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Electron paramagnetic resonance properties of the S2 state of the oxygen-evolving complex of photosystem II
Abstract
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 relative amplitudes of the HS-S 2 and LS-S 2 signals vary under different experimental conditions which indicates that the equilibrium between the HS-S 2 and LS-S 2 states can be perturbed (Zimmermann and Rutherford 1986;Pokhrel and Brudvig 2014;Boussac 2019;Amin et al. 2021). Notably, in spinach PSII, the EPR signal of the HS-S 2 state can be decreased below detection limits by a high concentration of glycerol (Zimmermann and Rutherford 1986;Pokhrel and Brudvig 2014) which is typically used during purification as a stabilizing agent and as a cryoprotectant Boussac et al. 2018). ...
... The relative amplitudes of the HS-S 2 and LS-S 2 signals vary under different experimental conditions which indicates that the equilibrium between the HS-S 2 and LS-S 2 states can be perturbed (Zimmermann and Rutherford 1986;Pokhrel and Brudvig 2014;Boussac 2019;Amin et al. 2021). Notably, in spinach PSII, the EPR signal of the HS-S 2 state can be decreased below detection limits by a high concentration of glycerol (Zimmermann and Rutherford 1986;Pokhrel and Brudvig 2014) which is typically used during purification as a stabilizing agent and as a cryoprotectant Boussac et al. 2018). Indeed, a 1.90 Å resolution X-ray crystal structure of PSII from the cyanobacteria Thermosynechococcus vulcanus (Umena et al. 2011) reports several glycerol molecules bound throughout the structure, but at sites distant to the OEC and unlikely to perturb the mechanism of water oxidation. ...
... The reported results are consistent with the lack of the HS-S 2 EPR signal from wild-type cyanobacterial PSII (Yachandra et al. 1996;Boussac et al. 2018). A similar perturbation of the hydrogen-bond network may also explain the effect of glycerol on spinach PSII, which shifts the S 2 state equilibrium to favor the LS-S 2 state as observed in spectral amplitude shifts between the HS g = 4.1 signal and the LS g = 2.0 multiline signal (Fig. 2) (Zimmermann and Rutherford 1986). However, an important difference between cyanobacterial and plant-type PSII is found in the narrow channel. ...
The oxygen-evolving complex (OEC) of photosystem II (PSII) cycles through redox intermediate states Si (i = 0–4) during the photochemical oxidation of water. The S2 state involves an equilibrium of two isomers including the low-spin S2 (LS-S2) state with its characteristic electron paramagnetic resonance (EPR) multiline signal centered at g = 2.0, and a high-spin S2 (HS-S2) state with its g = 4.1 EPR signal. The relative intensities of the two EPR signals change under experimental conditions that shift the HS-S2/LS-S2 state equilibrium. Here, we analyze the effect of glycerol on the relative stability of the LS-S2 and HS-S2 states when bound at the narrow channel of PSII, as reported in an X-ray crystal structure of cyanobacterial PSII. Our quantum mechanics/molecular mechanics (QM/MM) hybrid models of cyanobacterial PSII show that the glycerol molecule perturbs the hydrogen-bond network in the narrow channel, increasing the pKa of D1-Asp61 and stabilizing the LS-S2 state relative to the HS-S2 state. The reported results are consistent with the absence of the HS-S2 state EPR signal in native cyanobacterial PSII EPR spectra and suggest that the narrow water channel hydrogen-bond network regulates the relative stability of OEC catalytic intermediates during water oxidation.
... [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.
... The high (S total = 5/2) and low spin (S total = ½) forms in the S 2 state are interrelated, on the basis of the observation of the amplitude conversion of the S 2 -g4 EPR signal to the S 2 -g2 EPR signal (Casey and Sauer 1984, Beck and Brudvig 1986, Zimmermann and Rutherford 1986, Hansson et al. 1987. The distribution of high spin and low spin species and the g-values and hyperfine coupling values of these spin states are sensitive to several parameters, such as (a) species (higher-plant, thermophile or non- thermophile cyanobacterial PSII) in different pHs, (b) the presence of chemical additives like alcohol (methanol or ethanol) or sucrose and glycerol (often used as cryo-protectant) in the sample, (c) substitution of the native Ca 2+ in the OEC (Ca 2+ -PSII) by Sr (Sr 2+ -PSII), and (d) halide substitution in PSII with Br -or I -replacing the Cl -of the native state (Fig. 1C). ...
... A detailed discussion can be found in several reviews ( Haddy 2007, Pokhrel and Brudvig 2014, Boussac et al. 2015, Boussac et al. 2018). Briefly, in spinach PSII samples illuminated at 200 K, both S 2 -g2 and S 2 -g4 signals are observed in the presence of sucrose, while with glycerol, ethylene glycerol, or ethanol, the MLS is enhanced and the S 2 -g4 EPR signal is suppressed ( Zimmermann and Rutherford 1986). Some treatments such as (c) and (d) instead stabilize the S total = 5/2 state. ...
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.
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... For efficiency, the cluster was considered to comprise ferromagnetically coupled Mn atoms, where the total spin, S, was 12/2 for the Mn 3 [M]O 2 cluster and 10/2 for the Mn 3 [M]O 4 cluster. We note that, in the calculation of the native Mn 4 CaO 5 of PSII, the difference in S (e.g., S = 1/2 in S 2 (Zimmermann and Rutherford 1986), high, low, ferromagnetic, and antiferromagnetic) did not affect the (i) resulting geometry (Ames et al. 2011;Isobe et al. 2012), (ii) potential energy profile of proton transfer (Kawashima et al. 2018b), (iii) redox potential of each Mn site (Mandal et al. 2020), or (iv) pK a values of ligand water molecules W1-W4 (Saito et al. 2020c Table S2). E m (Mn1 III/IV ) was calculated from the HOMO energies, since the value of E m for one-electron oxidation is correlated with the energy of the highest occupied molecular orbital (HOMO) (Mendez-Hernandez et al. 2013;Igarashi and Seefeldt 2003;Mandal et al. 2020). ...
Photosystem II (PSII) contains Ca ²⁺ , which is essential to the oxygen-evolving activity of the catalytic Mn 4 CaO 5 complex. Replacement of Ca ²⁺ with other redox-inactive metals results in a loss/decrease of oxygen-evolving activity. To investigate the role of Ca ²⁺ in this catalytic reaction, we investigate artificial Mn 3 [M]O 2 clusters redox-inactive metals [M] ([M] = Mg ²⁺ , Ca ²⁺ , Zn ²⁺ , Sr ²⁺ , and Y ³⁺ ), which were synthesized by Tsui et al. (Nat Chem 5:293, 2013). The experimentally measured redox potentials ( E m ) of these clusters are best described by the energy of their highest occupied molecular orbitals. Quantum chemical calculations showed that the valence of metals predominantly affects E m (Mn III/IV ), whereas the ionic radius of metals affects E m (Mn III/IV ) only slightly.
... In the g=4.1 EPR state Mn1, Mn2, Mn3 are in the IV oxidation state, while Mn4 is in the III state (Fig 1). 17 In the g=2 redox isomer M1 is Mn 3+ while M4 is Mn 4+ . In addition, timeresolved photothermal beam deflection measurements suggest that a proton is released from the OEC or surroundings when the nearby Tyr, Yz, is oxidized before Mn oxidation in the S2-S3 transition. ...
Understanding the water oxidation mechanism in Photosystem II (PSII) stimulates the design of biomimetic artificial systems that can convert solar energy into hydrogen fuel efficiently. The Sr2+ substituted PSII is active but slower than with the native Ca2+ as an oxygen evolving catalyst. Here, we use Density Functional Theory (DFT) to compare the energetics of the S2 to S3 transition in the Mn4O5Ca2+ and Mn4O5Sr2+ clusters. The calculations show that deprotonation of the water bound to Ca2+ (W3), required for the S2 to S3 transition, is energetically more favorable in Mn4O5Ca2+ than Mn4O5Sr2+. In addition, we have calculated the pKa of the water that bridges Mn4 and the Ca2+/Sr2+ in the S2 using continuum electrostatics. The calculations show that the pKa is higher by 4 pH units in the Mn4O5Sr2+.
... EPR signals can be induced and studied in every S state of the Kok cycle [46][47][48], and are sensitive to the conformation of the Mn 4 Ca cluster. Two of the most prominent signals in PSII are the S 2 state low spin (LS) multiline (ML) signal around g ~ 2 and its high spin (HS) counterpart in the g ~ 4 region [49][50][51][52] [37,43,45,57], or to (iii) have a protonated O4 bridge (S 2 PI ) [58]. ...
