87D variant, whereas the cycling efficiency of the D1-N87A PSII variant was similar to that of wild-type PSII. Steady-state O2-evolution activity assays revealed that substitution of the D1-87 residue with alanine perturbs the chloridebinding site in the proton-exit channel. These findings provide new insight into the role of the D1-N87 site in the water-oxidation mechanism and explain the difference in the chloride-binding properties of cyanobacterial and higher-plant PSII.
... Previous experiments have shown that the S states do not, however, correspond to single homogenous spin and conformational states. Electron paramagnetic resonance (EPR) studies showed that the Mn 4 O 5 Ca can exist in alternative high and low spin states in the S 2 state and that these are close in energy [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29]. The spin state change was attributed to the Mn(III) valence being localized on a different Mn ion within the cluster [17,25]. ...
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 high-spin S2 state of the photosynthetic oxygen-evolving cluster Mn4CaO5, corresponding to the g = 4.1 signal for X-band electron paramagnetic resonance (EPR), was investigated using Q-band pulsed EPR, which detected a main peak at g = 3.10 and satellite peaks at 5.25, 4.55, and 2.80. We evaluated the spin state as the zero-field splitting of D = 0.465 cm-1 and E/D = 0.245 with S = 5/2. The temperature dependence of the T1 relaxation time revealed that the excited-state energy was 28.7 cm-1 higher than that of the S = 5/2 ground state. By comparing present quantum mechanical (QM) calculation models, a closed-cubane structure with the protonation state of two oxygens, W1 (= OH-) and W2 (= H2O), was the most probable structure for the S = 5/2 state. The three-pulse electron spin-echo envelope modulation (ESEEM) detected the nuclear signal, which was assigned to nitrogen as His332 ligated to the Mn1 ion. The obtained hyperfine constant for the nitrogen signal was significantly reduced from that in the S = 1/2 low-spin state. These results indicate that the S = 5/2 spin state arises from the closed-cubane structure.
Recent advances in serial femtosecond crystallography (SFX) of photosystem II (PSII), enabled by X-ray free electron lasers (XFEL), provided the first geometric models of distinct intermediates in the catalytic S-state cycle of the oxygen-evolving complex (OEC). These models are obtained by flash-advancing the OEC from the dark-stable state (S1) to more oxidized intermediates (S2 and S3), eventually cycling back to the most reduced S0. However, the interpretation of these models is controversial because geometric parameters within the Mn4CaO5 cluster of the OEC do not exactly match those expected from coordination chemistry for the spectroscopically verified manganese oxidation states of the distinct S-state intermediates. Here we focus on the first catalytic transition, S1 → S2, which represents a one-electron oxidation of the OEC. Combining geometric and electronic structure criteria, including a novel effective oxidation state approach, we analyze existing 1-flash (1F) SFX-XFEL crystallographic models that should depict the S2 state of the OEC. We show that the 1F/S2 equivalence is not obvious, because the Mn oxidation states and total unpaired electron counts encoded in these models are not fully consistent with those of a pure S2 state and with the nature of the S1 → S2 transition. Furthermore, the oxidation state definition in two-flashed (2F) structural models is practically impossible to elucidate. Our results advise caution in the extraction of electronic structure information solely from the literal interpretation of crystallographic models and call for re-evaluation of structural and mechanistic interpretations that presume exact correspondence of such models to specific catalytic intermediates of the OEC.
The photosystem II (PSII)-catalyzed water oxidation is crucial for maintaining life on earth. Despite the extensive experimental and computational research that has been conducted over the past two decades, the mechanisms of O-O bond formation and oxygen release during the S3 ∼ S0 stage remain disputed. While the oxo-oxyl radical coupling mechanism in the "open-cubane" S4 state is widely proposed, recent studies have suggested that O-O bond formation may occur from either the high-spin water-unbound S4 state or the "closed-cubane" S4 state. To gauge the various mechanisms of O-O bond formation proposed recently, the comprehensive QM/MM and QM calculations have been performed. Our studies show that both the nucleophilic O-O coupling from the Mn4 site of the high-spin water-unbound S4 state and the O5-O6 or O5-OW2 coupling from the "closed-cubane" S4 state are unfavorable kinetically and thermodynamically. Instead, the QM/MM studies clearly favor the oxyl-oxo radical coupling mechanism in the "open-cubane" S4 state. Furthermore, our comparative research reveals that both the O-O bond formation and O2 release are dictated by (a) the exchange-enhanced reactivity and (b) the synergistic coordination interactions from the Mn1, Mn3, and Ca sites, which partially explains why nature has evolved the oxygen-evolving complex cluster for the water oxidation.
Multifrequency (128 and 256 GHz) high-field electron paramagnetic resonance measurements up to 14.5 T over the temperature range 8.0 to 30.0 K were performed on powder samples of a recently reported salt of the cluster cation [Mn5O4(phth)3(phthH)(bpy)4]⁺ (1; Mn2IVMn2IIIMnII\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\mathrm{Mn}}_{2}^{\mathrm{IV}}{\mathrm{Mn}}_{2}^{\mathrm{III}}{\mathrm{Mn}}^{\mathrm{II}}$$\end{document}). Spectral simulations were performed to quantify the zero-field splitting parameters of 1, further supporting the previously assigned S = ⁷/2 ground state. 1 possesses a highly biaxial zero-field splitting tensor (E/D = 0.227) with overall easy-plane anisotropy (D > 0) arising from the near-perpendicular angle between the Jahn–Teller axes of the two MnIII that contribute a majority of the magnetic anisotropy. A microscopic model has been developed that relates the sign of D and the degree of ortho-rhombicity, E/D, to the angle between the two Jahn–Teller axes. The additional fine structure and peak-splitting features not represented by the simulations were attributed to population of excited states or the weak intermolecular interactions previously observed in the crystal structure.
The understanding of light‐induced biological water oxidation in oxygenic photosynthesis is of great importance both for biology and (bio)technological applications. The chemically difficult multistep reaction takes place at a unique protein‐bound tetra‐manganese/calcium cluster in photosystem II whose structure has been elucidated by X‐ray crystallography (Umena et al. Nature, 2011). The cluster moves through several intermediate states in the catalytic cycle. A detailed understanding of these intermediates requires information about the spatial and electronic structure of the Mn4Ca complex; the latter is only available from spectroscopic techniques. Here the important role of Electron Paramagnetic Resonance (EPR) and related double resonance techniques (ENDOR, EDNMR), complemented by quantum chemical calculations, is described. This has led to the elucidation of the cluster´s redox and protonation states, the valence and spin states of the manganese ions and the interactions between them, and contributed substantially to the understanding of the role of the protein surrounding, as well as the binding and processing of the substrate water molecules, the O‐O bond formation and dioxygen release. Based on these data models for the water oxidation cycle are developed. Light‐induced water oxidation and dioxygen release in photosynthesis is catalyzed by a paramagnetic μ‐oxo‐bridged Mn4Ca cofactor. It passes through five metastable states (S0 ‐ S4) whose structure is described focusing on the essential electronic structure obtained from spectroscopy, especially EPR techniques, supported by quantum chemistry. A catalytic cycle is presented which describe structural isomers of key S‐state intermediates facilitating substrate binding and cofactor activation.
The primary coordination sphere of the multinuclear cofactor (Mn4CaOx) in the oxygen-evolving complex (OEC) of photosystem II is absolutely conserved to maintain its structure and function. Recent time-resolved serial femtosecond crystallography identified large reorganization of the primary coordination sphere in the S2 to S3 transition, which elicits a cascade of events involving Mn oxidation and water molecule binding to a putative catalytic Mn site. We examined how the crystallographic fields, created by transient conformational states of the OEC at various time points, affect the thermodynamics of various isomers of the Mn cluster using DFT calculations, with an aim of comprehending the functional roles of the flexible primary coordination sphere in the S2 to S3 transition and in the recovery of the S2 state. The results show that the relative movements of surrounding residues change the size and shape of the cavity of the cluster and thereby affect the thermodynamics of various catalytic intermediates as well as the ability to capture a new water molecule at a coordinatively unsaturated site. The implication of these findings is that the protein dynamics may serve to gate the catalytic reaction efficiently by controlling the sequence of Mn oxidation/reduction and water binding/release. This interpretation is consistent with EPR experiments; g ∼ 5 and g ∼ 3 signals obtained after near-infrared (NIR) excitation of the S3 state at 4 K and a g ∼ 5 only signal produced after prolonged incubation of the S3 state at 77 K can be best explained as originating from water-bound S2 clusters (Stotal = 7/2) under a S3 ligand field, i.e., the immediate one-electron reduction products of the oxyl-oxo (Stotal = 6) and hydroxo-oxo (Stotal = 3) species in the S3 state.
The solar water-splitting protein complex, photosystem II (PSII), catalyzes one of the most energetically demanding reactions in nature by using light energy to drive a catalyst capable of oxidizing water. The water oxidation reaction is catalyzed at the Mn4Ca-oxo cluster in the oxygen-evolving complex (OEC), which cycles through five light-driven S-state intermediates (S0-S4). A detailed mechanism of the reaction remains elusive as it requires knowledge of the delivery and binding of substrate water in the higher S-state intermediates. In this study, we use two-dimensional (2D) hyperfine sublevel correlation spectroscopy, in conjunction with quantum mechanics/molecular mechanics (QM/MM) and density functional theory (DFT), to probe the binding of the substrate analog, methanol, in the S2 state of the D1-N87A variant of PSII from Synechocystis sp. PCC 6803. The results indicate that the size and specificity of the "narrow" channel is altered in D1-N87A PSII, allowing for the binding of deprotonated 13C-labeled methanol at the Mn4(IV) ion of the catalytic cluster in the S2 state. This has important implications on the mechanistic models for water oxidation in PSII.
