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Multifrequency EPR investigations into the origin of the S2-state signal at g = 4 of the O2-evolving complex
Abstract
The low-temperature S2-state EPR signal at g = 4 from the oxygen-evolving complex (OEC) of spinach Photosystem-II-enriched membranes is examined at three frequencies, 4 GHz (S-band), 9 GHz (X-band) and 16 GHz (P-band). While no hyperfine structure is observed at 4 GHz, the signal shows little narrowing and may mask underlying hyperfine structure. At 16 GHz, the signal shows g-anisotropy and a shift in g-components. The middle Kramers doublet of a near rhombic S = 5/2 system is found to be the only possible origin consistent with the frequency dependence of the signal. Computer simulations incorporating underlying hyperfine structure from an Mn monomer or dimer are employed to characterize the system. The low zero field splitting (ZFS) of D = 0.43 cm-1 and near rhombicity of E/D = 0.25 lead to the observed X-band g value of 4.1. Treatment with F- or NH3, which compete with Cl- for a binding site, increases the ZFS and rhombicity of the signal. These results indicate that the origin of the OEC signal at g = 4 is either an Mn(II) monomer or a coupled Mn multimer. The likelihood of a multimer is favored over that of a monomer.
... [50][51][52][53][54] However, an additional EPR signal centered at g ~ 4.1 has also been observed under a variety of sample preparation conditions. 40,[55][56][57][58][59][60] It has been suggested that the g ~ 4.1 EPR signal corresponds to a high spin (electron spin S = 5/2) state of the OEC 40,57,58 and originates from either slight changes in the spin coupling scheme of the Mn 4 Ca-oxo cluster 40,61 or a redistribution of valence states between the manganese ions within the cluster. 56 Interestingly, the two forms of the S 2 state are interconvertible by near-infrared irradiation at cryogenic temperatures. ...
... [50][51][52][53][54] However, an additional EPR signal centered at g ~ 4.1 has also been observed under a variety of sample preparation conditions. 40,[55][56][57][58][59][60] It has been suggested that the g ~ 4.1 EPR signal corresponds to a high spin (electron spin S = 5/2) state of the OEC 40,57,58 and originates from either slight changes in the spin coupling scheme of the Mn 4 Ca-oxo cluster 40,61 or a redistribution of valence states between the manganese ions within the cluster. 56 Interestingly, the two forms of the S 2 state are interconvertible by near-infrared irradiation at cryogenic temperatures. ...
The solar water-splitting protein complex, photosystem II (PSII), catalyzes one of the most energetically demanding reactions in Nature by using light energy to drive a catalyst capable of oxidizing water. The water oxidation reaction takes place at the tetra-nuclear manganese calcium-oxo (Mn4Ca-oxo) cluster at the heart of the oxygen-evolving complex (OEC) of PSII. Previous studies have determined the magnetic interactions between the paramagnetic Mn4Ca-oxo cluster and its environment in the S2 state of the OEC. The assignments for the electron-nuclear magnetic interactions that were observed in these studies were facilitated by the use of synthetic dimanganese di-μ-oxo complexes. However, there is an immense need to understand the effects of the protein environment on the coordination geometry of the Mn4Ca-oxo cluster in the OEC of PSII. In the present study, we use a proteinaceous model system to examine the protein ligands that are coordinated to the dimanganese catalytic center of manganese catalase from Lactobacillus plantarum. We utilize two-dimensional hyperfine sublevel correlation (2D HYSCORE) spectroscopy to detect the weak magnetic interactions of the paramagnetic dinuclear manganese catalytic center of superoxidized manganese catalase with the nitrogen and proton atoms of the surrounding protein environment. We obtain a complete set of hyperfine interaction parameters for the protons of a water molecule that is directly coordinated to the dinuclear manganese center. We also obtain a complete set of hyperfine and quadrupolar interaction parameters for two histidine ligands as well as a coordinated azide ligand, in azide-treated superoxidized manganese catalase. On the basis of the values of the hyperfine interaction parameters of the dimanganese model, manganese catalase, and those of the S2 state of the OEC of PSII, for the first time, we discuss the impact of a proteinaceous environment on the coordination geometry of multinuclear manganese clusters.