In photosynthesis, the oxygen-evolving complex (OEC) of the pigment-protein complex photosystem II (PSII) orchestrates the oxidation of water. Introduction of the V185N mutation into the D1 protein was previously reported to drastically slow O2-release and strongly perturb the water network surrounding the Mn4Ca cluster. Employing time-resolved membrane inlet mass spectrometry, we measured here the H218O/H216O-exchange kinetics of the fast (Wf) and slow (Ws) exchanging substrate waters bound in the S1, S2 and S3 states to the Mn4Ca cluster of PSII core complexes isolated from wild type and D1-V185N strains of Synechocystis sp. PCC 6803. We found that the rate of exchange for Ws was increased in the S1 and S2 states, while both Wf and Ws exchange rates were decreased in the S3 state. Additionally, we used EPR spectroscopy to characterize the Mn4Ca cluster and its interaction with the redox active D1-Tyr161 (YZ). In the S2 state, we observed a greatly diminished multiline signal in the V185N-PSII that could be recovered by addition of ammonia. The split signal in the S1 state was not affected, while the split signal in the S3 state was absent in the D1-V185N mutant. These findings are rationalized by the proposal that the N185 residue stabilizes the binding of an additional water-derived ligand at the Mn1 site of the Mn4Ca cluster via hydrogen bonding. Implications for the sites of substrate water binding are discussed.
... Isomer B is "closed" cuboid where the oxidation states of Mn1 and Mn4 are switched to IV and III, respectively. S 2A exhibits a characteristic multiline EPR signal at g = 2.0 [4], while S 2B has a broad EPR band at g = 4.1 [5,6]. A second flash moves the OEC into the S 3 state, where all four Mn centers have formal 4+ charges and an additional water is bound between Mn1 and Mn4, setting the complex up for oxygen evolution [7]. ...
The influence of the environment on the functionality of the oxygen-evolving complex (OEC) of photosystem II has long been a subject of great interest. In particular, various water channels, which could serve as pathways for substrate water diffusion, or proton translocation, are thought to be critical to catalytic performance of the OEC. Here, we address the dynamical nature of hydrogen bonding along the water channels by performing molecular dynamics (MD) simulations of the OEC and its surrounding protein environment in the S1 and S2 states. Through the eigenvector centrality (EC) analysis, we are able to determine the characteristics of the water network and assign potential functions to the major channels, namely that the narrow and broad channels are likely candidates for proton/water transport, while the large channel may serve as a path for larger ions such as chloride and manganese thought to be essential during PSII assembly.
... 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. ...
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.
... EPR spectroscopy indicates an S= 1/2 spin ground state for the S 2 state [48,49]. These The EPR results of different research groups support either the LV [56,57] [59,72,73]. ...
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.
... 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 identity and insertion pathway of the substrate oxygen atoms that are coupled to dioxygen by the oxygen-evolving complex (OEC) remains a central question toward understanding Nature’s water oxidation mechanism. In several studies, ammonia has been used as a small “water analogue” to elucidate the pathway of substrate access to the OEC and to aid in determining which of the oxygen ligands of the tetramanganese cluster are substrates for O–O bond formation. On the basis of structural and spectroscopic investigations, five first-sphere binding modes of ammonia have been suggested, involving either substitution of an existing H2O/OH–/O2– group or addition as an extra ligand to a metal ion of the Mn4CaO5 cluster. Some of these modes, specifically the ones involving substitution, have already been subject to spectroscopy-oriented quantum chemical investigations, whereas more recent suggestions that postulate the addition of ammonia have not been examined so far with quantum chemistry for their agreement with spectroscopic data. Herein, we use a common structural framework and theoretical methodology to evaluate structural models of the OEC that represent all proposed modes of first-sphere ammonia interaction with the OEC in its S2 state. Criteria include energetic, magnetic, kinetic, and spectroscopic properties compared against available experimental EPR, ENDOR, ESEEM, and EDNMR data. Our results show that models featuring ammonia replacing one of the two terminal water ligands on Mn4 align best with experimental data, while they definitively exclude substitution of a bridging μ-oxo ligand as well as incorporation of ammonia as a sixth ligand on Mn1 or Mn4.
Photosystem II, the water splitting enzyme of photosynthesis, utilizes the energy of sunlight to drive the four-electron oxidation of water to dioxygen at the oxygen-evolving complex (OEC). The OEC harbors a Mn4CaO5 cluster that cycles through five oxidation states Si (i = 0–4). The S3 state is the last metastable state before the O2 evolution. Its electronic structure and nature of the S2 → S3 transition are key topics of persisting controversy. Most spectroscopic studies suggest that the S3 state consists of four Mn(IV) ions, compared to the Mn(III)Mn(IV)3 of the S2 state. However, recent crystallographic data have received conflicting interpretations, suggesting either metal- or ligand-based oxidation, the latter leading to an oxyl radical or a peroxo moiety in the S3 state. Herein, we utilize high-energy resolution fluorescence detected (HERFD) X-ray absorption spectroscopy to obtain a highly resolved description of the Mn K pre-edge region for all S-states, paying special attention to use chemically unperturbed S3 state samples. In combination with quantum chemical calculations, we achieve assignment of specific spectroscopic features to geometric and electronic structures for all S-states. These data are used to confidently discriminate between the various suggestions concerning the electronic structure and the nature of oxidation events in all observable catalytic intermediates of the OEC. Our results do not support the presence of either peroxo or oxyl in the active configuration of the S3 state. This establishes Mn-centered storage of oxidative equivalents in all observable catalytic transitions and constrains the onset of the O–O bond formation until after the final light-driven oxidation event.
Existence of the alternative charge separation pathway in Photosystem II under the far-red light was induced photochemistry in Photosystem II was proposed by us on the basis of induced electron transfer reactions at cryogenic temperature 5 K. Here we extend these studies to the higher temperature range of 77-295 K with help of electron paramagnetic resonance spectroscopy. Induction of the S2 state multiline signal, oxidation of cytochrome b559 and chlorophyllZ was studied in Photosystem II membrane preparations from spinach after application of a laser flashes in visible (532 nm) or far-red (730-750 nm) spectral regions. Temperature dependence of the S2 state multiline signal induction after single flash at 730-750 nm (Tinhibition ~ 240 K) was found to be different than that at 532 nm (Tinhibition ~ 157 K). No contaminant oxidation of the secondary electron donors cytochrome b559 or chlorophyllZ was observed. Photoaccumulation experiments with extensive flashing at 77 K showed similar results, with no or very little induction of the secondary electron donors. Thus, the partition ratio defined as (yield of YZ/CaMn4O5-cluster oxidation):(yield of Cytb559/ChlZ/CarD2 oxidation) was found to be 0.4 at under visible light and 1.7 at under far-red light at 77 K. Our data show that different products of charge separation after far-red light exists in the wide temperature range which further support the model of the different primary photochemistry in PSII with localization of hole on the ChlD1 molecule.
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.
Ca2+, which provides binding sites for ligand water molecules W3 and W4 in the Mn4CaO5 cluster, is a prerequisite for O2 evolution in photosystem II (PSII). We report structural changes in the H-bond network and the catalytic cluster itself upon the replacement of Ca2+ with other alkaline earth metals, using a quantum mechanical/molecular mechanical approach. The small radius of Mg2+ makes W3 donate an H-bond to D1-Glu189 in Mg2+-PSII. If an additional water molecule binds at the large surface of Ba2+, it donates H-bonds to D1-Glu189 and the ligand water molecule at the dangling Mn, altering the H-bond network. The potential energy profiles of the H-bond between D1-Tyr161 (TyrZ) and D1-His190 and the interconversion between the open- and closed-cubane S2 conformations remain substantially unaltered upon the replacement of Ca2+. Remarkably, the O5⋯Ca2+ distance is shortest among all O5⋯metal distances irrespective of the radius being larger than that of Mg2+. Furthermore, Ca2+ is the only alkaline earth metal that equalizes the O5⋯metal and O2⋯metal distances and facilitates the formation of the symmetric cubane structure.
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 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.
Energy shortage and environmental pollution limit the sustainable development of human beings. Water, as a clean and renewable resource, provides a solution for sustainable energy conversion from water oxidation catalysis. Lessons should be learned from nature to explore efficient artificial catalysts. In this chapter, we will review recent major progress in natural photosynthesis, from the structure and functions to the mechanisms for the water-oxidizing center of the biological enzyme. Later, the development of molecular and material water oxidation catalysts is discussed, with a focus on the mechanisms and rational catalyst design.