The Photosystem II (PSII) water oxidation to molecular oxygen process is catalyzed by an oxygen-bridged Mn4CaO5 cluster, known as Oxygen Evolving Complex (OEC) of PSII. The Mn4CaO5 cluster undergoes periodically four one-electron oxidation steps, S0 → S1, S1 → S2, S2 → S3, S3 → S0. The O2 release takes place during the S3-[S4]-S0 transition, S4 being a transient. The intermediates of the S-state transitions are known as metalloradical intermediate states (SiYz) and involve the free radical of Tyrosine Z (Yz, TyrZ). In most metalloradical states it was established that Yz interacts magnetically with the Mn4CaO5 cluster. However, in Ca²⁺- depleted PSII preparations the spin-spin interaction between the Mn4O5 and Yz, during the formation of the S2Yz intermediate state was not strongly supported experimentally. In our effort to investigate the existence of the aforementioned interaction, we took advantance of the NIR sensitivity of the S2 state of Mn4 and examined whether the S2Yz EPR spectrum can be modified. Our EPR experiments, combined with the simulation analysis of the spectra show that reversible modification of the S2 EPR spectrum by NIR irradiation results in a reversible change of the Yz EPR signal shape. These observations strongly support the idea of the magnetic interaction between the Mn4 and tyrosyl radical, upon the formation of the S2Yz metalloradical state in Ca²⁺- depleted PSII membranes.
We report the single crystal XRD and MicroED structure, magnetic susceptibility, and EPR data of a series of CaMn3IVO4 and YMn3IVO4 complexes as structural and spectroscopic models of the cuboidal subunit of the oxygen‐evolving complex (OEC). The effect of changes in heterometal identity, cluster geometry, and bridging oxo protonation on the spin‐state structure was investigated. In contrast to previous computational models, we show that the spin ground state of CaMn3IVO4 complexes and variants with protonated oxo moieties need not be S=9/2. Desymmetrization of the pseudo‐C3‐symmetric Ca(Y)Mn3IVO4 core leads to a lower S=5/2 spin ground state. The magnitude of the magnetic exchange coupling is attenuated upon oxo protonation, and an S=3/2 spin ground state is observed in CaMn3IVO3(OH). Our studies complement the observation that the interconversion between the low‐spin and high‐spin forms of the S2 state is pH‐dependent, suggesting that the (de)protonation of bridging or terminal oxygen atoms in the OEC may be connected to spin‐state changes.
To better describe the Sn-state intermediates of Photosystem II, a ferromagnetically coupled CaMn3IVO4 subunit with an S=9/2 ground state has been proposed. This assignment has played a key role in the mechanism of water oxidation, but electronic-structure studies of CaMn3IVO4 complexes remain rare. Through cluster desymmetrization or oxo protonation, lower spin ground states are found to be accessible, challenging prior models.
Abstract
We report the single crystal XRD and MicroED structure, magnetic susceptibility, and EPR data of a series of CaMn3IVO4 and YMn3IVO4 complexes as structural and spectroscopic models of the cuboidal subunit of the oxygen-evolving complex (OEC). The effect of changes in heterometal identity, cluster geometry, and bridging oxo protonation on the spin-state structure was investigated. In contrast to previous computational models, we show that the spin ground state of CaMn3IVO4 complexes and variants with protonated oxo moieties need not be S=9/2. Desymmetrization of the pseudo-C3-symmetric Ca(Y)Mn3IVO4 core leads to a lower S=5/2 spin ground state. The magnitude of the magnetic exchange coupling is attenuated upon oxo protonation, and an S=3/2 spin ground state is observed in CaMn3IVO3(OH). Our studies complement the observation that the interconversion between the low-spin and high-spin forms of the S2 state is pH-dependent, suggesting that the (de)protonation of bridging or terminal oxygen atoms in the OEC may be connected to spin-state changes.
We derive a model that provides an exact solution to the substrate-water exchange kinetics in a double-conformation system and use this model to interpret recently published data for Ca²⁺- and Sr²⁺-containing PSII in the S2 state, in which the g = 2.0 and g = 4.1 conformations coexist. The component concentrations derived from the kinetic model provide an analytic description of the substrate-water exchange kinetics, allowing us to more accurately interpret the results. Based on this model and the previously reported data on the S2 state g = 2.0 conformation, we obtain the substrate-water exchange rates of the g = 4.1 conformation and the conformational change rates. Two conclusions are made from the analyses. First, contrary to previous reports, there is no significant effect of substituting Sr²⁺ for Ca²⁺ on any of the exchange rate constants. Second, the exchange rate of the slowly-exchanging water (Ws) in the S2 state g = 4.1 conformation is much faster than that in the S2 state g = 2.0 conformation. The second conclusion is consistent with the assignment of Ws to W1 or W2 bound as terminal ligands to Mn4; Mn4 has been proposed to undergo an oxidation state change from Mn(IV) in the g = 2.0 conformation to Mn(III) in the g = 4.1 conformation.
Quantum chemical approaches today are a powerful tool to study the properties and reactivity of metalloenzymes. In the field of solar fuels research these involve predominantly photosystem II and hydrogenases, which catalyze water oxidation and hydrogen evolution, as well as related biomimetic and bio-inspired models. Theoretical methods are extensively used to better comprehend the nature of catalytic intermediates, establish important structure-function and structure-property correlations, elucidate functional principles, and uncover the catalytic activity of these complex systems by unravelling the key steps of their reaction mechanism. Computations in the field of water oxidation and hydrogen evolution are used as predictive tools to elucidate structures, explain and synthesize complex experimental observations from advanced spectroscopic techniques, rationalize reactivity on the basis of atomistic models and electronic structure, and guide the design of new synthetic targets. This feature article covers recent advances in the application of quantum chemical methods for understanding the nature of catalytic intermediates and the mechanism by which photosystem II and hydrogenases achieve their function, and points at essential questions that remain only partly answered and at challenges that will have to be met by future advances and applications of quantum and computational chemistry.
World demand for energy is rapidly increasing and finding sufficient supplies of clean energy for the future is one of the major scientific challenges of today. This book presents the latest knowledge and chemical prospects in developing hydrogen as a solar fuel. Using oxygenic photosynthesis and hydrogenase enzymes for bio-inspiration, it explores strategies for developing photocatalysts to produce a molecular solar fuel. The book begins with perspective of solar energy utilization and the role that synthetic photocatalysts can play in producing solar fuels. It then summarizes current knowledge with respect to light capture, photochemical conversion, and energy storage in chemical bonds. Following chapters on the natural systems, the book then summarizes the latest developments in synthetic chemistry of photo- and reductive catalysts. Finally, important future research goals for the practical utilization of solar energy are discussed. The book is written by experts from various fields working on the biological and synthetic chemical side of molecular solar fuels to facilitate advancement in this area of research.
The g-factor shift of the g = 4.1 EPR signal was detected in spinach PsbO/P/Q-depleted PS II. The effective g-factor of the signal shifts up to ~4.9, depending on Ca²⁺ concentration. Hyperfine structure spacing with about 3 mT was de-tected in this g = 5 (4.9) signal. The shift to g = 5 (4.9) was related to the distortion of the manganese cluster, derived to the modification of the chemical bond or the crystalline field of the Mn4(III) in the manganese cluster. Based on the EPR analysis of the g = 5 (4.9) spin state, another molecular structure of the S2 state, a ‘distant Mn’ structure, was discussed as an intermediate state between the S2 and S3 states.
The identity of a key intermediate in the S2 to S3 transition of Nature’s water oxidising complex (WOC) in Photosystem 2 is presented. Broken symmetry density functional theory (BS-DFT) calculations and Heisenberg, Dirac, van Vleck (HDvV) spin ladder calculations show that an S2 state open cubane model of the WOC containing a µ-hydroxo O4 changes from an S=5/2 to an S=7/2, form on deprotonation of W1. This combined with X-band electron paramagnetic resonance (EPR) spectral analysis indicates that the g=4.1 EPR signal corresponds to an S=5/2 form of the WOC with W1 present as a water ligand to Mn4, while the g=4.8/4.9 form observed at high pH values corresponds to an S=7/2 form, with W1 as an hydroxo ligand. The latter is also likely to represent the form needed to progress to S3 in the functioning enzyme.
Natural photosynthesis is the only working system of solar energy storage that operates on a global scale. The water-oxidation reaction, carried out by the protein complex photosystem II (PSII), is the key reaction that initiates photosynthesis. However, the detailed mechanism of this reaction is yet to be fully understood. In this article, we review the current knowledge of how the essential components in PSII, including the oxygen-evolving complex and its surrounding environment, take part in the water-oxidation reaction, and finally present proposals for the water-oxidation mechanism.
The spin structure in the S2 state and the crystal structure of the manganese cluster of the oxygen evolving complex of plant photosystem II was combined by the quantitative evaluation of the magnetic anisotropy of the g = 4 signal. The g -values of 3.93 and 4.13 were obtained for the g = 4 signal in the directions parallel and perpendicular to the membrane normal, respectively. The peak-to-peak separations were 270 and 420 G for the parallel and perpendicular orientations to the membrane, respectively. By comparison with the crystal structure, the z-axis of the zero-field splitting was ascribed to the direction of the dangling Mn connecting water oxygen, Mn4-O(W1), in the manganese cluster. The results give the first experimental evidence that the valence of the dangling Mn is Mn(III) in the S2 high spin state. We showed the strong exchange coupling of Mn4 to Mn3 was required for g =4.1 spin state in the four spin couplings, estimated as > ~|-30 cm⁻¹|, indicating that the present closed cubane model in QM/MM calculation cannot explain the g = 4.1 spin structure. The onsite zero-field splitting of the dangling Mn was evaluated as –2.3 cm⁻¹ under the strong antiferromagnetic couplings (-50 cm⁻¹) with the dangling Mn to the cubane frame in the four coupled spin state. From the viewpoint of the arrangement of the Mn valences in the cluster, a closed cubane model is effective, but no large structural deviation from the S1 state crystal structure.
A new paradigm for the high and low spin forms of the S2 state of Nature’s water oxidising complex in Photosystem 2 is found. Broken symmetry density functional theory (BS-DFT) calculations combined with Hesienberg, Dirac, Van Vleck (HDvV) spin ladder calculations show that an open cubane form of the water oxidising complex changes from a low spin (LS), S=1/2, to a high spin (HS), S=5/2, form on protonation of the bridging O4 oxo. We show that such models are fully compatible with structural determinations of the S2 state by X-ray free electron laser (XFEL) crystallography and extended X-ray absorption fine structure (EXAFS) and provide a clear rationale for the effect of various treatments on the relative populations of each form observed experimentally in electron paramagnetic resonance (EPR) studies.