... It is likely that some NIR was present in the illuminations originally used by Casey et al. The 'ground' state g4.1 signal has been proposed to arise from a rhombic spin 5 2 center [31] or near axial spin 3 2 center [32]. A strong focus in this paper has been on the 'ground' state g4.1 signal, whether it is a rhombic 5 2 spin state signal or an axial 3 2 spin state signal. ...
The S2 state produces two basic electron paramagnetic resonance signal types due to the manganese cluster in oxygen-evolving complex, which are influenced by the solvents, and cryoprotectant added to the photosystem II samples. It is presumed that a single manganese center oxidation occurs on S1 → S2 state transition. The S2 state has readily visible multiline and g4.1 electron paramagnetic resonance signals and hence it has been the most studied of all the Kok cycle intermediates due to the ease of experimental preparation and stability. The S2 state was studied using electron paramagnetic resonance spectroscopy at X-band frequencies. The aim of this study was to determine the spin states of the g4.1 signal. The multiline signal was observed to arise from a ground state spin ½ centre while the g4.1 signal generated at ≈140 K NIR illumination was proposed to arise from a spin 52 center with rhombic distortion. The 'ground' state g4.1 signal was generated solely or by conversion from the multiline. The data analysis methods used involved numerical simulations of the experimental spectra on relevant models of the oxygen-evolving complex cluster. A strong focus in this paper was on the 'ground' state g4.1 signal, whether it is a rhombic 52 spin state signal or an axial 32 spin state signal. The data supported an X-band CW-EPR-generated g4.1 signal as originating from a near rhombic spin 5/2 of the S2 state of the PSII manganese cluster.
... (1) The presence of MeOH (or other small, primary alcohols) shifts the equilibrium between the low-spin (S = 1/2) and high-spin (S = 5/2) conformations of S 2 strongly to the low-spin conformation. This equilibrium is also affected by the inclusion of a number of other chemical additives to the sample buffer, some favoring the low-spin conformation (50% glycerol, 30% polyethylene glycol) with others favoring the high-spin conformation (sucrose, 49,98 certain amines, 99 F − , 100 and other inhibitors of Cl − binding [101][102][103] ). These two signals represent distinct ground states, as opposed to sublevels of the same electron spin manifold, and they can be interconverted by near-infrared illumination at cryogenic temperatures. ...
The binding of the substrate analogue methanol to the catalytic Mn4CaO5 cluster of the water-oxidizing enzyme photosystem II is known to alter the electronic structure properties of the oxygen-evolving complex without retarding O2-evolution under steady-state illumination conditions. We report the binding mode of 13C-labeled methanol determined using 9.4 GHz (X-band) hyperfine sublevel-correlation (HYSCORE) and 34 GHz (Q-band) electron spin-echo electron nuclear double resonance (ESE-ENDOR) spectroscopies. These results are compared to analogous experiments on a mixed-valence Mn(III)Mn(IV) complex (2-OH-3,5-Cl2-salpn)2Mn(III)Mn(IV) (salpn = N,N'-bis(3,5-dichlorosalicylidene)-1,3-diamino-2-hydroxypropane) in which methanol ligates to the Mn(III) ion (Larson, et al. 1992 J. Am. Chem. Soc. 114:6263).1 In the mixed-valence Mn(III,IV) complex, the hyperfine coupling to the 13C of the bound methanol (Aiso = 0.65 MHz, T = 1.25 MHz) is appreciably larger than that observed for 13C methanol associated with the Mn4CaO5 cluster poised in the S2 state, where only a weak dipolar hyperfine interaction (Aiso = 0.05 MHz, T = 0.27 MHz) is observed. An evaluation of the 13C hyperfine interaction using the x-ray structure coordinates of the Mn4CaO5 cluster indicates that methanol does not bind as a terminal ligand to any of the manganese ions in the OEC. We favor methanol binding in place of a water ligand to the Ca2+ in the Mn4CaO5 cluster or in place of one of the waters that form hydrogen bonds with the oxygen bridges of the cluster.