The [8Fe-7S] P-cluster of nitrogenase MoFe protein mediates electron transfer from nitrogenase Fe protein during the catalytic production of ammonia. The P-cluster transitions between three oxidation states, PN, P+, P2+ of which PN↔P+ is critical to electron exchange in the nitrogenase complex during turnover. To dissect the steps in formation of P+ during electron transfer, photochemical reduction of MoFe protein at 231-263 K was used to trap formation of P+ intermediates for analysis by EPR. In complexes with CdS nanocrystals, illumination of MoFe protein led to reduction of the P-cluster P2+ that was coincident with formation of three distinct EPR signals: S = 1/2 axial and rhombic signals, and a high-spin S = 7/2 signal. Under dark annealing the axial and high-spin signal intensities declined, which coincided with an increase in the rhombic signal intensity. A fit of the time-dependent changes of the axial and high-spin signals to a reaction model demonstrates they are intermediates in the formation of the P-cluster P+ resting state and defines how spin-state transitions are coupled to changes in P-cluster oxidation state in MoFe protein during electron transfer.
Understanding the water oxidation mechanism in Photosystem II (PSII) stimulates the design of biomimetic artificial systems that can convert solar energy into hydrogen fuel efficiently. The Sr²⁺ substituted PSII is active but slower than with the native Ca²⁺ as an oxygen evolving catalyst. Here, we use Density Functional Theory (DFT) to compare the energetics of the S2 to S3 transition in the Mn4O5Ca²⁺ and Mn4O5Sr²⁺ clusters. The calculations show that deprotonation of the water bound to Ca²⁺ (W3), required for the S2 to S3 transition, is energetically more favorable in Mn4O5Ca²⁺ than Mn4O5Sr²⁺. In addition, we have calculated the pKa of the water that bridges Mn4 and the Ca²⁺/Sr²⁺ in the S2 using continuum electrostatics. The calculations show that the pKa is higher by 4 pH units in the Mn4O5Sr²⁺.
Virtually all life on our planet is powered by the sun through the process of photosynthesis. Understanding the design and operating principles of natural photosynthesis is a central challenge for fundamental research because biology presents a unique paradigm and possible blueprint for the realization of artificial alternatives. All processes of natural photosynthesis, including light harvesting, charge separation and accumulation, water oxidation, and carbon fixation, embody “solutions” optimized through billions of years of evolution to challenges currently faced by scientists developing components for artificial photosynthetic devices. The present chapter provides an overview of the most important aspects of natural photosynthesis and discusses the ways in which they motivate biomimetic and bioinspired research into artificial photosynthesis.
A new synthesized and structurally characterized phenolato bridged di-nuclear manganese(II) complex, formulated as [Mn2L(OAc)2(DMF)2]PF6 (1) (where HL = 4-methyl-2,6-bis-[(2-morpholin-4-yl-ethylimino)-methyl]-phenol and OAc = acetate anion), can act as a turn-on Zn²⁺ sensing probe through the in situ formation of the corresponding dinuclear Zn(II) complex, [Zn2L(OAc)2]PF6 (2) via direct Mn²⁺ ion replacement in MeOH/DMF (1/14) medium. The selective and specific fluorescent change due to addition of Zn²⁺ ions to a solution of 1 enables the detection of Zn²⁺ ions in dimethylformamide (DMF). The fluorescent dinuclear zinc(II) complex 2 has also been produced through the direct reaction between Zn²⁺ ions and HL. Both complexes 1 and 2 have been characterized by using physico-chemical and spectroscopic tools, and finally by single crystal X-ray crystallography for a detailed structural determination. The crystallographic investigation indicates that the Mn(II) and Zn(II) ions in the complexes exhibit a distorted octahedral and distorted trigonal bipyramidal coordination geometry, respectively. Interestingly, complex 1 showed very weak fluorescence in solution and no emission in the solid state, whereas complex 2 gave a strong bluish-green coloured fluorescence in both solution and in the solid state, which may be due to the distinct packing of complex 2 compared to 1, as well as to intermolecular hydrogen bonding and C–H---π, C–H---N and C–H---O interactions in the crystal packing, likely playing crucial roles for the fluorescence behavior.
The S3 state is the last semi-stable state in the water splitting reaction that is catalyzed by the Mn4O5Ca cluster that makes up the oxygen-evolving complex (OEC) of photosystem II (PSII). Recent high-field/frequency (95 GHz) electron paramagnetic resonance (EPR) studies of PSII isolated from the thermophilic cyanobacterium Thermosynechococcus elongatus have found broadened signals induced by chemical modification of the S3 state. These signals are ascribed to an S3 form that contains a five-coordinate MnIV center bridged to a cuboidal MnIV3O4Ca unit. High-resolution X-ray free-electron laser studies of the S3 state have observed the OEC with all-octahedrally coordinated MnIV in what is described as an open cuboid-like cluster. No five-coordinate MnIV centers have been reported in these S3 state structures. Here, we report high-field/frequency (130 GHz) pulse EPR of the S3 state in Synechocystis sp. PCC 6803 PSII as isolated in the presence of glycerol. The S3 state of PSII from Synechocystis exhibits multiple broadened forms (≈69% of the total signal) similar to those seen in the chemically modified S3 centers from T. elongatus. Field-dependent ELDOR-detected nuclear magnetic resonance resolves two classes of 55Mn nuclear spin transitions: one class with small hyperfine couplings (|A| ≈ 1-7 MHz) and another with larger hyperfine couplings (|A| ≈ 100 MHz). These results are consistent with an all-MnIV4 open cubane structure of the S3 state and suggest that the broadened S3 signals arise from a perturbation of Mn4A and/or Mn3B, possibly induced by the presence of glycerol in the as-isolated Synechocystis PSII.
Release of the protons from the substrate water molecules is prerequisite for O2 evolution in photosystem II (PSII). Proton-releasing water molecules with low pKa values at the catalytic moiety can be the substrate water molecules. In some studies, one of the ligand water molecules, W2, is regarded as OH−. However, the PSII crystal structure shows neither proton acceptor nor proton-transfer pathway for W2, which is not consistent with the assumption of W2 = OH−. Here we report the pKa values of the four ligand water molecules, W1 and W2 at Mn4 and W3 and W4 at Ca2+, of the Mn4CaO5 cluster. pKa(W1) ≈ pKa(W2) << pKa(W3) ≈ pKa(W4) in the Mn4CaO5 cluster in water. However, pKa(W1) ≈ pKa(D1-Asp61) << pKa(W2) in the PSII protein environment. These results suggest that in PSII, deprotonation of W2 is energetically disfavored as far as W1 exists. Water ligands found in the oxygen-evolving Mn4CaO5 cluster in photosystem II are thought to be important during photosynthesis, but the nature of the proton-releasing substrate water molecules is disputed. Here the pKa values of four key water molecules are estimated, with implications for the mechanism of water oxidation.
In photosystem II, water oxidation occurs at the oxygen-evolving complex (OEC). The presence of a hydronium ion (H3O+) was proposed at the Cl– binding site and Ca2+-depleted OEC. Using a quantum mechanical/molecular mechanical (QM/MM) approach, we report the stability of H3O+ in the PSII protein environment. Neither release of the proton from the ligand water molecule W2 at OEC nor formation of H3O+ at Cl– is energetically favorable. In contrast, H3O+ can exist at the Ca2+-depleted OEC. As far as H3O+ exists in Ca2+-depleted PSII, the H-bond network of the redox-active tyrosine (TyrZ) remains unaltered, retaining the unusually short low-barrier H-bond with D1-His190, and the redox potential of TyrZ, Em(TyrZ), remains unaltered. These explain why the oxidation of the Ca2+-depleted Mn4O5 cluster by TyrZ (i.e., the S2 to S3 transition) is not inhibited at low pH. It seems likely that Ca2+ plays a role not only in (i) maintaining the H-bond network and facilitating TyrZ oxidation (tuning Em(TyrZ)) but also in (ii) providing the valence of +2, decreasing pKa of the ligand molecule (W1), and facilitating the release of the proton from W1 in the S2 to S3 transition together with Cl–.