To better understand the biol. water-oxidn. catalyst, the pentanuclear oxygen-evolving complex (OEC) of Photosystem II (PSII), synthetic model compds. have been utilized to benchmark spectroscopic signatures and proposed mechanisms of the enzyme. Due to the complexity and low symmetry of the OEC, accurate models remain rare. Although systematic changes to the coordination environment of metal centers in clusters can be informative regarding effects on spectroscopy and reactivity, synthetic methods to a series of structurally related clusters are limited. Starting from Mn_4O_4 cubanes supported by a multinucleating triarylbenzene ligand framework and acetates, a family of related complexes was prepd. through ligand-exchange with phosphinate and tethered diamidate ligands. These clusters, in the MnIIIMnIV_3 redox state, show EPR features reminiscent of the S_2 state of the OEC. SQUID magnetometry, EPR spectroscopy, and reactivity studies of these cubane clusters will be presented in the context of modeling the properties of the oxygen-evolving complex in Photosystem II.
Significance
Recent results have shown that nature’s water splitting catalyst inserts an additional water molecule into what appears to be a solvent inaccessible site late in its reaction cycle. The emerging consensus of the field is that this water molecule is one of the substrates of the reaction. Here, we show that this water molecule does not come directly from solvent. It instead represents an earlier bound water, which is inserted into this site via facile structural tautomerism. The trigger for this process is cofactor oxidation. This then allows an additional water to bind from solvent to a more open site of the cofactor. In this way the cofactor carefully regulates water uptake, preventing water insertion earlier in the reaction cycle.
The catalytic cycle of photosynthetic water oxidation occurs at the Mn4CaO5 oxygen-evolving complex (OEC) of Photosystem II (PSII). Extensive spectroscopic data have been collected on the intermediates, especially the S2 (Kok) state, although the proton and electron inventories (Mn oxidation states) are still uncertain. The “high-oxidation” paradigm, assigns S2 Mn oxidation level (III, IV, IV, IV) or (IV, IV, IV, III), whereas a “low-oxidation” paradigm posits two additional electrons. Here we investigate the geometric (XRD, EXAFS) and spectroscopic (EPR, ENDOR) properties of the S2 state using quantum chemical DFT calculations, focusing on the neglected low paradigm. Two interconvertible electronic spin configurations are predicted as ground states, producing multiline (S = 1/2) and broad (S = 5/2) EPR signals in the low paradigm oxidation state (III, IV, III, III) and with W2 as OH– and O5 as OH–. They have “open” (S = 5/2) and “closed” (S = 1/2) Mn3CaO4-cubane geometries. Other energetically accessible isomers with ground spin states 1/2, 7/2, 9/2, or 11/2 can be obtained through perturbations of hydrogen-bonding networks (e.g. H⁺ from His337 to O3 or W2), consistent with experimental observations. Conformers with the low oxidation state configuration (III, IV, IV, II) also become energetically accessible when the protonation state is O5 (OH–), W2 (H2O) and neutral His337. Configuration with (III, IV, III, III) agrees well with earlier low temperature EPR and ENDOR interpretations, while the MnII-containing configuration agrees partially with recent ENDOR data. We conclude that the low Mn oxidation state proposal for the OEC can closely fit nearly all the available structural and electronic data for S2 at accessible energies.
The evolution of aerobic life on earth is depended on proceeding water splitting accomplished through photosynthesis in plants, algae, and cyanobacteria. Photosystem II (PSII), with a catalytic center CaMn4O5 located on the lumenal surface, is responsible for water splitting and generating molecular oxygen through a four-step photocatalytic cycle. So far, the structure of the catalytic center and its ligation environments have been studied by different methods mostly relied on various spectroscopic techniques, disclosing unknowing aspects of the PSII components. Over the last half-decade, the experimental methods have extensively been coupled with quantum mechanics/molecular mechanics (QM/MM) methods to explore diverse aspects of PSII structure and water oxidation mechanism. However, despite the progress made in the past years, distinguishing a generally accepted mechanism on the O–O bond formation is still a challenge. This substantial challenge, if resolved, would provide a widespread criterion for development of globally deployable biomimetic model systems for water splitting catalysts. Here, we highlight some latest studies performed on the structure and function of PSII, the information that tells us how to establish new artificial catalytic systems to deliver maximum performance through water splitting in research labs.
Ascorbic acid (AA) and hydroxyl peroxide (H2O2) redox pair induced free radical grafting reaction is a promising approach to conjugate phenolic groups with chitosan (CS). In order to reveal the exact mechanisms of AA/H2O2 redox pair induced grafting reaction, free radicals generated in AA/H2O2 redox system were compared with hydroxyl radical (•OH) produced in Fe2+/H2O2 redox system. Moreover, the structural and physicochemical properties caffeic acid grafted CS (CA-g-CS) synthesized in these two redox systems were compared. Results showed only ascorbate radical (Asc•−) was produced in AA/H2O2 system. The reaction between Asc•− and CS produced novel carbon-centered radicals, whereas no new free radicals was detected when •OH reacted with CS. Thin layer chromatography, UV–vis, Fourier transform infrared and nuclear magnetic resonance spectroscopic analyses all confirmed CA was successfully grafted onto CS through Asc•−. However, CA could be hardly grafted onto CS via •OH. CA-g-CS synthesized through Asc•− exhibited lower thermal stability and crystallinity than the reaction product obtained through •OH. For the first time, our results demonstrated the synthesis of CA-g-CS in AA/H2O2 redox system was mediated by Asc•− rather than •OH.
The structural polymorphism of the oxygen-evolving complex is of great significance to photosynthetic water oxidation. Employing density functional theory calculations, we have made further advisement on the interconversion mechanism of O5 transfer in the S2 state, mainly focusing on the potentiality of multi-state reactivity and spin transitions. Then, O5 protonation is proven impossible in S2 for irreversibility of the interconversion, which serves as an auxiliary judgment for that in S1. Besides, the structural polymorphism could also be archived by alternative mechanisms involving Mn3 ligand exchange, one of which with Mn3(III) makes sense to substrate water exchange in S2, although being irresponsible for the derivations of the observed EPR signals. During the water exchange, high-spin states would prevail to facilitate electron transfer between the ferromagnetically coupled Mn centers. In addition, water exchange in S1 could account for the closed-cubane structure as the initial form entering S2 at cryogenic temperatures. With regard to water oxidation, the structural flexibility and variability in both S2 and S3 guarantee smooth W2-O5 coupling in S4, according to the substrate assignments from water exchange kinetics. Within this theoretical framework, the new XFEL findings on S1-S3 can be readily rationalized. Finally, an alternative mechanistic scenario for O-O bond formation with ·OH radical near O4 is presented, followed by water binding to the pivot Mn4(III) from O4 side during S4-S0. This may diversify the substrate sources combined with the Ca channel in water delivery for the forthcoming S-cycle.
A recently reported synthetic complex with a Mn4CaO4 core represents a remarkable structural mimic of the Mn4CaO5 cluster in the oxygen-evolving complex (OEC) of photosystem II (Zhang et al., Science 2015, 348, 690). Oxidized samples of the complex show electron paramagnetic resonance (EPR) signals at g ≈ 4.9 and 2, similar to those associated with the OEC in its S2 state (g ≈ 4.1 from an S = (5)/2 form and g ≈ 2 from an S = (1)/2 form), suggesting similarities in the electronic as well as geometric structure. We use quantum-chemical methods to characterize the synthetic complex in various oxidation states, to compute its magnetic and spectroscopic properties, and to establish connections with reported data. Only one energetically accessible form is found for the oxidized "S2 state" of the complex. It has a ground spin state of S = (5)/2, and EPR simulations confirm it can be assigned to the g ≈ 4.9 signal. However, no valence isomer with an S = (1)/2 ground state is energetically accessible, a conclusion supported by a wide range of methods, including density matrix renormalization group with full valence active space. Alternative candidates for the g ≈ 2 signal were explored, but no low-spin/low-energy structure was identified. Therefore, our results suggest that despite geometric similarities the synthetic model does not mimic the valence isomerism that is the hallmark of the OEC in its S2 state, most probably because it lacks a coordinatively flexible oxo bridge. Only one of the observed EPR signals can be explained by a structurally intact high-spin one-electron-oxidized form, while the other originates from an as-yet-unidentified rearrangement product. Nevertheless, this model provides valuable information for understanding the high-spin EPR signals of both the S1 and S2 states of the OEC in terms of the coordination number and Jahn-Teller axis orientation of the Mn ions, with important consequences for the development of magnetic spectroscopic probes to study S-state intermediates immediately prior to O-O bond formation.
Efficient photoelectrochemical water oxidation may open a way to produce energy from renewable solar power. In biology, generation of fuel due to water oxidation happens efficiently on an immense scale during the light reactions of photosynthesis. To oxidize water, photosynthetic organisms have evolved a highly conserved protein complex, Photosystem II. Within that complex, water oxidation happens at the CaMn4O5 inorganic catalytic cluster, the so-called oxygen-evolving complex (OEC), which cycles through storage “S” states as it accumulates oxidizing equivalents and produces molecular oxygen. In recent years, there has been significant progress in understanding the OEC as it evolves through the catalytic cycle. Studies have combined conventional and femtosecond X-ray crystallography with extended X-ray absorption fine structure (EXAFS) and quantum mechanics/molecular mechanics (QM/MM) methods and have addressed changes in protonation states of μ-oxo bridges and the coordination of substrate water through the analysis of ammonia binding as a chemical analog of water. These advances are thought to be critical to understanding the catalytic cycle since protonation states regulate the relative stability of different redox states and the geometry of the OEC. Therefore, establishing the mechanism for substrate water binding and the nature of protonation/redox state transitions in the OEC is essential for understanding the catalytic cycle of O2 evolution.
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.
Nature’s water-splitting catalyst moves through a reaction cycle with five catalytic intermediates characterized by different spin ground states, the origin of which is connected to their geometric structures and intermetallic magnetic couplings. The early “inactive” intermediates have low-spin ground states, while the later “active” intermediates transition to high-spin states. The cofactor’s ability to switch from lower to higher-spin states via an open-to-closed cubane conversion is critical for substrate water binding.
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.