The understanding of light‐induced biological water oxidation in oxygenic photosynthesis is of great importance both for biology and (bio)technological applications. The chemically difficult multistep reaction takes place at a unique protein‐bound tetra‐manganese/calcium cluster in photosystem II whose structure has been elucidated by X‐ray crystallography (Umena et al. Nature, 2011). The cluster moves through several intermediate states in the catalytic cycle. A detailed understanding of these intermediates requires information about the spatial and electronic structure of the Mn4Ca complex; the latter is only available from spectroscopic techniques. Here the important role of Electron Paramagnetic Resonance (EPR) and related double resonance techniques (ENDOR, EDNMR), complemented by quantum chemical calculations, is described. This has led to the elucidation of the cluster´s redox and protonation states, the valence and spin states of the manganese ions and the interactions between them, and contributed substantially to the understanding of the role of the protein surrounding, as well as the binding and processing of the substrate water molecules, the O‐O bond formation and dioxygen release. Based on these data models for the water oxidation cycle are developed. Light‐induced water oxidation and dioxygen release in photosynthesis is catalyzed by a paramagnetic μ‐oxo‐bridged Mn4Ca cofactor. It passes through five metastable states (S0 ‐ S4) whose structure is described focusing on the essential electronic structure obtained from spectroscopy, especially EPR techniques, supported by quantum chemistry. A catalytic cycle is presented which describe structural isomers of key S‐state intermediates facilitating substrate binding and cofactor activation.
The aim of this review is to elucidate geometric structures of the catalytic CaMn4Ox (x = 5, 6) cluster in the Kok cycle for water oxidation in the oxygen evolving complex (OEC) of photosystem II (PSII) based on the high-resolution (HR) X-ray diffraction (XRD) and serial femtosecond crystallography (SFX) experiments using the X-ray free-electron laser (XFEL). Quantum mechanics (QM) and QM/molecular mechanics (MM) computations are performed to elucidate the electronic and spin structures of the CaMn4Ox (x = 5, 6) cluster in five states Si (i = 0 ∼ 4) on the basis of the X-ray spectroscopy, electron paramagnetic resonance (EPR) and related experiments. Interplay between the experiments and theoretical computations has been effective to elucidate the coordination structures of the CaMn4Ox (x = 5, 6) cluster ligated by amino acid residues of the protein matrix of PSII, valence states of the four Mn ions and total spin states by their exchange-couplings, and proton-shifted isomers of the CaMn4Ox (x = 5, 6) cluster. The HR XRD and SFX XFEL experiments have also elucidated the biomolecular systems structure of OEC of PSII and the hydrogen bonding networks consisting of water molecules, chloride anions, etc., for water inlet and proton release pathways in PSII. Large-scale QM/MM computations have been performed for elucidation of the hydrogen bonding distances and angles by adding invisible hydrogen atoms to the HR XRD structure. Full geometry optimizations by the QM and QM/MM methods have been effective for elucidation of the molecular systems structure around the CaMn4Ox (x = 5, 6) cluster in OEC. DLPNO-CCSD(T0) method has been applied to elucidate relative energies of possible intermediates in each state of the Kok cycle for water oxidation. Implications of these results are discussed in relation to the blueprint for developments of artificial catalysts for water oxidation.
DLPNO-CCSD(T) calculations coupled with the exact diagonalization of spin Hamiltonian matrix elucidated two right (R)-opened structures with the low spin (LS) (S = 1/2, g = 2) and intermediate spin (IS) (S = 5/2, g > 4) state and two left (L)-opened structures with the IS (S = 5/2, g = 4; S = 7/2, g > 4) and high spin (HS) (S = 13/2) state. Multiple intermediates in the S2 state revealed by DLPNO-CCSD(T) are compatible with the complex EPR, EXAFS, and XFEL results for the S2 state, indicating proton transfer coupled spin transitions of the CaMn4Ox cluster in OEC of PSII.