In photosynthesis, dioxygen formation from water is catalyzed by the oxygen evolving complex (OEC) in Photosystem II (PSII) that harbours the Mn4Ca cluster. During catalysis, the OEC cycles through five redox states, S0 to S4. In the S2 state, the Mn4Ca cluster can exist in two conformations, which are signified by the low-spin (LS) g = 2 EPR multiline signal and the high-spin (HS) g = 4.1 EPR signal. Here, we employed time-resolved membrane inlet mass spectrometry to measure the kinetics of H218O/H216O exchange between bulk water and the two substrate waters bound at the Mn4Ca cluster in the S2LS, S2HS, and the S3 states in both Ca-PSII and Sr-PSII core complexes from T. elongatus. We found that the slowly exchanging substrate water exchanges 10-times faster in the S2HS than in the S2LS state, and that the S2LS S2HS conversion has at physiological temperature an activation barrier of 17 1 kcal/mol. Of the presently suggested S2HS models, our findings are best in agreement with a water exchange pathway involving a S2HS state that has an open cubane structure with a hydroxide bound between Ca and Mn1. We also show that water exchange in the S3 state is governed by different equilibrium than in S2, and that the exchange of the fast substrate water in the S2 state is unaffected by Ca/Sr substitution. These findings support that (i) O5 is the slowly exchanging substrate water, with W2 being the only other option, and (ii) either W2 or W3 is the fast exchanging substrate. The three remaining options for O-O bond formation in PSII are discussed.
In photosystem II (PSII), water oxidation occurs in the Mn4CaO5 cluster with the release of electrons via the redox-active tyrosine (TyrZ) to the reaction-center chlorophylls (PD1/PD2). Using a quantum mechanical/molecular mechanical approach, we report the redox potentials (Em) of these cofactors in the PSII protein environment. The E values suggest that the Mn4CaO5 cluster, TyrZ, and PD1/PD2 form a downhill electron transfer pathway. E for the first oxidation step, Em(S0/S1), is uniquely low (730 mV) and is ~100 mV lower than that for the second oxidation step, Em(S1/S2) (830 mV) only when the O4 site of the Mn4CaO5 cluster is protonated in S0. The O4-water chain, which directly forms a low-barrier H-bond with the Mn4CaO5 cluster and mediates proton-coupled electron transfer in the S0 to S1 transition, explains why the second lowest oxidation state, S1, is the most stable and S0 is converted to S1 even in the dark.
The biological generation of oxygen by the oxygen-evolving complex (OEC) in photosystem II (PS II) is one of nature's most important reactions. The OEC is a Mn4Ca-cluster that has multiple Mn-O-Mn and Mn-O-Ca bridges and binds four water molecules. Previously binding of an additional oxygen was detected in the S2 to S3 transition. Here we demonstrate that early binding of the substrate oxygen to the 5-coordinate Mn1 center in the S2 state is likely responsible for the S2 high-spin EPR signal. Substrate binding in the Mn1-OH form explains the prevalence of the high spin S2-state at higher pH and its low temperature conversion into the S3-state. The given interpretation was confirmed by X-ray absorption spectroscopic measurements, DFT and broken symmetry DFT calculations of structures and magnetic properties. Structural, electronic and spectroscopic properties of the high spin S2 state model are provided and compared with the available S3-state models. New interpretation of the high spin S2 state opens opportunity for analysis of factors controlling the oxygen substrate binding in PS II.
Heterogeneity in intermediate catalytic states of the oxygen-evolving complex (OEC) of Photosystem II is known from a wide range of experimental and theoretical data, but its potential implications for the mechanism of water oxidation remain unexplored. We delineate the consequences of structural heterogeneity for the final step of the catalytic cycle by tracing the evolution of three spectroscopically relevant and structurally distinct components of the last metastable S3 state to the transient O2-evolving S4 state of the OEC. Using quantum chemical calculations, we show that each S3 isomer leads to a different electronic structure formulation for the active S4 state. Crucially, in addition to previously hypothesized Mn(IV)-oxyl species, we establish for the first time, how a genuine Mn(V)-oxo can be obtained in the catalytically active S4 state: this takes the form of a five-coordinate and locally high-spin (SMn = 1) Mn(V) site. This formulation for the S4 state evolves naturally from a preceding S3-state structural intermediate that contains a quasi-trigonal-bipyramidal Mn(IV) ion. The results strongly suggest that water binding in the S3 state is not prerequisite for reaching the oxygen-evolving S4 state of the complex, supporting the notion that both substrates are preloaded at the beginning of the catalytic cycle. This scenario allows true four-electron metal-centered hole accumulation to precede OO bond formation and hence the latter can proceed via a genuine even-electron mechanism. This can occur as intramolecular nucleophilic coupling of two oxo units synchronously with the binding of a water substrate for the next catalytic cycle.
The sunlight-powered oxidation of water by photosystem II (PSII) of algae, plants, and cyanobacteria underpins the energy conversion processes that sustain most of life on our planet. Understanding the structure and function of the “engine of life”, the oxygen-evolving complex (OEC) in the active site of PSII, has been one of the great and persistent challenges of modern science. Immense progress has been achieved in recent years through combined contributions of diverse disciplines and research approaches, yet the challenge remains. The improved understanding of the tetramanganese-calcium cluster of the OEC for the experimentally accessible catalytic states often creates a more complex picture of the system than previously imagined, while the various strands of evidence cannot always be unified into a coherent model. This review focuses on selected current problems that relate to structural–electronic features of the OEC, emphasizing conceptual aspects and highlighting topics of structure and function that remain uncertain or controversial. The Mn4CaOx cluster of the OEC cycles through five redox states (S0–S4) to store the oxidizing equivalents required for the final step of dioxygen evolution in the spontaneously decaying S4 state. Remarkably, even the dark-stable state of the OEC, the S1 state, is still incompletely understood because the available structural models do not fully explain the complexity revealed by spectroscopic investigations. In addition to the nature of the dioxygen-evolving S4 state and the precise mechanism of O–O bond formation, major current open questions include the type and role of structural heterogeneity in various intermediate states of the OEC, the sequence of events in the highly complex S2–S3 transition, the heterogeneous nature of the S3 state, the accessibility of substrate or substrate analogues, the identification of substrate oxygen atoms, and the role of the protein matrix in mediating proton removal and substrate delivery. These open questions and their implications for understanding the principles of catalytic control in the OEC must be convincingly addressed before biological water oxidation can be understood in its full complexity on both the atomic and the systemic levels.
The Mn4CaO5 oxygen-evolving complex (OEC) of photosystem II catalyzes the light-driven oxidation of two substrate waters to molecular oxygen. An ELDOR-detected NMR and computational study has indicated that ammonia, a substrate analogue, binds as a terminal ligand to the Mn4A ion trans to the O5 μ4 oxido bridge.[Perez Navarro M., et al., Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 15561−15566] A subsequent electron spin echo envelope modulation (ESEEM) study confirmed this result and showed that ammonia hydrogen bonds to the carboxylate sidechain of D1-Asp61.[Oyala P.H., et al., J. Am. Chem. Soc. 2015, 137, 8829−8837] Here we further probe the environment of OEC with an emphasis on the proximity of exchangeable protons, comparing ammonia-bound and unbound forms. Our ESEEM and electron nuclear double resonance (ENDOR) results indicate that ammonia substitutes for the W1 terminal water ligand without significantly altering the electronic structure of the OEC.
The oxygen evolving complex (OEC) of photosystem II (PS II) is composed of a four manganese cluster (1) which cycles through 5 different redox (S-) states, So to S4 (Kok model); S1 is the dark stable state. The best-characterized EPR signals of the OEC, recorded in the standard perpendicular-polarization mode (referring to the orientation of the static magnetic (H-) field relative to the H-component of the microwave) are the g=2 ‘multiline’ signal and the featureless g = 4.1 signal of the S2 state which are believed to originate from two half-integer spin configurations of the exchange-coupled Mn4 cluster (1). Since the Kok model predicts integer-spin states for S1 and S3 (given the half-integer spin of S2), parallel-polarization mode signals are expected from these states, unless they are diamagnetic. Indeed a broad featureless S1-specific signal in this EPR mode (geff = 4.8) was reported from plant PSII (2). We recently reported a S1-specific parallel-mode multiline signal (geff = 12) from both cyanobacterial PSII (3) and plant PSII depleted of the 17 and 23 kDa proteins (4) which shield the OEC at the lumenal peripherie of the plant PSII. On this congress we present further characterization of this signal such as temperature dependence, correlation with the S2 signals and modulation by ligand binding.
The source of molecular oxygen on Earth is the oxidation of water by Photosystem II in oxygenic photosynthesis. The water oxidising complex is composed of a Mn-cluster ligated to a protein environment and Cl⁻ and Ca²⁺ ions. During water oxidation the Mn-cluster cycles through five different oxidation states (S0-S4).