Ca2+-depleted and Ca2+-reconstituted spinach photosystem II was studied using polarized X-ray absorption spectroscopy of oriented PS II preparations to investigate the structural and functional role of the Ca2+ ion in the Mn4O5Ca cluster of the oxygen-evolving complex (OEC). Samples were prepared by low pH/citrate treatment as one-dimensionally ordered membrane layers, and poised in the Ca2+-depleted S1 (S1'), S2 (S2') and S2'YZ• states, at which point the catalytic cycle of water oxidation is inhibited, and the Ca2+-reconstituted S1 state. Polarized Mn K-edge XANES and EXAFS spectra exhibit pronounced dichroism. Polarized EXAFS data of all states of Ca2+-depleted PS II investigated show only minor changes in distances and orientations of the Mn-Mn vectors compared to the Ca2+-containing OEC, which may be attributed to some loss of rigidity of the core structure. Thus, removal of the Ca2+ ion does not lead to fundamental distortion or rearrangement of the tetranuclear Mn cluster, which indicates that the Ca2+ ion in the OEC is not critical for structural maintenance of the cluster at least in the S1 and S2 states, but fulfills a crucial catalytic function in the mechanism of the water oxidation reaction. On the basis of this structural information, reasons for the inhibitory effect of Ca2+ removal are discussed, attributing to the Ca2+ ion a fundamental role in organizing the surrounding (substrate) water framework and in proton-coupled electron transfer to YZ• (D1-Tyr161).
The solar water-splitting protein complex, photosystem II (PSII), catalyzes one of the most energetically demanding reactions in Nature by using light energy to drive a catalyst capable of oxidizing water. The water oxidation reaction takes place at the tetra-nuclear manganese calcium-oxo (Mn4Ca-oxo) cluster at the heart of the oxygen-evolving complex (OEC) of PSII. Previous studies have determined the magnetic interactions between the paramagnetic Mn4Ca-oxo cluster and its environment in the S2 state of the OEC. The assignments for the electron-nuclear magnetic interactions that were observed in these studies were facilitated by the use of synthetic dimanganese di-μ-oxo complexes. However, there is an immense need to understand the effects of the protein environment on the coordination geometry of the Mn4Ca-oxo cluster in the OEC of PSII. In the present study, we use a proteinaceous model system to examine the protein ligands that are coordinated to the dimanganese catalytic center of manganese catalase from Lactobacillus plantarum. We utilize two-dimensional hyperfine sublevel correlation (2D HYSCORE) spectroscopy to detect the weak magnetic interactions of the paramagnetic dinuclear manganese catalytic center of superoxidized manganese catalase with the nitrogen and proton atoms of the surrounding protein environment. We obtain a complete set of hyperfine interaction parameters for the protons of a water molecule that is directly coordinated to the dinuclear manganese center. We also obtain a complete set of hyperfine and quadrupolar interaction parameters for two histidine ligands as well as a coordinated azide ligand, in azide-treated superoxidized manganese catalase. On the basis of the values of the hyperfine interaction parameters of the dimanganese model, manganese catalase, and those of the S2 state of the OEC of PSII, for the first time, we discuss the impact of a proteinaceous environment on the coordination geometry of multinuclear manganese clusters.
Water oxidation at the oxygen-evolving complex (OEC) of photosystem II (PSII) is fine tuned for turnover rates unmatched by any artificial system. Efficient proton removal from the OEC and activation of substrate water molecules are some of the key aspects optimized in the OEC for such high turnover rates. The hydrogen-bonding network around the OEC is critical for efficient proton transfer and for tuning the position and pKas of the substrate water/hydroxo/oxo molecules. The D1-N181 residue is part of the hydrogen-bonding network on the active face of the OEC. D1-N181 is also associated with the chloride ion in the D2-K317 site and is the closest residue to a putative substrate water molecule bound as a terminal ligand to Mn4. We studied the effect of the D1-N181A and the D1-N181S mutations on the water-oxidation chemistry at the OEC. PSII core complexes isolated from the D1-N181A,S mutants have lower steady-state O2-evolution rates than wild-type PSII core complexes. FTIR spectroscopy indicates slight perturbations of the hydrogen-bonding network in D1-N181A,S PSII core complexes, similar to some other mutations in the same region, but to a lesser extent. Unlike in wild-type PSII core complexes, a g = 4 signal was observed in the S2-state EPR spectra of D1-N181A,S PSII core complexes in addition to the normal g = 2 multiline signal. The S-state cycling of D1-N181A,S PSII core complexes was similar to wild-type PSII core complexes, whereas the O2-release kinetics of D1-N181A,S PSII core complexes were much slower than the O2-release kinetics of wild-type PSII core complexes. Based on these results, we conclude that proton transfer is not compromised in the D1-N181A,S mutants, but that the O-O bond formation step is retarded in these mutants.
(36)Cl(-) was used to study the slow exchange of chloride at a binding site associated with Photosystem II (PS II). When PS II membranes were labeled with different concentrations of (36)Cl(-), saturation of binding at about I chloride/PS II was observed. The rate of binding showed a clear dependence on the concentration of chloride approaching a limiting value of about 3·10(-4) s(-1) at high concentrations, similar to the rate of release of chloride from labeled membranes. These rates were close to that found earlier for the release of chloride from PS II membranes isolated from spinach grown on (36)Cl(-), which suggests that we are observing the same site for chloride binding. The similarity between the limiting rate of binding and the rate of release of chloride suggests that the exchange of chloride with the surrounding medium is controlled by an intramolecular process. The binding of chloride showed a pH-dependence with an apparent pKa of 7.5 and was very sensitive to the presence of the extrinsic polypeptides at the PS II donor side. The binding of chloride was competitively inhibited by a few other anions, notably Br(-) and NO3 (-). The slowly exchanging Cl(-) did not show any significant correlation with oxygen evolution rate or yield of EPR signals from the S2 state. Our studies indicate that removal of the slowly exchanging chloride lowers the stability of PS II as indicated by the loss of oxygen evolution activity and S2 state EPR signals.
A light-induced g = 4.1 EPR signal of the manganese cluster in the oxygen-evolving complex of plant photosystem II was investigated by an electron-spin-echo method. The dependence of the ESE signal amplitude on the intensity of the microwave magnetic field H1 was consistent with the model assuming a manganese electron spin of with EPR spectrum characteristics determined by nearly rhombic symmetry of the crystalline field.
Previous research (Sandusky, P.O. and Yocum, C.F. (1983) FEBS Lett. 162, 339–343 and (1984) Biochim. Biophys. Acta 766, 603–611) has documented a competition between chloride and ammonia or Tris for a binding site within the oxygen-evolving complex of Photosystem II. This competition is in fact a general property of inhibitory amines which is related to their nucleophilicity; this in turn suggests that the binding site is associated with a metal. Only ammonia, of all amines tested, is able to occupy a second binding site which is unrelated to the site of chloride binding; this sterically hindered site may be identical to the site already described for binding of hydroxylamine, hydrazine, and certain of their derivatives (Radmer, R. and Ollinger, O. (1983) FEBS Lett. 152, 39–43). When the interaction between amines, chloride and the inhibitory halide fluoride was examined, steady-state kinetic plotting procedures revealed that amines and fluoride compete for the chloride binding site; binding of one inhibitor precludes the binding of the other. It was also observed that the intensity of inhibitor binding to the oxygen-evolving complex was influenced by the electron acceptor present during assays; stronger inhibition was observed with a PS II-specific electron acceptor (2,5-dichloro-p-benzoquinone) than with an acceptor (ferricyanide) which requires electron transport to the reducing terminus of Photosystem I. These results are interpreted in terms of a model which proposes that the binding site for chloride on the oxidizing side of Photosystem II resides within the pool of functional manganese associated with the oxygen-evolving complex of Photosystem II.
The process of water oxidation and dioxygen evolution by the photosystem II (PSII) component of plant photosynthesis is cyclic, with intermediate states of the oxygen-evolving complex (OEC) designated Sâ through Sâ. Two electron paramagnetic resonance (EPR) signals have been assigned to the Sâ state of the complex. A multiline' EPR signal centered at the g = 2 region of the spectrum shows 16 or more partially resolved Mn hyperfine transitions and arises from a cluster with a minimum of two exchange-coupled mixed-valence Mn atoms. The other Sâ EPR signal occurs in the g = 4.1 region of the spectrum. The lack of resolved Mn hyperfine couplings has prevented conclusive assignment of the g = 4.1 EPR signal to a Mn center. However, a shift of the Mn X-ray K edge to higher energies is correlated with the appearance of the g = 4.1 signal in PSII membranes illuminated at 140 K. A considerable body of experimental work, including measurements of the temperature dependence of the EPR signals and observations of the interconversion between the multiline and the g = 4.1 signals, has given rise to two different models involving S = 3/2 Mn origins for the g = 4.1 signal. In this communication, the authors present direct spectral evidence of a multinuclear Mn origin for the Sâ g = 4.1 signal.
Signal II of plant photosynthesis, which is generally thought to be connected to the secondary donor complex of Photosystem II, has been investigated with EPR spectroscopy at 9 and 35 GHz. From the spectrum at 35 GHz of deuterated Chlorella vulgaris, the principle values of the g-tensor are determined to be gxx = 2.0074, gyy = 2.0044 and gzz = 2.0023. Proton hyperfine coupling tensor elements and orientations were determined from spectral simulation of random and oriented samples, assuming that Signal II is due to a plastose-miquinone cation having its π-electrons in an antisymmetric orbital as proposed by P.J. O'Malley, G.T. Babcock and R.C. Prince (Biochim. Biophys. Acta 765 (1984) 370–379). In contrast to their work, it is found that most hyperfine interaction is due to the methylene group at ring position 5 and to both hydroxyl groups. One of the hydroxyl groups shows bond bending of 35°. We presume that this is due to hydrogen bonding and that this bond stabilizes the antisymmetric orbital of the π-electrons.