We derive a model that provides an exact solution to the substrate-water exchange kinetics in a double-conformation system and use this model to interpret recently published data for Ca²⁺- and Sr²⁺-containing PSII in the S2 state, in which the g = 2.0 and g = 4.1 conformations coexist. The component concentrations derived from the kinetic model provide an analytic description of the substrate-water exchange kinetics, allowing us to more accurately interpret the results. Based on this model and the previously reported data on the S2 state g = 2.0 conformation, we obtain the substrate-water exchange rates of the g = 4.1 conformation and the conformational change rates. Two conclusions are made from the analyses. First, contrary to previous reports, there is no significant effect of substituting Sr²⁺ for Ca²⁺ on any of the exchange rate constants. Second, the exchange rate of the slowly-exchanging water (Ws) in the S2 state g = 4.1 conformation is much faster than that in the S2 state g = 2.0 conformation. The second conclusion is consistent with the assignment of Ws to W1 or W2 bound as terminal ligands to Mn4; Mn4 has been proposed to undergo an oxidation state change from Mn(IV) in the g = 2.0 conformation to Mn(III) in the g = 4.1 conformation.
World demand for energy is rapidly increasing and finding sufficient supplies of clean energy for the future is one of the major scientific challenges of today. This book presents the latest knowledge and chemical prospects in developing hydrogen as a solar fuel. Using oxygenic photosynthesis and hydrogenase enzymes for bio-inspiration, it explores strategies for developing photocatalysts to produce a molecular solar fuel. The book begins with perspective of solar energy utilization and the role that synthetic photocatalysts can play in producing solar fuels. It then summarizes current knowledge with respect to light capture, photochemical conversion, and energy storage in chemical bonds. Following chapters on the natural systems, the book then summarizes the latest developments in synthetic chemistry of photo- and reductive catalysts. Finally, important future research goals for the practical utilization of solar energy are discussed. The book is written by experts from various fields working on the biological and synthetic chemical side of molecular solar fuels to facilitate advancement in this area of research.
Full geometry optimization of all S2 intermediates in OEC (oxygen-evolving complex) of PSII (photosystem II) were carried out by using UB3LYP-D3/Def2-TZVP with COSMO solvation effects. Our detail calculations yielded meta-stable structures of six (=3 × 2 (HS (high-spin), IS (intermediate-spin)) intermediates for W1 = W2 = H2O and eight (=4 × 2 (HS, IS)) intermediates for W1 = OH⁻ or W2 = OH⁻, and were named to H2O and OH models, respectively. In the next step, relative stability among these intermediate structures were investigated by hybrid-DFT and DLPNO-CC methods. UB3LYP methods show that right (R)-opened structures (open-cubane) are more stable than left (L)-opened structures (closed-cubane) by about 3.5 kcal/mol, though decreasing of DFT-weights suppresses such energy gaps. DLPNO-CCSD(T0) methods promote stabilization of (L)-structures and finally reproduce near degeneration or more stable (L)-structure. All pattern of spin configurations in four Mn(III) and Mn(IV) ions were assumed and BS (broken-symmetry) solutions were successfully obtained to find the most stable spin structures. This complete sets of all spin conformations enabled us evaluate sets of effective exchange integrals J as magnetic coupling parameters. The calculated J values for the spin Hamiltonian elucidated one g2 (S = 1/2) and two g4 (S = 5/2) molecular structures in the S2 state in accord with the recent EXAFS results.
The identity of a key intermediate in the S2 to S3 transition of Nature’s water oxidising complex (WOC) in Photosystem 2 is presented. Broken symmetry density functional theory (BS-DFT) calculations and Heisenberg, Dirac, van Vleck (HDvV) spin ladder calculations show that an S2 state open cubane model of the WOC containing a µ-hydroxo O4 changes from an S=5/2 to an S=7/2, form on deprotonation of W1. This combined with X-band electron paramagnetic resonance (EPR) spectral analysis indicates that the g=4.1 EPR signal corresponds to an S=5/2 form of the WOC with W1 present as a water ligand to Mn4, while the g=4.8/4.9 form observed at high pH values corresponds to an S=7/2 form, with W1 as an hydroxo ligand. The latter is also likely to represent the form needed to progress to S3 in the functioning enzyme.
Natural photosynthesis is the only working system of solar energy storage that operates on a global scale. The water-oxidation reaction, carried out by the protein complex photosystem II (PSII), is the key reaction that initiates photosynthesis. However, the detailed mechanism of this reaction is yet to be fully understood. In this article, we review the current knowledge of how the essential components in PSII, including the oxygen-evolving complex and its surrounding environment, take part in the water-oxidation reaction, and finally present proposals for the water-oxidation mechanism.