Chloride is a well-known activator of oxygen evolution activity in photosystem II. Its effects have been characterized over several decades of research, as methods have developed and improved. By replacing chloride with other small anions with a range of chemical properties, a picture of the requirements of a successful anion activator can be formulated. In this review, the results of experiments on the chloride effect using enzyme kinetics methods and electron paramagnetic resonance spectroscopy are described, with summaries for the major anion activators and inhibitors that have been studied.
Sunlight is absorbed and converted to chemical energy by photosynthetic organisms. At the heart of this process is the most fundamental reaction on Earth, the light-driven splitting of water into its elemental constituents. In this way molecular oxygen is released, maintaining an aerobic atmosphere and creating the ozone layer. The hydrogen that is released is used to convert carbon dioxide into the organic molecules that constitute life and were the origin of fossil fuels. Oxidation of these organic molecules, either by respiration or combustion, leads to the recombination of the stored hydrogen with oxygen, releasing energy and reforming water. This water splitting is achieved by the enzyme photosystem II (PSII). Its appearance at least 3 billion years ago, and linkage through an electron transfer chain to photosystem I, directly led to the emergence of eukaryotic and multicellular organisms. Before this, biological organisms had been dependent on hydrogen/electron donors, such as H2S, NH3, organic acids and Fe2+, that were in limited supply compared with the oceans of liquid water. However, it is likely that water was also used as a hydrogen source before the emergence of PSII, as found today in anaerobic prokaryotic organisms that use carbon monoxide as an energy source to split water. The enzyme that catalyses this reaction is carbon monoxide dehydrogenase (CODH). Similarities between PSII and the iron- and nickel-containing form of this enzyme (Fe-Ni CODH) suggest a possible mechanism for the photosynthetic O–O bond formation.
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.
The S2 redox intermediate of the oxygen-evolving complex in Photosystem II is present as two spin isomers. The S = 1/2 isomer gives rise to a multiline EPR signal at g = 2, while the S = 5/2 isomer exhibits a broad EPR signal at g = 4.1. The electronic structures of these isomers are known, but their role in the catalytic cycle of water oxidation remains unclear. We show that formation of the S = 1/2 state from the S = 5/2 state is exergonic at temperatures above 160 K. However, the S = 1/2 isomer decays to S1 more slowly than the S = 5/2 isomer. These differences support the hypotheses that the S3 state is formed via the S2 state S = 5/2 isomer and that the stabilized S2 state S = 1/2 isomer plays a role in minimizing S2QA(-) decay in light-limiting conditions.
Methanol has long being used as a substrate analogue to probe access pathways and investigate water delivery at the oxygen-evolving complex (OEC) of photosystem-II. In this contribution we study the interaction of methanol with the OEC by assembling available spectroscopic data into a quantum mechanical treatment that takes into account the local channel architecture of the active site. The effect on the magnetic energy levels of the Mn4Ca cluster in the S2 state of the catalytic cycle can be explained equally well by two models that involve either methanol binding to the calcium ion of the cluster, or a second-sphere interaction in the vicinity of the "dangler" Mn4 ion. However, consideration of the latest ¹³C hyperfine interaction data shows that only one model is fully consistent with experiment. In contrast to previous hypotheses, methanol is not a direct ligand to the OEC, but is situated at the end-point of a water channel associated with the O4 bridge. Its effect on magnetic properties of plant PS-II results from disruption of hydrogen bonding between O4 and proximal channel water molecules, thus enhancing superexchange (antiferromagnetic coupling) between the Mn3 and Mn4 ions. The same interaction mode applies to the dark-stable S1 state and possibly to all other states of the complex. Comparison of protein sequences from cyanobacteria and plants reveals a channel-altering substitution (D1-Asn87 versus D1-Ala87) in the proximity of the methanol binding pocket, explaining the species-dependence of the methanol effect. The water channel established as the methanol access pathway is the same that delivers ammonia to the Mn4 ion, supporting the notion that this is the only directly solvent-accessible manganese site of the OEC. The results support the pivot mechanism for water binding at a component of the S3 state and would be consistent with partial inhibition of water delivery by methanol. Mechanistic implications for enzymatic regulation and catalytic progression are discussed.
The Mn4CaO5 cluster in photosystem II catalyzes the four-electron redox reaction of water oxidation in natural photosynthesis. This catalytic reaction cycles through four intermediate states (Si, i = 0 to 4), involving changes in the redox state of the four Mn atoms in the cluster. Recent studies suggest the presence and importance of isomorphous structures within the same redox/intermediate S-state. It is highly likely that geometric and electronic structural flexibility play a role in the catalytic mechanism. Among the catalytic intermediates that have been identified experimentally thus far, there is clear evidence of such isomorphism in the S2 state, with a high-spin (5/2) (HS) and a low spin (1/2) (LS) form, identified and characterized by their distinct electron paramagnetic resonance (EPR spectroscopy) signals. We studied these two S2 isomers with Mn extended X-ray absorption fine structure (EXAFS) and absorption and emission spectroscopy (XANES/XES) to characterize the structural and electronic structural properties. The geometric and electronic structure of the HS and LS S2 states are different as determined using Mn EXAFS and XANES/XES, respectively. The Mn K-edge XANES and XES for the HS form are different from the LS and indicate a slightly lower positive charge on the Mn atoms compared to the LS form. Based on the EXAFS results which are clearly different, we propose possible structural differences between the two spin states. Such structural and magnetic redox-isomers if present at room temperature, will likely play a role in the mechanism for water-exchange/oxidation in photosynthesis.
Ammonia binds to two sites in the oxygen-evolving complex (OEC) of Photosystem II (PSII). The first is as a terminal ligand to Mn in the S2 state and the second is at a site outside the OEC that is competitive with chloride. Binding of ammonia in this latter secondary site results in the S2 state S = 5/2 spin isomer being favored over the S = 1/2 spin isomer. Using EPR spectroscopy, we find that ammonia binds to the secondary site in wild-type Synechocystis sp. PCC 6803 PSII, but not in D2-K317A mutated PSII that does not bind chloride. By combining these results with quantum mechanics/molecular mechanics calculations, we propose that ammonia binds in the secondary site in competition with D1-D61 as a hydrogen-bond acceptor to the OEC terminal water ligand, W1. Implications for the mechanism of ammonia binding in its primary site directly to Mn4 in the OEC are discussed.
This article highlights mixed valence oxidation states of clusters comprised of 2, 3 or 4 Mn ions and how these can be distinguished by EPR spectroscopy. The vectorcoupling theory which enables the prediction of the spin dependent properties of these clusters is presented. Examples from the literature which are covered included synthetic inorganic Mn complexes and Mn enzymes. The latter group includes the binuclear Mn catalases and the tetranuclear Mn site responsible for photosynthetic water oxidation. A discussion of possible functional consequences which formation of a mixed valence state has on the mechanism of catalysis by these enzymes is also given.
Manganese is an essential element in many biological processes. Two functional values can be distinguished; the Mn2+ as a Lewis acid, like divalent ions, Magnesium, Calcium, Zinc and in higher oxidation states(Mn3+, Mn4+) as an oxidation catalyst, like Copper, Iron, Cobalt. The most well known Mn(II) proteins are: Arginase, containing 4 Mn(II) ions per enzyme; Concanavalin A, a manganese-calcium metalloprotein; Glutamine-Synthatase, requiring two Mn(II) ions; Phosphoenolpyruvate Carboxykinase, converting irreversible cytoplasmic oxaloacetate to phosphoenolpyruvate; a manganese ribonucleotide reductase isolated from B. ammoniagemes; Mn Thiosulfate Oxidase containing a binuclear Mn(II) site; Isopropylmalate Synthase, with the Mn(II) bound to the S-H group near site; Pyruvate carboxylase, the first metalloenzyme shown to contain manganese. Manganese redox enzymes with manganese in oxidation states 3+ and 4+ are: Manganese SOD catalysing the dismutasion of superoxide radicals to oxygen and hydrogen peroxide with a single Mn(III) center; The Manganese Peroxidase(MnP) is one of the two known enzymes capable for the oxidative degradation of lignin containing protoporphyrin IX heme prosthetic group; non heme manganese catalase containing two manganese per subunit and the Oxygen Evolving Complex, catalysing one of the most important reactions occurring in the plants, the light driven oxidation of water to oxygen and protons, containing four manganese atoms while the presence of calcium and chloride ions is required for proper functioning. Open structure mixed valence trinuclear complexes with the formula Mn(II)/Mn(III)2(Schiff-base)2(OOCR)4(L)2 were prepared and structurally characterised in an attempt to mimic the active site of OEC.