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 multiline and g= 4.1 EPR signals from the manganese-containing water oxidation site of plant photosystem II have been studied at Q-band (35 GHz). Comparisons with X-band spectra show a significant g anisotropy in the multiline signal, which is inequivalent for the plus and minus alcohol forms. Provisional values for the plus alcohol form are g∥= 1.970, g⊥= 1.984. The Q-band 4.1 spectrum indicates that the signal arises from a quasi axial, probably spin- system, with a slight splitting of the g⊥ components into g⊥x= 4.35 and g⊥y= 4.14. Each component has a (peak-to-peak) width of ca. 30 mT, similar to that of the (unresolved) signal at X-band. The 4.1 signal from one dimensionally ordered photosystem II samples has also been studied at X-band. This shows a variation of the apparent g⊥ value with sample orientation in the magnetic field, consistent with the above limits from the powder-pattern Q-band data. Assuming the transitions around g= 4 arise from the ⊥ components of a quasi-axial spin- system, the Q- and X-band results indicate that |D| > 5 cm–1 and |E/D|≈ 0.017 for the zero field terms of the state. The oriented X-band data then show that the D(∥) axis is nearly parallel to the thylakoid membrane plane. Further, Mn hyperfine structure is resolved on the oriented X-band 4.1 signals, the first such detection in unmodified enzyme. The spacing (ca. 4 mT) is similar to that reported recently for structure on the 4.1 signal of NH3 inhibited enzyme (Kim et al., J. Am. Chem. Soc., 1990, 112, 9389), but the lines are less distinct.
The Mn-containing catalytic site for photosynthetic water oxidation undergoes changes in oxidation states during the catalytic cycle. One of these intermediates, the S2 state, can be studied directly by e.s.r. at liquid-helium temperatures. Two distinct e.s.r. signals from the S2 state are produced when dark-adapted Photosystem II membranes are illuminated in the 130–200 K range: a g= 4.1 signal or a signal centred at g= 2.0 with many hyperfine lines, referred to as the multiline e.s.r. signal. The yields and magnetic properties of these e.s.r. signals are found to depend on the temperature at which the S2 state is formed and the choice of cryoprotectant (ethylene glycol or sucrose). The intensity of the g= 4.1 e.s.r. signal obeys the Curie law in the 4.0–20.0 K temperature range. The S2-state multiline e.s.r. signal exhibits an intensity maximum at 7.0 K which is independent of microwave powers below 2 mW, if the samples contain 30 % ethylene glycol. This non-Curie behaviour is not observed in samples containing 0.4 mol dm–3 sucrose. A model is presented in which the S2 state e.s.r. signals arise from an exchange-coupled Mn tetramer, where both ferromagnetic and antiferromagnetic exchange occur. According to our model, the multiline e.s.r. signal observed in samples suspended in 30 % ethylene glycol originates from the thermally populated first excited s= 1/2 state of the exchange-coupled Mn tetramer, whereas the g= 4.1 e.s.r. signal arises from the ground s= 3/2 state of the Mn tetramer in a configuration that makes the s= 1/2 state thermally inaccessible. The different behaviour of the S2-state multiline e.s.r. signal in samples containing sucrose can be explained by a small conformational change of the Mn complex which alters the exchange couplings. In support of our assignment of the multiline e.s.r. signal, we present spectral simulations at S-, X- and Q-bands. The fits to the experimental spectra at X- and Q-bands are improved if a small degree of anisotropy is introduced in the g tensor of the Mn complex.
The effects of selective removal of extrinsic proteins on donor side electron transport in oxygen-evolving PS II particles were examined by monitoring the decay time of the EPR signal from the oxidized secondary donor, Z+, and the amplitude of the multiline manganese EPR signal. Removal of the 16 and 24 kDa proteins by washing with 1 M NaCl inhibits oxygen evolution, but rapid electron transfer to Z+ still occurs as evidenced by the near absence of Signal IIf. The absence of a multiline EPR signal shows that NaCl washing induces a modification of the oxygen-evolving complex which prevents the formation of the S2 state. This modification is different from the one induced by chloride depletion of PS II particles, since in these a large multiline EPR signal is found. After removal of the 33 kDa protein with 1 M MgCl2, Signal IIf is generated after a light flash. Readdition of the 33 kDa component to the depleted membranes accelerates the reduction of Z+. Added calcium ions show a similar effect. These findings suggest that partial advancement through the oxygen-evolving cycle can occur in the absence of the 16 and 24 kDa proteins. The 33 kDa protein, on the other hand, may be necessary for such reactions to take place.
The positions of powder lines in the electron paramagnetic spectra of high‐spin ferric systems (d5,S = ) have been calculated by solving the spin Hamiltonian = gβB⋅S + ⅓D[3Sz2 − S(S + 1)] + E(Sx2 − Sy2) for a broad range of parameters. Powder lines are obtained for every transition when the magnetic field points along the principal axes of the fine structure tensor. However, it was found that for most transitions extra powder lines are often found when the field lies in any of the principal planes but not along the axes. Particular attention is directed to the transition responsible for the g′ ≈ 4.2 absorption in nearly rhombic (E / D ∼ ⅓) ferric complexes. The calculations show that, depending on the value of the ratio between the microwave quantum and the parameter D, this transition may consist of 3–6 powder lines near g′ = 4.2. The g′ values for all these powder lines were also obtained from a third‐order perturbation calculation which assumes nearly rhombic symmetry and D > gβB. The 9.2‐ and 34‐GHz spectra of Fe(III)–EDTA diluted in the corresponding diamagnetic Co(III) compound and the 2.7‐, 9.2‐, and 34‐GHz spectra of native human serum transferrin have been analyzed by the aid of the calculations. It was determined that for FeEDTA∣ D ∣ = 0.83cm−1 and ∣ E / D ∣ = 0.31, while for transferrin ∣ D ∣ = 0.27cm−1 and ∣ E / D ∣ = 0.31–0.32.
The ESR spectra of Mn2+ ions have been observed in borate glasses of the composition (MnO)x(B2O3+0.04 K2O)1−x with x from 0.001 up to 0.030. For small concentrations of Mn2+ the central fine‐structure transitions (M = +☒↔—☒), although broadened by the random orientation of small noncubic crystalline fields, show a well‐resolved hyperfine structure. In addition to the Δm=0 transitions, ``forbidden'' Δm=±1 have been observed. The spectra prove that the coordination of the O2− ions around the Mn2+ ions is mainly cubic, but that small deformations of the cubic symmetry exist. The deformations vary from ion to ion, and range up to average values of the order of 5% in the interionic distances and 10 deg in the angles. The discussion has been based on a spin Hamiltonian involving an isotropic g factor, fine structure term D, and hyperfine structure term A. The line positions have been calculated up to the third order in D and A. The numerical results are: g=2.002±0.002, ∣ A ∣ /hc= (87–2)×10−4 cm−1, and DAv/hc= (89±10)×10−4 cm−1, the latter value representing an average over all deviations from cubic symmetry.
Electron paramagnetic resonance (EPR) and electron spin resonance (ESR) are synonymous terms for describing the resonant absorption of microwave radiation by a paramagnetic substance in a static magnetic field. A paramagnetic substance consists of weakly interacting ions or free radicals that possess permanent magnetic moments originating from electron spin and also, in most cases, including contributions from the electron orbital angular momentum. This chapter discusses basic principles of continuous wave EPR (CW-EPR) as applied to metalloproteins. The information obtained from an EPR spectrum can be divided into two classes: (1) structural information obtained from the spin Hamiltonian parameters and (2) the quantification of the EPR signal intensity. Although most researchers use EPR spectroscopy for the elucidation of structural information, spin quantitation can be used to determine (1) the number of EPR active centers present and the spin state, (2) the redox potentials of a paramagnetic center, and (3) the rate constants for biochemical reactions.
An ESEEM (electron spin−echo envelope modulation) spectroscopic study employing a series of 2H-labeled alcohols provides direct evidence that small alcohols (methanol and ethanol) ligate to the Mn cluster of the oxygen evolving complex (OEC) of Photosystem II in the S2-state of the Kok cycle. A numerical method for calculating the through-space hyperfine interactions for exchange-coupled tetranuclear Mn clusters is described. This method is used to calculate hyperfine interaction tensors for protons [deuterons] in the vicinity of two different arrangements of Mn ions in a tetranuclear cluster: a symmetric cubane model and the EXAFS-based Berkeley “dimer-of-dimers” model. The Mn−H distances derived from the spectroscopically observed coupling constants for methanol and ethanol protons [deuterons] and interpreted with these cluster models are consistent with the direct ligation of these small alcohols to the OEC Mn cluster. Specifically, for methanol we can simulate the three-pulse ESEEM time domain pattern with three dipolar hyperfine interactions of 2.92, 1.33, and 1.15 MHz, corresponding to a range of maximal Mn−H distances in the models of 3.7−5.6 Å (dimer-of-dimers) and 3.6−4.9 Å (symmetric cubane). We also find evidence for limited access of n-propanol, but no evidence for 2-propanol or DMSO access. Implications for substrate accessibility to the OEC are discussed.
The two forms of the g ≈ 4.1 signal recently identified in photosystem II (Smith, P. J.; Pace, R. J. Biochim. Biophys. Acta 1996, 1275, 213) have been simulated at several frequencies as near-axial spin 3/2 centers. In both cases, an explicit spin coupling model is assumed, involving two magnetically isolated Mn pairs, one for each signal type. For that pair assumed to give rise to the spin 1/2 multiline signal as the ground state, the modeling of the first-excited-state 4.1 signal gives estimates of the fine structure parameters for the individual Mn centers and the exchange coupling constant for the pair. The fine structure terms suggest that one Mn ion is a conventional MnIII ion in a highly axially distorted environment. The other Mn center, which is formally spin 3/2, is unlikely to be a conventional MnIV ion, but rather a MnIII−radical ligand pair, strongly antiferromagnetically coupled to give a net spin 3/2 state. The coupling between this Mn−radical center and the other MnIII is weak (J = −2.3 cm-1) in the absence of alcohol in the buffer medium, as determined earlier (Smith and Pace). The model is shown to be quantitatively consistent with the behavior of other signals proposed to arise from this coupled dimer. Comparison of our own data with those of others (Haddy, A.; et al. Biochim. Biophys. Acta 1992, 1099, 25−34) on one-dimensionally ordered photosystem II samples shows a generally consistent orientation of the molecular axis system for the dimer in the membrane plane. The second 4.1 signal, which exhibits ground-state behavior, may be simulated at X- and Q-band frequencies as an isolated system with D = +1.1 cm-1 and E/D = 0.037. The spin center is suggested to arise from a radical-bridged Mn homodimer, and the modeling parameters have been interpreted within this framework. The resulting proposal, involving two isolated dimers for the Mn organization within the oxygen evolving center, is critically examined in the light of recent work from other groups.