Domain-based local pair natural orbital (DLPNO) coupled cluster single and double (CCSD) with triple perturbation (T) correction methods were applied for fourteen different S2 structures of the CaMn4O5 cluster in oxygen evolving complex (OEC) of photosystem II (PSII). The DLPNO-CCSD(T0) calculations elucidated that the right (R)-opened S2aYZ structure (a = O²⁻ at the O(5) site, Y = W2 and Z = W1) with the low spin (LS) (S = 1/2, g = 2) state and two left (L)-opened S2aYZ structures with the high spin (HS) (S = 5/2, g = 4; g > 4) state were nearly degenerated in energy, supporting previous one LS-two HS model for the S2 state in compatible with recent EXAFS and EPR results.
A new paradigm for the high and low spin forms of the S2 state of Nature’s water oxidising complex in Photosystem 2 is found. Broken symmetry density functional theory (BS-DFT) calculations combined with Hesienberg, Dirac, Van Vleck (HDvV) spin ladder calculations show that an open cubane form of the water oxidising complex changes from a low spin (LS), S=1/2, to a high spin (HS), S=5/2, form on protonation of the bridging O4 oxo. We show that such models are fully compatible with structural determinations of the S2 state by X-ray free electron laser (XFEL) crystallography and extended X-ray absorption fine structure (EXAFS) and provide a clear rationale for the effect of various treatments on the relative populations of each form observed experimentally in electron paramagnetic resonance (EPR) studies.
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.
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.
Syntheses and magnetic functionalities of exchange-coupled magnetic systemsin a controlled fashion of molecular basis have been the focus of the current topics in chemistry and materials science; particularly extremely large spins in molecular frames and molecular high-spin clusters have attracted much attention among the diverse topics of molecule-based magnetics and high spin chemistry. Magnetic characterizations of molecule-based exchange-coupled high-spin clusters are described in terms of conventional as well as highfield/high-frequency ESR spectroscopy. Off-principal-axis extra lines as a salient feature of fine structure ESR spectroscopy in non-oriented media are emphasized in the spectral analyses. Pulse-ESR-based two-dimensional electron spin transient nutation spectroscopy applied to molecular high-spin clusters is also dealt with, briefly. Solution-phase fine-structure ESR spectroscopy is reviewed in terms of molecular magnetics. In addition to finite molecular high-spin clusters, salient features of molecule-based low-dimensional magnetic materials are dealt with. Throughout the chapter, electron spin resonance for high-spin systems is treated in a general manner in terms of theory. Hybrid eigenfield method is formulated in terms of direct products, and is described as a powerful and facile approach to the exact numerical calculation of resonance fields and transition probabilities for molecular high spin systems. Exact analytical expressions for resonance fields of high spin systems in their principal orientations are for the first time given.
This chapter provides an overview of oxygenic photosynthesis, with primary focus on the oxygen-evolving complex (OEC) of photosystem II (PSII). The introduction includes a general discussion of photosynthesis and a brief overview of the metals involved. This is followed by an in-depth description of the metal centers within PSII, in which the structural and electronic characterizations of the OEC are reviewed in detail. The role of chloride in PSII and the proton exit pathway are discussed. Several mechanisms proposed for the O. O bond formation in the OEC are presented, along with a few detailed mechanisms for the complete catalytic cycle obtained from quantum mechanics/molecular mechanics and density functional theory calculations. Model complexes that are functional mimics of the OEC are also briefly discussed. General discussion on plastocyanin, iron-sulfur centers, [FeFe] and [NiFe] hydrogenases, and ribulose-1,5-bisphosphate carboxylase-oxygenase is included. The chapter concludes with the implications of natural photosynthesis on the development of an artificial photosynthetic framework.