Photosynthetic water oxidation to dioxygen takes place in a Mn-containing protein in PSII via Kok’s intermediates, labeled S0–S4[1]. These S-states probably reflect different redox states of some or all of four Mn ions involved in the enzyme-substrate(H2O) complex. The molecular structure and oxidation states of this Mn-cluster in each S-state has been the subject of considerable recent spectroscopic studies involving EPR[2–5] and X-ray absorption[6,7]. In this paper, we present high-quality Mn K-edge XANES spectra measured for spinach PSII membranes in S1 and S2 states, which exhibit informative pre-edge feature due to 1s to 3d transition as well as four fine structures superimposed on the principal absorption band. These features are semi-empirically analized in comparison with those of authentic Mn complexes. Based on the results obtained, we propose a hypothetical model for the Mn-cluster which is compatible with EPR data[2–5] in S2-state.
Chloroplasts and cyanobacterial cells exposed to short light flashes release oxygen in a characteristic oscillatory manner with maximum release on every fourth flash. This was explained as resulting from cyclic transitions between different states (S0 – S4) of Photosystem II characterized by different redox configurations of the donor and acceptor sides with O2 released on the S3 to S4/S0 transition (1). The intimate details of this process are still unknown but various spectroscopic techniques have revealed that the transitions are coupled with redox changes of the manganese associated with PS II (2). Investigations utilizing EPR have shown that the S2 state exhibits two EPR signals from manganese, the multiline signal, which owes its complex appearance to interacting manganese ions, and the simpler-looking g=4.1 signals (Figs. 1A, 2A) which are proposed to arise from different conformations of the oxygen-evolving complex (3). Both signals show the same characteristic oscillating behavior as a response to light flashes as the O2 yield with maxima appearing in a period of four fashion (4). Both O2 yield and EPR amplitudes demonstrate damping.
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.
One of the noise mechanisms experienced by passive towed sonar arrays is that of convective flow noise due to boundary layer turbulence generated as the array moves through the water at a fixed tow speed. The purpose of the present work is to arrive at quantitative predictions of the effects of convective flow noise using relatively simple model calculations. Line arrays are modeled as homogeneous, layered cylinders while turbulent eddies are modeled as random pressure fluctuations traveling at the convective speed of the eddies (about 80%) of the tow speed. The qualitative difference betwen solid and liquid fills is explained with this analysis. Solid-filled arrays are more susceptible to convective flow noise than are liquid-filled arrays because the noise-carrying shear waves are highly attentuated in the liquid. The detailed analysis is presented both for homogeneous cylinders and for cylinders with multiple homogeneous layers. Examples are presented to illustrate the analysis and the numerical methods employed in the calculations.
We have studied the Fe protein (Av2) of the Azotobacter vinelandii nitrogenase system with Mössbauer and EPR spectroscopies and magnetic susceptometry. In the oxidized state the protein exhibits Mössbauer spectra typical of diamagnetic [4Fe-4S]2+ clusters. Addition of Mg.ATP or Mg.ADP causes a pronounced decline in the quadrupole splitting of the Mössbauer spectra of the oxidized protein. Our studies show that reduced Av2 in the native state is heterogeneous. Approximately half of the molecules contain a [4Fe-4S]1+ cluster with electronic spin S = 1/2 and half contain a [4Fe-4S]1+ cluster with spin S = 3/2. The former yields the characteristic g = 1.94 EPR signal whereas the latter exhibits signals around g = 5. The magnetization of reduced Av2 is dominated by the spin S = 3/2 form of its [4Fe-4S]1+ clusters. These results explain a long standing puzzle, namely why the integrated spin intensity of the g = 1.94 EPR signal is substantially less than 1 spin/4 Fe atoms. In 50% ethylene glycol, 90% of the clusters are in the spin S = 1/2 form whereas, in 0.4 M urea, 85% are in the S = 3/2 form. In 0.4 M urea, the EPR spectrum of reduced Av2 exhibits well defined resonances at g = 5.8 and 5.15, which we assign to the S = 3/2 system. The EPR and Mössbauer studies yield a zero-field splitting of 2D approximately equal to -5 cm-1 for this S = 3/2 state.
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.
Extraction conditions have been found which result in the retention of managanese to the 33–34 kDa protein, first isolated as an apoprotein by Kuwabara and Murata (Kuwabara, T. and Murata, N. (1979) Biochim. Biophys Acta 581, 228–236). By maintaining an oxidizing-solution potential, with hydrophilic and lipophilic redox buffers during protein extraction of spinach grana-thylakoid membranes, the 33–34 kDa protein is observed to bind a maximum of 2 Mn/protein which are not released by extended dialysis versus buffer. This manganese is a part of the pool of 4 Mn/Photosystem II normally associated with the oxygen-evolving complex. The mechanism for retention of Mn to the protein during isolation appears to be by suppression of chemical reduction of natively bound, high-valent Mn to the labile Mn(II) oxidation state. This protein is also present in stoichiometric levels in highly active, O2-evolving, detergent-extracted PS-II particles which contain 4–5 Mn/PS II. Conditions which result in the loss of Mn and O2 evolution activity from functional membranes, such as incubation in 1.5 mM NH2OH or in ascorbate plus dithionite, also release Mn from the protein. The protein exists as a monomer of 33 kDa by gel filtration and 34 kDa by gel electrophoresis, with an isoelectric point of 5.1 ± 0.1. The protein exhibits an EPR spectrum only below 12 K which extends over at least 2000 G centered at g = 2 consisting of non-uniformly separated hyperfine transitions with average splitting of 45–55 G. The magnitude of this splitting is nominally one-half the splitting observed in monomeric manganese complexes having O or N donor ligands. This is apparently due to electronic coupling of the two 55Mn nuclei in a presumed binuclear site. Either a ferromagnetically coupled binuclear Mn2(III,III) site or an antiferromagnetically coupled mixed-valence Mn2(II,III) site are considered as possible oxidation states to account for the EPR spectrum. Qualitatively similar hyperfine structure splittings are observed in ferromagnetically coupled binuclear Mn complexes having even-spin ground states. The extreme temperature dependence suggests the population of low-lying excited spin states such as are present in weakly coupled dimers and higher clusters of Mn ions, or, possibly, from efficient spin relaxation such as occurs in the Mn(III) oxidation state. Either 1.5 mM NH2OH or incubation with reducing agents abolishes the low temperature EPR signal and releases two Mn(II) ions to solution. This is consistent with the presence of Mn(III) in the isolated protein. The intrinsically unstable Mn2(II,III) oxidation state observed in model compounds favors the assignment of the stable protein oxidation state to the Mn2(III,III) formulation. This protein exhibits characteristics consistent with an identification with the long-sought Mn site for photosynthetic O2 evolution. An EPR spectrum having qualitatively similar features is observable in dark-adapted intact, photosynthetic membranes (Dismukes, G.C., Abramowicz, D.A., Ferris, F.K., Mathur, P., Upadrashta, B. and Watnick, P. (1983) in The Oxygen-Evolving System of Plant Photosynthesis (Inoue, Y., ed.), pp. 145–158, Academic Press, Tokyo) and in detergent-extracted, O2-evolving Photosystem-II particles (Abramowicz, D.A., Raab, T.K. and Dismukes, G.C. (1984) Proceedings of the Sixth International Congress on Photosynthesis (Sybesma, C., ed.), Vol. I, pp. 349–354, Martinus Nijhoff/Dr. W. Junk Publishers, The Hague, The Netherlands), thus establishing a direct link with the O2 evolving complex.
Redox titrations of the photo-induced pheophytin EPR signal in Photosystem II show two transitions which reflect the redox state of Q. The high potential wave (Em ⋍ −50 mV) can be photo-induced at 5 K and 77 K. The low potential wave (Em ⋍ −275 mV) required illumination at 200 K. This indicates the presence of two kinds of PS-II reaction centres differing in terms of the competence of their donors at low temperature and the Em-values of their acceptors. Measurements of the semiquinone-iron acceptor also demonstrate functional heterogeneity at low temperature. This is the first observation of the semiquinone-iron acceptor in a non-mutant species.
Divalent salt-washing of O2-evolving PS II particles caused total liberation of 33-, 24- and 16-kDa proteins, but the resulting PS II particles retained almost all amounts of Mn present in initial particles. The retained Mn was EPR-silent when the particles were kept in high concentrations of divalent salt. By divalent salt-washing, the activity of diphenylcarbazide (DPC) photooxidation was not affected at all, neither suppressed nor enhanced, while O2 evolution was totally inactivated. These results indicate that Mn can be kept associated with PS II particles even after liberation of the 33-kDa protein, and suggest that the 33-kDa protein is probably not responsible for binding Mn onto membranes, but is possibly responsible for maintaining the function of Mn atoms in the O2-evolving center.