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 process of water oxidation and dioxygen evolution by the photosystem II (PSII) component of plant photosynthesis is cyclic, with intermediate states of the oxygen-evolving complex (OEC) designated S{sub 0} through S{sub 4}. Two electron paramagnetic resonance (EPR) signals have been assigned to the S{sub 2} state of the complex. A multiline' EPR signal centered at the g = 2 region of the spectrum shows 16 or more partially resolved Mn hyperfine transitions and arises from a cluster with a minimum of two exchange-coupled mixed-valence Mn atoms. The other S{sub 2} EPR signal occurs in the g = 4.1 region of the spectrum. The lack of resolved Mn hyperfine couplings has prevented conclusive assignment of the g = 4.1 EPR signal to a Mn center. However, a shift of the Mn X-ray K edge to higher energies is correlated with the appearance of the g = 4.1 signal in PSII membranes illuminated at 140 K. A considerable body of experimental work, including measurements of the temperature dependence of the EPR signals and observations of the interconversion between the multiline and the g = 4.1 signals, has given rise to two different models involving S = 3/2 Mn origins for themore » g = 4.1 signal. In this communication, the authors present direct spectral evidence of a multinuclear Mn origin for the S{sub 2} g = 4.1 signal.« less
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.
The EPR spectrum at both X- and S-band (3.94 GHz) of the oxidized acceptor-side iron in photosystem II from spinach shows two absorption-type peaks at g = 8.0 and 5.6. The intensities of these peaks have been measured at X-band in the temperature range 2–10 K. All results can be fully described assuming that the EPR spectrum arises from high-spin Fe(III) with D = 1.0±0.3 cm−1 and E/D = 0.10 ±0.01. Quantifications show that the spectrum in our case represents 0.4–0.5 Fe(III) per reaction center. The EPR parameters are consistent with the iron having bicarbonate and/or tyrosine as ligands in addition to four imidazoles.
A unique capability of the Photosystem II (PS II) reaction center is the ability to extract electrons from water, producing
molecular oxygen as a byproduct. Four photon-induced charge separations at the site of the chlorophyll moiety P680 couple
sequentially to oxidation events at an Oxygen Evolving Complex (OEC), resulting in the formation of molecular oxygen. A tetranuclear
manganese cluster is at the heart of the OEC. Recent biochemical and spectroscopic results have given new insights into the
structure of this Mn cluster, its ligation to the PS II polypeptides, and the role of essential cofactors Ca2+ and Cl−. Models for the oxygen evolution mechanism are discussed, including new models that assign a direct role in water splitting
to the redox active tyrosine YZ•.
Four representative inhibitors of Photosystem II (PS II) Q−A to QB electron transfer were shown to bind, at high concentrations, to PS II reaction centers having the acceptor-side non-heme iron in the Fe(III) state. Three of the inhibitors studied, DCMU, o-phenanthroline and dinoseb, modified the EPR spectrum of the Fe(III) relative to that obtained by ferricyanide oxidation in the absence of inhibitor. o-Phenanthroline gave particularly axial symmetry, while DCMU and dinoseb gave more rhombic configurations. The herbicide inhibitor, atrazine and its analogue, terbutryn, had no effect. The dissociation constants for inhibitor binding to reaction centers in the Fe(III) state were measured directly and also estimated from shifts in the midpoint potential of the Fe(III)/Fe(II) couple and were shown to increase by factors of approx. 100, approx. 10 and 10–15 for DCMU (pH 7.5), atrazine (pH 7.0) and o-phenanthroline (pH 7.0), respectively, upon oxidation of the iron. Atrazine and o-phenanthroline, which induce the smallest changes in the midpoint potential of the Fe(III)/Fe(II) couple, were shown to inhibit light-induced oxidation of the Fe(II) by phenyl-p-BQ, described in the preceding paper (Petrouleas, V. and Diner, B.A. (1987) Biochim. Biophys. Acta 893, 126–137). The extent of inhibition was much greater than would be predicted from a simple shift in the midpoint potential for Fe(III)/Fe(II) and we conclude that phenyl-p-BQ and the other quinones, which show light-induced oxidation, act through the QB binding site. It is also argued that reduction and oxidation of the iron by ferro- and ferricyanide, respectively, occur through this site. The effects of these inhibitors and of various quinones on the Fe(III) environment are discussed with reference to the known contact points between the protein and o-phenanthroline and terbutryn in the QB binding pocket of Rhodopseudomonas viridis reaction centers (Michel, H., Epp, O. and Deisenhofer, J. (1986) EMBO J. 5, 2445–2451). The Fe(III) EPR spectrum is thus a new and sensitive probe of the contact points at which molecules bind to the QB binding site.
The role of chloride on the S-state transition in spinach Photosystem II (PS II) particles was investigated by EPR spectroscopy at low temperature and the following results were obtained. (1) After excitation by continuous light at 200 K, chloride-depleted particles did not show the EPR multiline signal associated with the S2 state, but only showed the broad signal at g = 4.1. The S2 multiline signal was completely restored upon chloride repletion. (2) In the absence of chloride the S2 multiline signal was not induced by a single flash excitation at 0°C. However, upon addition of chloride after the flash the signal was developed in darkness. (3) The amplitude of the multiline S2 signal thus developed upon chloride addition after flash illumination did not show oscillations dependent upon flash number. These results indicate that the O2-evolving complex in chloride-depleted PS II membranes is able to store at least one oxidizing equivalent, a modified S2 state, which does not give rise to the multiline signal. Addition of chloride converts this oxidizing equivalent to the normal S2 state which gives rise to the multiline signal. The modified S2 state is more stable than the normal S2 state, showing decay kinetics about 20-times slower than those of the normal S2 state, and the formation of higher S states is blocked.
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).
In the presence of Cl−, the severity of ammonia-induced inhibition of photosynthetic oxygen evolution is attenuated in spinach thylakoid membranes (Sandusky, P.O. and Yocum, C.F. (1983) FEBS Lett. 162, 339–343). A further examination of this phenomenon using steady-state kinetic analysis suggests that there are two sites of ammonia attack, only one of which is protected by the presence of Cl−. In the case of Tris-induced inhibition of oxygen evolution only the Cl− protected site is evident. In both cases the mechanism of Cl− protection involves the binding of Cl− in competition with the inhibitory amine. Anions (Br− and NO−3) known to reactive oxygen evolution in Cl−-depleted membranes also protect against Tris-induced inhibition, and reactivation of Cl−-depleted membranes by Cl− is competitively inhibited by ammonia. Inactivation of the oxygen-evolving complex by NH2OH is impeded by Cl−, whereas Cl− does not affect the inhibition induced by so-called ADRY reagents. We propose that Cl− functions in the oxygen-evolving complex as a ligand bridging manganese atoms to mediate electron transfer. This model accounts both for the well known Cl− requirement of oxygen evolution, and for the inhibitory effects of amines on this reaction.
Measurements of the area bounded by the variable fluorescence induction curve and the maximum fluorescence yield as a function of redox potential led I. Ikegami and S. Katoh ((1973) Plant Cell Physiol. 14, 829–836) to propose the existence of a high-potential electron acceptor, Q400 (Em7.8 = 360 mV), associated with Photosystem II (PS II). We have generated the oxidized form of this acceptor (Q+400) using ferricyanide and other oxidants in thylakoid membranes isolated from a mutant of Chlamydomonas reinhardtii lacking Photosystem I and the cytochrome complex. Q+400 was detected by a decrease in the extent of reduction of the primary quinone electron acceptor, QA, in a low-intensity light flash exciting PS II reaction centers only once. EPR measurements in the presence of Q+400 indicated the presence of new signals at g = 8, 6.4 and 5.5. These disappeared upon illumination at 200 K or upon reduction with ascorbate. Mössbauer absorption attributed to the Fe2+ of the QA-Fe2+ acceptor complex of PS II disappeared upon addition of ferricyanide due to the formation of Fe3+. The Fe2+ signal was restored by subsequent addition of ascorbate. All of these spectroscopic signals show similar pH-dependent (n = 1) midpoint potentials (approx. −60 mV / pH unit) and an Em7.5 = 370 mV. We assign the EPR signals to the Fe3+ state of the quinone-iron acceptor. Electron transfer to the Fe3+ is responsible for the decrease in QA reduction upon single-hit flash excitation. The properties of the redox couple are consistent with those of and we conclude that the iron of the QA-Fe acceptor complex is responsible for this species.
Two forms of the g = 4.1 signal in photosystem II (PS 1I) were identified from X-band and Q-band ESR signal shape and temperature dependence studies. Using ethylene glycol cryoprotected PS II illuminated at 130K, a g = 4.1 signal was generated which exhibited a temperature dependence consistent with it arising from a ground state species. Using sucrose cryoprotected PS II illuminated at 200K (in the absence of monoalcohols), a g = 4.1 signal was cogenerated with the multiline signal. At temperatures above ∼ 20K, a signal at g ∼ 6 became evident in these samples. The temperature dependencies of the multiline, g = 4.1 and g ∼ 6 signals were quantitatively consistent with them arising from the first 3 states (spin ) respectively of a weakly antiferromagnetically coupled Mn III-IV dimer. The temperature dependence of the signals in these samples indicated that the g = 4.1 signal now arose from a centre displaying excited state behaviour. The two types of g = 4.1 signal were very similar in shape at X-band but showed significantly different line shapes at Q-band. It is suggested that they arise from separate, near axial, centres in well-defined states. A model is proposed, based on the temperature dependencies, ESR line shapes and probable spin states, to suggest that the four Mn ions are arranged as two exchange coupled pairs and that each g = 4.1 signal arises from a separate manganese dimer. The ground state g = 4.1 signal then requires the involvement of at least one additional spin species, coupling to each Mn of a homodimer (probably IV-IV oxidation state). The spin centre may be an oxidised protein side chain, possibly acting as a bridging ligand between the two Mn ions. It is concluded that the Mn dimers are sufficiently spatially separated within the protein structure to exclude magnetic exchange between the dimers, but within range to allow rapid electron transfer.