Magneto-structural correlations in oxygen-evolving complex (OEC) of photosystem II (PSII) have been elucidated on the basis of theoretical and computational results in combination with available electron paramagnetic resonance (EPR) experimental results, and extended x-ray absorption fine structure (EXAFS) and x-ray diffraction (XRD) results. To this end, the computational methods based on broken-symmetry (BS) UB3LYP solutions have been developed to elucidate magnetic interactions in the active manganese catalyst for water oxidation by sunlight. The effective exchange interactions J for the CaMn(III)Mn(IV)3O5(H2O)3Y(Y = H2O or OH−) cluster (1) model of OEC of PSII have been calculated by the generalised approximate spin projection (GAP) method that eliminates the spin contamination errors of the BS UB3LYP solution. Full geometry optimisations followed by the zero-point energy (ZPE) correction have been performed for all the spin configurations of 1 to improve the J values that are compared with accumulated EPR in the S2 state of Kok cycle and magnetic susceptibility results of Christou model complex Ca2Mn(IV)3O4 (2). Using the calculated J values, exact diagonalisation of the spin Hamiltonian matrix has been carried out to obtain excitation energies and spin densities of the ground and lower excited states of 1. The calculated excitation energies are consistent with the available experimental results. The calculated spin densities (projection factors) are also compatible with those of the EPR results. The calculated spin densities have been used to calculate the isotropic hyperfine (Aiso) constants of 55Mn ions revealed by the EPR experiments. Implications of the computational results are discussed in relation to the structural symmetry breaking (SSB) in the S1, S2 and S3 states, spin crossover phenomenon induced by the near-infrared excitation and the right- and left-handed scenarios for the O–O bond formation for water oxidation.
Syntheses and magnetic functionalities of exchange-coupled magnetic systems in a controlled fashion of molecular basis have been the focus of the current topics in chemistry and materials science; particularly extremely large spins in molecular frames and molecular high-spin clusters have attracted much attention among the diverse topics of molecule-based magnetics and high spin chemistry. Magnetic characterizations of molecule-based exchange-coupled high-spin clusters are described in terms of conventional as well as high-field/high-frequency ESR spectroscopy. Off-principal-axis extra lines as a salient feature of fine-structure ESR spectroscopy in non-oriented media are emphasized in the spectral analyses. Pulse-ESR-based two-dimensional electron spin transient nutation spectroscopy applied to molecular high-spin clusters is also dealt with, briefly. Solution-phase fine-structure ESR spectroscopy is reviewed in terms of molecular magnetics. In addition to finite molecular high-spin clusters, salient features of molecule-based low-dimensional magnetic materials are dealt with. Throughout the chapter, electron spin resonance for high-spin systems is treated in a general manner in terms of theory. Hybrid eigenfield method is formulated in terms of direct products, and is described as a powerful and facile approach to the exact numerical calculation of resonance fields and transition probabilities for molecular high spin systems. Exact analytical expressions for resonance fields of high spin systems in their principal orientations are for the first time given.
Water oxidation at the oxygen-evolving complex (OEC) of photosystem II (PSII) is fine tuned for turnover rates unmatched by any artificial system. Efficient proton removal from the OEC and activation of substrate water molecules are some of the key aspects optimized in the OEC for such high turnover rates. The hydrogen-bonding network around the OEC is critical for efficient proton transfer and for tuning the position and pKas of the substrate water/hydroxo/oxo molecules. The D1-N181 residue is part of the hydrogen-bonding network on the active face of the OEC. D1-N181 is also associated with the chloride ion in the D2-K317 site and is the closest residue to a putative substrate water molecule bound as a terminal ligand to Mn4. We studied the effect of the D1-N181A and the D1-N181S mutations on the water-oxidation chemistry at the OEC. PSII core complexes isolated from the D1-N181A,S mutants have lower steady-state O2-evolution rates than wild-type PSII core complexes. FTIR spectroscopy indicates slight perturbations of the hydrogen-bonding network in D1-N181A,S PSII core complexes, similar to some other mutations in the same region, but to a lesser extent. Unlike in wild-type PSII core complexes, a g = 4 signal was observed in the S2-state EPR spectra of D1-N181A,S PSII core complexes in addition to the normal g = 2 multiline signal. The S-state cycling of D1-N181A,S PSII core complexes was similar to wild-type PSII core complexes, whereas the O2-release kinetics of D1-N181A,S PSII core complexes were much slower than the O2-release kinetics of wild-type PSII core complexes. Based on these results, we conclude that proton transfer is not compromised in the D1-N181A,S mutants, but that the O-O bond formation step is retarded in these mutants.