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.
We have investigated the effects of temperature on the formation and decay of the light-induced multiline EPR signal species associated with photosynthetic oxygen evolution (Dismukes, G.C. and Siderer, Y. (1980) FEBS Lett. 121, 78–80). (1) The decay rate following illumination is temperature dependent: at 295 K the half-time of decay is about 40 s, at 253 K the half-time is approx. 40 min. (2) A single intense flash of light becomes progressively less effective in generating the multiline signal below about 240 K. (3) Continuous illumination is capable of generating the signal down to almost 160 K. (4) Continuous illumination after a preilluminating flash generates less signal above 200 K than at lower temperatures. Our results support the conclusion of Dismukes and Siderer that the S2 state gives rise to this multiline signal; we find that the S1 state can be fully advanced to the S2 state at temperatures as low as 160 K. The S2 state is capable of further advancement at temperatures above about 210 K, but not below that temperature.
— Using isolated chloroplasts and techniques as described by Joliot and Joliot[6] we studied the evolution of O2 in weak light and light flashes to analyze the interactions between light induced O2 precursors and their decay in darkness. The following observations and conclusions are reported: 1. Light flashes always produce the same number of oxidizing equivalents either as precursor or as O2. 2. The number of unstable precursor equivalents present during steady state photosynthesis is ∼ 1.2 per photochemical trapping center. 3. The cooperation of the four photochemically formed oxidizing equivalents occurs essentially in the individual reaction centers and the final O2 evolution step is a one quantum process. 4. The data are compatible with a linear four step mechanism in which a trapping center, or an associated catalyst, (S) successively accumulates four + charges. The S4+ state produces O2 and returns to the ground state S0. 5. Besides S0 also the first oxidized state S+ is stable in the dark, the two higher states, S2+ and S3+ are not. 6. The relaxation times of some of the photooxidation steps were estimated. The fastest reaction, presumably S*1←S2, has a (first) half time ≤ 200 μsec. The S*2 state and probably also the S*0 state are processed somewhat more slowly (˜ 300–400 μsec).
Freezing of spinach or barley chloroplasts during continuous illumination results in the trapping of a paramagnetic state or a mixture of such states characterized by a multiline EPR spectrum. Added Photosystem II electron acceptor enhances the signal intensity considerably. Treatments which abolish the ability of the chloroplasts to evolve oxygen, by extraction of the bound manganese, prevent the formation of the paramagnetic species. Restoration of Photosystem II electron transport in inhibited chloroplasts with an artificial electron donor (1,5-diphenylcarbazide) does not restore the multiline EPR spectrum. The presence of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) results in a modified signal which may represent a second paramagnetic state. The paramagnetic forms appear to originate on the donor side in Photosystem II and are dependent on a functional oxygenevolving site and bound, intact manganese. It is suggested that magnetically interacting manganese ions in the oxygen-evolving site may be responsible for the EPR signals. This suggestion is supported by calculations.
Treatment of Photosystem II particles from spinach chloroplasts with Triton X-100 with 2.6 M urea in the presence of 200 mM NaCl removed 3 polypeptides of 33 kDa, 24 kDa and 18 kDa, but left Mn bound to the particles. The (urea + NaCl)-treated particles could evolve oxygen in 200 mM, but not in 10 mM NaCl. Mn was gradually released with concomitant loss of oxygen-evolution activity in 10 mM NaCl but not in 200 mM Cl−. The NaCl-treated particles, which contained Mn and the 33-kDa polypeptide but not the 24-kDa and 18-kDa polypeptides, did not lose Mn or oxygen-evolution activity in 10 mM NaCl. These observations suggest that the 33-kDa polypeptide maintains the binding of Mn to the oxygen-evolution system and can be functionally replaced by 200 mM Cl−.
We report the continuous power saturation and temperature dependence of three EPR signals which are generated by low-temperature illumination of dark-adapted Photosystem II (PSII) membranes and are associated with the S2 state of the O2-evolving complex of photosynthesis. PSII membranes which are dark-adapted for 4 h at 0°C and illuminated at 200 K for 2 min exhibit a S2 state EPR signal which saturates easily (P1/2 = 3.7 mW at 6.0 K) and has an intensity maximum at 6.9 K under nonsaturating conditions. The S2 state EPR signal obtained from 6-min dark-incubated samples illuminated at 160 K exhibits no intensity maximum in the 4.0-16.0 K range under nonsaturating conditions and saturates at higher microwave powers (P1/2 = 37.1 mW at 6.0 K). Finally, a third signal produced by 170 K illumination of 6-min dark-adapted membranes shows an intensity maximum at 5.9 K under nonsaturating conditions and is not saturated with our current experimental setup (P1/2 > 156 mW at 6.0 K). We conclude that each EPR spectrum originates from a thermally excited state of one of three distinct configurations of the manganese complex which is believed to make up the active site. The temperature dependence data are fitted to a model in which two paramagnetic sites are ferromagnetically exchange coupled. All three sets of data can be accounted for by varying the magnitude of the superexchange coupling constant.
An Mn-containing 33-kDa protein was isolated by phase partitioning with 50% n-butanol from an O2-evolving photosystem-II preparation. The Mn content in the 33-kDa protein increased when 1 mM potassium ferricyanide and 0.4 mM diaminodurene were present as oxidants during the butanol treatment and the following dialysis. Under these conditions, 0.1–0.25 atom Mn was detected in one 33-kDa protein molecule. EPR spectra of the Mn protein showed that the Mn atoms were bound to the protein.
Deoxycholate was used to solubilize the 16 and 24 kDa polypeptides from spinach thylakoids, resulting in the loss of oxygen evolution. Manganese was retained in the membrane. When the deoxycholate-extracted membranes were subjected to a mild heat treatment, the water-soluble 33 kDa protein was selectively released. Less than one manganese per reaction center was lost on heating but this loss was not correlated to the solubilization of protein. Most of the manganese bound to the membrane remained EPR-undetectable and could be released by 2-amino-2-hydroxymethylpropane-1,3-diol (Tris) or hydroxylamine treatments. This indicates that the manganese involved in oxygen evolution remains in its native binding site despite the loss of the 33 kDa protein. These results contradict the hypothesis that the 33 kDa protein is responsible for manganese binding at the photosynthetic oxygen-evolving site.
The light-induced EPR multiline signal is studied in O2-evolving PS II membranes. The following results are reported: (1) Its amplitude is shown to oscillate with a period of 4, with respect to the number of flashes given at room temperature (maxima on the first and fifth flashes). (2) Glycerol enhances the signal intensity. This effect is shown to come from changes in relaxation properties rather than an increase in spin concentration. (3) Deactivation experiments clearly indicate an association with the S2 state of the water-oxidizing enzyme. A signal at g = 4.1 with a linewidth of 360 G is also reported and it is suggested that this arises from an intermediate donor between the S states and the reaction centre. This suggestion is based on the following observations: (1) The g = 4.1 signal is formed by illumination at 200 K and not by flash excitation at room temperature, suggesting that it arises from an intermediate unstable under physiological conditions. (2) The formation of the g = 4.1 signal at 200 K does not occur in the presence of DCMU, indicating that more than one turnover is required for its maximum formation. (3) The g = 4.1 signal decreases in the dark at 220 K probably by recombination with Q−AFe. This recombination occurs before the multiline signal decreases, indicating that the g = 4.1 species is less stable than S2. (4) At short times, the decay of the g = 4.1 signal corresponds with a slight increase in the multiline S2 signal, suggesting that the loss of the g = 4.1 signal results in the disappearance of a magnetic interaction which diminishes the multiline signal intensity. (5) Tris-washed PS II membranes illuminated at 200 K do not exhibit the signal.
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).