Continuous illumination at 200 K of photosystem (PS) II-enriched membranes generates two electron paramagnetic resonance (EPR) signals that both are connected with the S(2) state: a multiline signal at g 2 and a single line at g = 4.1. From measurements at three different X-band frequencies and at 34 GHz, the g tensor of the multiline species was found to be isotropic with g = 1.982. It has an excited spin multiplet at approximately 30 cm(-1), inferred from the temperature-dependence of the linewidth. The intensity ratio of the g = 4.1 signal to the multiline signal was found to be almost constant from 5 to 23 K. Based on these findings and on spin quantitation of the two signals in samples with and without 4% ethanol, it is concluded that they arise from the ground doublets of paramagnetic species in different PS II centers. It is suggested that the two signals originate from separate PS II electron donors that are in a redox equilibrium with each other in the S(2) state and that the g = 4.1 signal arises from monomeric Mn(IV).
Mn2+-ESR spectra of soybean, wax bean and lima bean agglutinin at Q- and X-band frequencies show nearly axially symmetric zero field splitting (ZFS); the dominant anisotropic term of the spin hamiltonian is the quadratic ZFS interaction. There is a relatively large distribution of ZFS parameters. No effects of specific inhibitor (N-acetylgalactosamine) on the soybean agglutinin spectrum were observed. The stoichiometric complex obtained on addition of Mn2+ to a Mn2+-free sample of this protein has a spectrum similar to that of the native protein. The small changes in the spectrum are interpreted in terms of a wider distribution of the ZFS parameters at the Mn binding site. Addition of Ca2+ to Mn2+-soybean agglutinin sharpens the lines, possibly because Ca2+ increases the rigidity of the complex.
The g = 4 and g = 2 multiline EPR signals arising from the Mn cluster of the photosynthetic oxygen-evolving complex (OEC) in the S2 state were studied in preparations of oriented photosystem II (PSII) membranes. The ammonia-modified forms of these two signals were also examined. The g = 4 signal obtained in oriented PSII membranes treated with NH4Cl at pH 7.5 displays at least 16 partially resolved Mn hyperfine transitions with a regular spacing of 36 G [Kim, D.H., Britt, R.D., Klein, M.P., & Sauer, K. (1990) J. Am. Chem. Soc. 112, 9389-9391]. The observation of this g = 4 "multiline signal" provides strong spectral evidence for a tetranuclear Mn origin for the g = 4 signal and is strongly suggestive of a model in which different spin state configurations of a single exchange-coupled Mn cluster give rise to the g = 4 and g = 2 multiline signals. A simulation shows the observed spectrum to be consistent with an S = 3/2 or S = 5/2 state of a tetranuclear Mn complex. The resolution of hyperfine structure on the NH3-modified g = 4 signal is strongly dependent on sample orientation, with no resolved hyperfine structure when the membrane normal is oriented perpendicular to the applied magnetic field. The dramatic NH3-induced changes in the g = 4 signal resolved in the spectra of oriented samples are suggestive that NH3 binding at the Cl- site of the OEC may represent direct coordination of NH3 to the Mn cluster.(ABSTRACT TRUNCATED AT 250 WORDS)
The low-temperature S2-state EPR signal at g = 4 from the oxygen-evolving complex (OEC) of spinach Photosystem-II-enriched membranes is examined at three frequencies, 4 GHz (S-band), 9 GHz (X-band) and 16 GHz (P-band). While no hyperfine structure is observed at 4 GHz, the signal shows little narrowing and may mask underlying hyperfine structure. At 16 GHz, the signal shows g-anisotropy and a shift in g-components. The middle Kramers doublet of a near rhombic S = 5/2 system is found to be the only possible origin consistent with the frequency dependence of the signal. Computer simulations incorporating underlying hyperfine structure from an Mn monomer or dimer are employed to characterize the system. The low zero field splitting (ZFS) of D = 0.43 cm-1 and near rhombicity of E/D = 0.25 lead to the observed X-band g value of 4.1. Treatment with F- or NH3, which compete with Cl- for a binding site, increases the ZFS and rhombicity of the signal. These results indicate that the origin of the OEC signal at g = 4 is either an Mn(II) monomer or a coupled Mn multimer. The likelihood of a multimer is favored over that of a monomer.
The photochemistry in photosystem II of spinach has been characterized by electron paramagnetic resonance (EPR) spectroscopy in the temperature range of 77-235 K, and the yields of the photooxidized species have been determined by integration of their EPR signals. In samples treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a single stable charge separation occurred throughout the temperature range studied as reflected by the constant yield of the Fe(II)-QA-EPR signal. Three distinct electron donation pathways were observed, however. Below 100 K, one molecule of cytochrome b559 was photooxidized per reaction center. Between 100 and 200 K, cytochrome b559 and the S1 state competed for electron donation to P680+. Photooxidation of the S1 state occurred via two intermediates: the g = 4.1 EPR signal species first reported by Casey and Sauer [Casey, J. L., & Sauer, K. (1984) Biochim. Biophys. Acta 767, 21-28] was photooxidized between 100 and 160 K, and upon being warmed to 200 K in the dark, this EPR signal yielded the multiline EPR signal associated with the S2-state. Only the S1 state donated electrons to P680+ at 200 K or above, giving rise to the light-induced S2-state multiline EPR signal. These results demonstrate that the maximum S2-state multiline EPR signal accounts for 100% of the reaction center concentration. In samples where electron donation from cytochrome b559 was prevented by chemical oxidation, illumination at 77 K produced a radical, probably a chlorophyll cation, which accounted for 95% of the reaction center concentration. This electron donor competed with the S1 state for electron donation to P680+ below 100 K.(ABSTRACT TRUNCATED AT 250 WORDS)
Properties of the S2 state formed in photosystem II membranes in which Cl- had been replaced by various anions were investigated by means of thermoluminescence measurements and low temperature EPR spectroscopy. The Br--substituted membranes showed the normal thermoluminescence B-band arising from S2Q-B charge recombination, whereas the SO2-4-, F--, CH3COO--, and NO-3-substituted membranes showed modified B-bands with variously upshifted peak temperatures. The extent of the peak temperature upshift varied in parallel with the extent of inhibition of O2 evolution depending on the anion species. A normal EPR S2 multiline signal was induced in Br--substituted membranes, but its amplitude was reduced to less than 10% in F--, NO-3-, CH3COO--, and SO2-4-substituted membranes, In contrast, the g = 4.1 signal from S2 was markedly enhanced in F-- and NO-3-substituted membranes, not much affected in CH3COO-- and SO2-4-substituted membranes, and decreased to 70% in Br--substituted membranes. Based on these data, the effect of various types of S2 modification on the O2-evolving activity was discussed. It was suggested that anions have an important role in regulating the interaction between the Mn atoms, and thereby adjust the redox properties of the S2 state to enable further transitions beyond S2.
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.
Photosystem II membranes, dialyzed against a Cl(-)-free buffer to remove bound Cl-, lost about 65% of the control activity. A light-intensity study of the Cl(-)-free membranes showed that all PS II centers were able to evolve oxygen at about 35% of the control rate when measured in Cl(-)-free medium. The Cl(-)-depleted membranes were immediately (< 15 s) reactivated to 85-90% of the original activity by the addition of fairly high concentrations of Cl- (Kd = 0.5 mM), but both Cl- and the activity were promptly lost when the membranes immediately after reactivation were diluted in a Cl(-)-free medium. However, stabilization of Cl(-)-binding could be accomplished by prolonged incubation in the presence of Cl-. The transition to stable binding, followed using 36Cl-, occurred over several minutes. The stable binding was further characterized by a Kd of 20 microM and a t1/2 for dissociation of about 1h [Lindberg et al. (1993) Photosynth. Res. 38, 401-408]. The effects on S2 signals of removal of Cl- were studied using EPR. The depletion of Cl- was accompanied by a shift in intensity toward the g = 4.1 signal at the expense of the multiline signal. When Cl- or Br- but not F- was added to the depleted PS II membranes, the original distribution of the signals was immediately (< 30 s) restored. We propose that Cl(-)-binding responsible for high oxygen-evolution activity and normal EPR properties of the S2 state may occur either as high affinity (Kd = 20 microM) and slowly exchanging (t1/2 = 1 h), or as low affinity (Kd = 0.5 mM) and rapidly exchanging (t1/2 < 15 s). Our results suggest that Br- but not F- has a mode of binding similar to that of Cl-. The high-affinity state is the normal state of binding, but once Cl- has been removed, it will first rebind as low-affinity, rapidly exchanging followed by conversion into a high-affinity, slowly exchanging mode of binding.
Experiments are described which allow the detection and characterization of new EPR signals in photosystem II (PSII). PSII has been extensively studied with the water oxidising complex (WOC) poised in the S1 and S2 states. Other stages in the cycle of water oxidation lack characteristic EPR signals for use as probes. In this study, experiments use multiple turnovers of PSII from an initial S1 state to allow new states of PSII to be studied. The first EPR signal detected, centered at g = 4.85 and termed the g = 5 signal, is suggested to be a new form of S2 probably formed by decay of S3 at cryogenic temperatures, but a novel form of oxidized non-heme iron cannot be fully excluded at present. The second signal is split around g = 2 and shows characteristics of signals formed by spin-spin interaction between two paramagnetic species. The split g = 2 signal is reversibly formed by illumination at <30 K of a sample containing the g = 5 signal. The g = 2 signal may be a form of the "S3" EPR signal previously only found in a variety of PSII preparations where oxygen evolution has been inhibited. Those "S3" signals are thought to arise from the interaction of an oxidized amino acid radical and the S2 state, i.e., S2X+. Illumination at higher temperatures or illumination at <30 K, followed by dark-adaptation at 77 K, removes the g = 5 signal and prevents subsequent detection of the g = 2 signal on illumination at <30 K. The most likely explanation of our data is that illumination at <30 K of centers containing the g = 5 species allows accumulation of an oxidized intermediate and that at higher temperatures electron transfer proceeds to re-form an EPR-silent S state equivalent to that initially trapped during sample preparation. Study of these signals should provide an important new insight into the WOC and PSII.