Two Mn2(mu-O)2 units linked by another two oxo bridges make up the cluster core of 1 (see structure on the right; two bipyridine ligands have been omitted from both Mn1 and Mn4). The Mn-Mn distances and magnetic properties of 1 indicate that this cluster may be a model of the reaction center for oxygen evolution in plants. The question whether the Mn4 unit has a chainlike arrangement or a cubane structure in the naturally occurring enzyme is nevertheless still unanswered.
Full geometry optimizations followed by the vibrational analysis were performed for eight spin configurations of the CaMn4O4X(H2O)3Y (X = O, OH; Y = H2O, OH) cluster in the S1 and S3 states of the oxygen evolution complex (OEC) of photosystem II (PSII). The energy gaps among these configurations obtained by vertical, adiabatic and adiabatic plus zero-point-energy (ZPE) correction procedures have been used for computation of the effective exchange integrals (J) in the spin Hamiltonian model. The J values are calculated by the (1) analytical method and the (2) generalized approximate spin projection (AP) method that eliminates the spin contamination errors of UB3LYP solutions. Using J values derived from these methods, exact diagonalization of the spin Hamiltonian matrix was carried out, yielding excitation energies and spin densities of the ground and lower-excited states of the cluster. The obtained results for the right (R)- and left (L)-opened structures in the S1 and S3 states are found to be consistent with available optical and magnetic experimental results. Implications of the computational results are discussed in relation to (a) the necessity of the exact diagonalization for computations of reliable energy levels, (b) magneto-structural correlations in the CaMn4O5 cluster of the OEC of PSII, (c) structural symmetry breaking in the S1 and S3 states, and (d) the right- and left-handed scenarios for the O-O bond formation for water oxidation.
This review describes the progress in our understanding of the structure of the Mn complex in Photosystem II over the last two decades. Emphasis is on the research from our laboratory, especially the results from X-ray absorption spectroscopy, low temperature electron paramagnetic resonance and electron spin echo envelope modulation studies. The importance of the interplay between electron paramagnetic resonance studies and X-ray absorption studies, which has led to a description of the oxidation states of manganese as the enzyme cycles through the Kok cycle, is described. Finally, the path, by which our group has utilized these two important methods to arrive at a working structural model for the manganese complex that catalyzes the oxidation of water to dioxygen in higher plants and cyanobacteria, is explained.
Flash-induced absorption changes at 515 nm observed as a function of flash number are examined in relation to the flash-induced fluorescence yields in inside-out thylakoids. After partial dissipation of the delocalized transmembrane electric field by adding gramicidin, the analysis of period 4 oscillations and of the kinetics in the 10 ms-1 s range suggest that the variation of the absorption changes at 515 nm as a function of flash number is the result of at least two processes:1) an electric field increase related to the S2 state and 2) the fact that the field generated by the water protons inside the membrane decreases when these protons are released outside the membrane. The former field correlates with the flash-induced fluorescence yield increase induced by the donor side of Photosystem II. Both measurements show similar oscillations as a function of flash number, with maxima on the 1st, 5th and 9th flash. These oscillations, after a shift of two flashes, appear to be different from those of the O2 yield observed under similar conditions. It is proposed that, in a population of centers the electric field during the S2 state reflects the presence of a stabilized positive equivalent in the protein close to the Mn complex.
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A light-induced g = 4.1 EPR signal of the manganese cluster in the oxygen-evolving complex of plant photosystem II was investigated by an electron-spin-echo method. The dependence of the ESE signal amplitude on the intensity of the microwave magnetic field H1 was consistent with the model assuming a manganese electron spin of with EPR spectrum characteristics determined by nearly rhombic symmetry of the crystalline field.