EPR signals arising from components in oriented multilayers of Photosystem II (PS II) membranes have been studied and the following results have been obtained. (1) The EPR signals arising from the primary semiquinone-iron complex (Q−AFe) were highly oriented, with features at g = 1.90, g = 1.82 and g = 1.66 showing maxima when the membranes were perpendicular to the magnetic field. (2) The EPR signal, arising from the reduced pheophytin acceptor interacting with Q−AFe, showed an orientation-dependent splitting, ranging from 39 G when the membranes were parallel to the magnetic field to 27 G when the membranes were perpendicular to the magnetic field. (3) The S2 multiline signal associated with the O2-evolving enzyme showed an orientation dependence. This was most marked as position shifts in the low-field wings of the spectrum. These effects indicate that the component is oriented within the membrane and has some magnetically anisotropic character. (4) The component at g ⋍ 4, though to be due to an oxidized charge carrier close to S2, showed a slight orientation dependence in its amplitude, but a significant orientation-dependent field-position shift was present, indicating that this is a magnetically anisotropic centre with a fixed geometry in the membrane. (5) Cytochrome b-559 in its oxidized form showed large highly oriented signals. The gz 2.97 feature was maximum when the membranes were oriented parallel to the magnetic field, while the gy 2.22 was maximum when the membrane plane was perpendicular to the magnetic field. This indicates that the haem plane is perpendicular to the plane of the membrane, in agreement with previous reports using chloroplasts. Ageing of the sample brings about a change from low- to high-spin state accompanied by a change in orientation of the haem relative to the membrane (from perpendicular to approximately 45°). (6) Signal II slow, which is present in the dark and which arises from a component which acts as an electron donor in PS II, is highly oriented. The signal becomes resolved into two different symmetrical four-line spectra. When the membranes were parallel to the magnetic field a narrow signal centred at g ≈ 2.0032 was present, while when the membranes were perpendicular a wider signal centred at g ≈ 2.0061 was present. The g-shift may be taken as an indication that the semiquinone ring is perpendicular to the membrane. (7) The spin-polarized triplet state of P-680, the primary donor chlorophyll, can be photoinduced in oriented PS II multilayers. The Z transition was maximum when the membranes were oriented perpendicular to the magnetic field, while the X and Y transitions were maximum when the membranes were parallel to the magnetic field. This indicates that the plane of the chorophyll ring is parallel to the plane of the membrane.
The line-shape of the EPR signal around g 6 of yellow lipoxygenase-1, obtained upon addition of I molar equivalent of 13-,11-trans-octadecadienoic acid to the native enzyme (linoleate:oxygen oxidoreductase, EC 1.13.11.12), is strongly affected by alcohols. NMR spectra of solutions of alcohols to which lipoxygenase has been added show a line-broadening of the proton resonances which is due to proton relaxation enhancement from magnetic interaction between iron and protons. This can be taken as direct evidence for binding of alcohols in the vicinity of iron. For unbranched alcohols the line-broadening gradually increases, going from the methyl protons to the protons on carbon atom 1, indicating that the latter are closer to iron. Titrations of yellow lipoxygenase with ethanol, 1-butanol and 1-hexanol reveal that the affinity of the alcohols increases with longer carbon chain length; their binding constants were found to be 260, 30 and approx. 3 mM respectively. The distances between protons of bound alcohol and iron were calculated with the Solomon-Bloembergen equation, leading to values of approx. 6 Å, for the distance between iron and the methyl protons. A hydrophobic binding of the alcohols to the enzyme is proposed in line with the mode of binding of the natural substrates, polyunsaturated fatty acids.
The yellow form of soybean lipoxygenase-1 (linoleate:oxygen oxidoreductase, EC 1.13.11.12), obtained upon addition of one molar equivalent of acid (13--HPOD) to the native enzyme, shows a complex EPR signal around g 6 which results from contributions of different high-spin Fe(III) species with rhombic or axial symmetry. The signal cannot be attributed to different enzyme-product complexes because removal of the products or variation of the concentration of the products in the enzyme solution does not lead to an EPR spectrum characteristic for one particular species. Upon varying the pH of the enzyme solution from 7 to 11 changes in the line-shape are observed, but no distinct spectrum of either a rhombic or an axial form could be observed. The relative amounts of the different species visible in the signal around g 6 are strongly affected by cyanide, primary alcohols or 13--hydroxyoctadecadienoic acid (reduced 13-- HPOD). The presence of either of these substances causes a shift to an axial type of spectrum, t-Butanol and sodium dodecyl sulfate induce a shift towards a more rhombic line-shape. A shift to an axial type of spectrum is observed after storage of the enzyme in the yellow form at 4°C. Storage of the native form at 4°C also leads to changes which become apparent after oxidation in a similar axial type of spectrum. Reversion to the original, more rhombic, spectrum is possible by ammonium sulfate precipitation. It is concluded that the species giving rise to the EPR signal around g 6 are enzyme species differing only in the structure of the environment of the iron atom.
The magnetic state of iron in haem proteins has long been recognized as a convenient indicator of chemical coordination as well as more subtle biochemical properties. In the trivalent case in particular, there are three magnetically distinct configurations of the five 3 d electrons, yielding spin states of S = 1/2, 3/2 and 5/2. The first and third are extremely familiar, and often lie close enough in energy so that a thermal mixture of low-spin S = 1/2 and high-spin S = 5/2 states exists in an ensemble of molecules. Selection rules for common perturbations (Δ S = ∘, ± 1 for the spin-orbit interaction and Δ S = ∘ for the electronic Zeeman interaction) ensure that quantum mixtures, in which the wave function is a true combination of S = 1/2 and S = 5/2 components, are not observed. The mid-spin, S = 3/2, state, and allowed 5/2–3/2 and 1/2–3/2 quantum mixtures including it, are much less well known. These have only rarely been invoked in recent years as an explanation for experimental haem protein magnetic data.
During dark adaptation, a change in the O2-evolving complex (OEC) of spinach photosystem II (PSII) occurs that affects both the structure of the Mn site and the chemical properties of the OEC, as determined from low-temperature electron paramagnetic resonance (EPR) spectroscopy and O2 measurements. The S2-state multiline EPR signal, arising from a Mn-containing species in the OEC, exhibits different properties in long-term (4 h at 0 degrees C) and short-term (6 min at 0 degree C) dark-adapted PSII membranes or thylakoids. The optimal temperature for producing this EPR signal in long-term dark-adapted samples is 200 K compared to 170 K for short-term dark-adapted samples. However, in short-term dark-adapted samples, illumination at 170 K produces an EPR signal with a different hyperfine structure and a wider field range than does illumination at 160 K or below. In contrast, the line shape of the S2-state EPR signal produced in long-term dark-adapted samples is independent of the illumination temperature. The EPR-detected change in the Mn site of the OEC that occurs during dark adaptation is correlated with a change in O2 consumption activity of PSII or thylakoid membranes. PSII membranes and thylakoid membranes slowly consume O2 following illumination, but only when a functional OEC and excess reductant are present. We assign this slow consumption of O2 to a catalytic reduction of O2 by the OEC in the dark. The rate of O2 consumption decreases during dark adaptation; long-term dark-adapted PSII or thylakoid membranes do not consume O2 despite the presence of excess reductant.(ABSTRACT TRUNCATED AT 250 WORDS)
In addition to their g = 1.94 EPR signal, nitrogenase Fe-proteins from Azotobacter vinelandii, Azotobacter chroococcum and Klebsiella pneumoniae exhibit a weak EPR signal with g approximately equal to 5. Temperature dependence of the signal was consistent with an S = 3/2 system with negative zero-field splitting, D = -5 +/- 0.7 cm-1. The ms = +/- 3/2 ground state doublet gives rise to a transition with geff = 5.90 and the transition within the excited ms = +/- 1/2 doublet has a split geff = 4.8, 3.4. Quantitation gave 0.6 to 0.8 spin . mol-1 which summed with the spin intensity of the S = 1/2 g = 1.94 line to roughly 1 spin/mol. MgATP and MgADP decreased the intensity of the S = 3/2 signal with no concomitant changes in intensity of the S = 1/2 signal.
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)
Cytochrome c′, a heme protein isolated from photosynthetic and denitrifying bacteria, has previously been shown to have unusual chemical and physical properties. EPR and magnetic susceptibility measurements reported here indicate that in the pH range 1–11, the oxidized form of the protein can exist in four magnetically distinguishable states. Reversible transitions between these states can be induced by changing the pH of the protein solution. The two protein states which exist at physiological pH have magnetic properties unlike any other known heme protein. We show that these unique magnetic properties can best be explained by postulating iron electronic states which are quantum mechanical admixtures of an intermediate spin state and a high spin state. The suggestion, previously made, that the unusual magnetic properties of the protein are due to a thermal mixture of high and low spin states, is shown to be inconsistent with the magnetic data. The protein states at pH 1 and 11, though slightly dissimilar in symmetry at the iron site, are both typical ferric high spin states, quite similar in magnetic properties to the acid forms of metmyoglobin and methemoglobin. In the pH range 1–11 small anions are unable to bind to the iron site, indicating the presence of a strong hydrophobic region in the vicinity of the heme. Possible ligand-iron configurations corresponding to the four different protein states are discussed.