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.
The Mn cluster of Photosystem II (PSII) from Synechococcus elongatus was studied using EPR. A signal with features between g = 5 and g = 9 is reported from the S2-state. The signal is attributed to the manganese cluster in a state with a spin 5/2 state. Spectral simulations of the signal indicate zero field splitting parameters where the |E/D| was 0.13. The new signal is formed by irradiating PSII samples which contain the spin = 1/2 S2-state using 813 nm light below 200 K. This effect is attributed to a spin-state change in the manganese cluster due to absorption of the IR light by the Mn-cluster itself. The signal is similar to that reported recently in PSII of plants [Boussac, A., Un, S., Horner, O., and Rutherford, A. W. (1998) Biochemistry 37, 4001-4007]. In plant PSII the comparable signal is formed at a lower temperature (optimally below 77 K), and gradual warming of the sample in the dark leads to the formation of the state responsible for the well-known g = 4.1 signal prior to formation of the spin 1/2 multiline signal. In the present work using cyanobacterial PSII, warming of the sample in the dark leads to the formation of the spin 1/2 multiline signal without formation of the g = 4 type signal as an intermediate. These observations provide a partial explanation for the long-standing "mystery of the missing g = 4 state" in cyanobacterial PSII. The observations are rationalized in terms of three possible states which can exist for S2: (i) the spin 1/2 multiline signal, (ii) the state responsible for the g = 4.1 signal, and (iii) the new spin 5/2 state. The relative stability of these states differs between plants and cyanobacteria.
Oxygen evolution by higher plants requires chloride, which binds to a site associated with the oxygen-evolving complex of photosystem II (PSII). In this study, the inhibitory effect of the anion azide was characterized using steady state measurements of oxygen evolution activity in PSII-enriched thylakoid membranes. N3- (7.8 mM) inhibited O2 evolution activity by 50% when a standard buffer containing chloride was used. By considering Cl- as the substrate in O2 evolution assays, we found azide to be primarily competitive with Cl- with an inhibitor dissociation constant Ki of about 0.6 mM. An uncompetitive component with a Ki ' of 11 mM was also found. Removal of the 17 and 23 kDa polypeptides resulted in a decrease in each inhibition constant. A pH dependence study of O2 evolution activity showed that the pH maximum became narrower and shifted to a higher pH in the presence of azide. Analysis of the data indicated that an acidic residue defined the low side of the pH maximum with an apparent pKa of 6.7 in the presence of azide compared with 5.5 for the control. A basic residue was also affected, exhibiting an apparent pKa of 7.1 compared with a value of 7.6 for the control. This result can be explained by a simple model in which azide binding to the chloride site moves negative charge of the anion away from the basic residue and toward the acidic residue relative to chloride. As a competitor of chloride, azide may provide an interesting probe of the oxygen-evolving complex in future studies.
The Mn-derived electron paramagnetic resonance (EPR) multiline signal from the S(0) state of the water-oxidizing complex is observable only in the presence methanol. In the present study, we have characterized the effect of methanol on the EPR signals from the S(0) and S(2) states as well as on the EPR Signal II(slow) originating from the Tyrosine(D)(ox) radical. The amplitudes of the S(0) and S(2) multiline signals increase with the methanol concentration in a similar way, whereas the S(2) g=4.1 excited state signal amplitude shows a concomitant decrease. The methanol concentration at which half of the spectral change has occurred is approximately 0.2% and the effect is saturating around 5%. Methanol has an effect on the microwave power saturation of the S(2) multiline signal, as well. The microwave power at half saturation (P(1/2)) is 85 mW in the presence of methanol, whereas the signal relaxes much slower (P(1/2) approximately 27 mW) without. The relaxation of Signal II(slow) in the presence of methanol has also been investigated. The P(1/2) value of Signal II(slow) oscillates with the S cycle in a similar way as without methanol, but the P(1/2) values are consistently lower in the methanol-containing samples. From the results, we conclude that methanol modifies the magnetic properties of the S(0) and S(2) states in a similar way. The possible site and nature of methanol binding is discussed.
The Mn(4) complex which is involved in water oxidation in photosystem II is known to exhibit three types of EPR signals in the S(2) state, one of the five redox states of the enzyme cycle: a multiline signal (spin 1/2), signals at g5 (spin 5/2) and a signal at g=4.1 (or g=4.25). The g=4.1 signal could be generated under two distinct sets of conditions: either by illumination at room temperature or at 200 K in certain experimental conditions (g4(S) signal) or by near-infrared illumination between approximately 77 and approximately 160 K of the S(2)-multiline state (g4(IR) signal). The two g=4.1 signals arise from states which have quite different stability in terms of temperature. In the present work we have compared these two signals in order to test if they originate from the same or from different chemical origins. The microwave power saturation properties of the two signals measured at 4.2 K were found to be virtually identical. Their temperature dependencies measured at non-saturating powers were also identical. The presence of Curie law behavior for the g4(S) and g4(IR) signals indicates that the states responsible for both signals are ground states. The orientation dependence, anisotropy and resolved hyperfine structure of the two g4 signals were also found to be virtually indistinguishable. We have been unable to confirm the behavior reported earlier indicating that the g4(S) signal is an excited state, nor were we able to confirm the presence of signal from a higher excited state in samples containing the g4(S), nor a radical signal in samples containing the g4(IR). These findings are best interpreted assuming that the two signals have a common origin i.e. a spin 5/2 ground state arising from a magnetically coupled Mn-cluster of 4 Mn ions.
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 water-oxidizing complex of photosystem II cycles through five oxidation states, denoted S(i)() (i = 0-4), during water oxidation to molecular oxygen, which appears at the (transient) S(4) state. The recent detection of bimodal EPR signals from the S(3) state [Matsukawa, T., Mino, H., Yoneda, D., Kawamori, A. (1999) Biochemistry 38, 4072-4077] has drawn significant attention to this critical state. An interesting property of the S(3) state is the sensitivity to near-IR (NIR) light excitation. Excitation of the S(3) state by near-IR light at cryogenic temperatures induces among other signals a derivative-shaped EPR signal at g= 5 [Ioannidis, N., and Petrouleas, V. (2000) Biochemistry 39, 5246-5254]. The signal bears unexpected similarities to a signal observed earlier in samples that had undergone multiple turnovers and subsequently had been stored at 77 K for a week or longer [Nugent, J. H. A., Turconi, S., and Evans, M. C. W. (1997) Biochemistry 36, 7086-7096]. Recently, both signals were assigned to an S = 7/2 configuration of the Mn cluster [Sanakis, Y., Ioannidis, N., Sioros, G., and Petrouleas, V. (2001) J. Am. Chem. Soc. 123, 10766-10767]. In the present study, we employ bimodal EPR spectroscopy to investigate the pathways of formation of this unusual state. The following observations are made: (i) The g = 5 signal evolves in apparent correlation with the diminution of the S(3) state signals during the slow (tens of hours to several days range) charge recombination of S(3) with Q(A)(-) at 77 K. The tyrosyl radical D* competes with S(3) for recombination with Q(A)(-), the functional redox couple at cryogenic temperatures inferred to be D*/D(-). Transfer to -50 degrees C and above results in the relaxation of the g = 5 to the multiline and g = 4.1 signals of the normal S(2) state. (ii) The transition of S(3) to the state responsible for the g = 5 signal can be reversed by visible light illumination directly at -30 degrees C or by illumination at 4.2 K followed by brief (2 min) transfer to -50 degrees C in the dark. The latter step is required in order to overcome an apparent thermal activation barrier (charge recombination appears to be faster than forward electron transfer at 4.2 K). (iii) The "g = 5" state can be reached in a few tens of minutes at 4.2 K by near-IR light excitation of the S(3) state. This effect is attributed to the transfer of the positive hole from the Mn cluster to a radical (probably tyr Z), which recombines much faster than the Mn cluster with Q(A)(-). (iv) The above properties strongly support the assignment of the configuration responsible for the g = 5 signal to a modified S(2) state, denoted S(2)'. Evidence supporting the assignment of the S(2)' to a proton-deficient S(2) configuration is provided by the observation that the spectrum of S(2) at pH 8.1 (obtained by illumination of the S(1) state at -30 degrees C) contains a g = 5 contribution.
ESEEM studies of alcohol binding to the manganese cluster of the oxygen evolving complex of Photosystem II Q-Band EPR of the S2State of PSII 2895 Isolation of a photosystem 2 preparation from higher plants with highly enriched oxygen evolution activity
- D A Force
- D W Randall
- G A Lorigan
- K L Clemens
Force, D. A., D. W. Randall, G. A. Lorigan, K. L. Clemens, and R. D. Britt. 1998. ESEEM studies of alcohol binding to the manganese cluster of the oxygen evolving complex of Photosystem II. J. Am. Chem. Soc. 120:13321–13333. Q-Band EPR of the S2State of PSII 2895 Biophysical Journal 87(4) 2885–2896 rFord, R. C., and M. C. W. Evans. 1983. Isolation of a photosystem 2 preparation from higher plants with highly enriched oxygen evolution activity. FEBS Lett. 160:159–164
The manganese and calcium ions of photosynthetic oxygen evolution Studies of the manganese site of Photosystem II by electron spin resonance spectroscopy Electron transfer in Photosystem II at cryogenic temperatures Electron spin resonance of manganese in borate glasses
- R J Debus
- J C Paula
- W F Beck
- A.-F Miller
- R B Wilson
- G W Brudvig
Debus, R. J. 1992. The manganese and calcium ions of photosynthetic oxygen evolution. Biochim. Biophys. Acta. 1102:269–352. de Paula, J. C., W. F. Beck, A.-F. Miller, R. B. Wilson, and G. W. Brudvig. 1987. Studies of the manganese site of Photosystem II by electron spin resonance spectroscopy. J. Chem. Soc., Faraday Trans. 1. 83: 3635–3651. de Paula, J. C., J. B. Innes, and G. W. Brudvig. 1985. Electron transfer in Photosystem II at cryogenic temperatures. Biochemistry. 24:8114–8120. de Wijn, H. W., and R. F. van Balderen. 1967. Electron spin resonance of manganese in borate glasses. J. Chem. Phys. 46:1381–1387