The tetranuclear species [Mn[sup IV] [sub 4]O[sub 6] (bpy)[sub 6]] (ClO[sub 4])[sub 4][center dot]H[sub 2]O was isolated from an aqueous nitric acid solution (pH = 2) of Mn[sup III] (bpy) Cl[sub 3](H[sub 2]O) upon addition of NaClO[sub 4]. It crystallizes in the triclinic space group P[bar l] with a = 20.213(7) [angstrom], b = 13.533(7) [angstrom], c = 13.411(8) [angstrom], [alpha] = 112.01(8)[degree], [beta] = 96.72(11)[degree], [gamma] = 100.34(12)[degree], V = 3276.9 [angstrom][sup 3], and Z = 2. The cation has a nonrectilinear chain structure [(bpy)[sub 2]Mn[sup IV[sub a]]O[sub 2]Mn[sup IV[sub b]](bpy)O[sub 2]Mn[sup IV[sub c]](bpy)O[sub 2]Mn[sup IV[sub d]](bpy)[sub 2]][sup 4+]. The metal-metal distances are Mn[sub a]-Mn[sub b] = 2.746(5) [angstrom], Mn[sub b]-Mn[sub c] = 2.760(5) [angstrom], Mn[sub c]-Mn[sub d] = 2.735(4) [angstrom], Mn[sub a]-Mn[sub c] = 4.899(10) [angstrom], Mn[sub b]-Mn[sub d] = 4.897(6) [angstrom], and Mn[sub a]-Mn[sub d] = 6.419(11) [angstrom]. The variable temperature magnetic susceptibility data for [Mn[sup IV[sub 4]]O[sub 6](bpy)[sub 6]](ClO[sub 4])[sub 4][center dot]H[sub 2]O in the range 12-294 K were fit using the spin Hamiltonian H[sub S] = J[sub ab]S[sub a]S[sub b] -J[sub bc]S[sub b]S[sub c] - J[sub cd]S[sub c]S[sub d], with J[sub ab] = J[sub cd] = -176 cm[sup [minus]1] and J[sub bc] = [minus]268 cm[sup [minus]1]. 55 refs., 11 figs., 3 tabs.
Single atoms of Ca2+and Cl?are closely associated with the tetranuclear Mn cluster of Photosystem II (PS II). Extraction of either cofactor blocks advancement of the S-state cycle beyond S2. In the case of Cl?depletion, this modification has been proposed to result from replacement of the anion as a Mn ligand. This would cause a decrease of the potential, and thereby localization of the Mn(IV) state on the Mn to which Cl?was bound. The implications of this hypothesis, and of the notion that the ligand replacing Cl?in the modified S2state is most likely hydroxyl (OH?), are discussed and found to provide a plausible explanation for apparently conflicting reports in the literature. The location of Ca2+with respect to the Mn cluster is less certain. Although the metal is positioned so as to interfere with the attack of a small ligand, such as hydroxylamine (NH2OH), on the Mn cluster, the distance between Ca2+and atoms of the Mn cluster is not resolved at the present time. Calcium can be shown to reinforce the stability of Mn ligation by PS II. However, its role in water oxidation must extend beyond structural effects to account for the block in electron transfer at S2observed in Ca2+-depleted PS II, and the upward shift in the minimum temperature at which the transition from S1to S2occurs. The inactivation of PS II caused by replacement of Ca2+with lanthanides suggests that it functions as a site for binding of water molecules destined for oxidation at the Mn site in PS II.
Aus zwei Mn 2 (μ‐O) 2 ‐Einheiten , die wiederum über zwei Oxobrücken verknüpft sind, besteht der Clusterkern von 1 (Strukturbild rechts, Je zwei Bipyridin(bpy)‐Liganden an Mn1 und Mn4 weggelassen). Dessen Mn‐Mn‐Abstände und magnetische Eigenschaften deuten an, daß der Cluster ein Modell für das Reaktionszenturm der Sauerstofferzeugung in pflanzen sein könnte. Ob die Mn 4 ‐Anordnung im natürlich vorkommenden Enzym kettenförmig oder Teil einer Cubanstruktur ist, bleibt jedoch weiter offen. magnified image
The effects of a training procedure and two maintenance contingencies on consequence-dispensing behavior were investigated. Four peer behavior managers were trained to supervise small groups of subjects (four to six per group) working in programmed math materials and were compared with a teacher skilled in the use of social and point reinforcement and response cost. Manager training was differentially effective in accelerating manager's rates of appropriate social and point dispensing. Having manager reinforcement contingent upon manager consequence-dispensing resulted in moderately higher rates of appropriate social and point dispensing for three of four subjects than did having manager reinforcement contingent upon group study behavior. Two managers exposed to the group performance contingency before the manager performance contingency increased inappropriate social and point-dispensing behaviors to pretraining baseline levels. Subsequent change to the manager performance contingency was effective in reducing the inappropriate dispensing behavior of only one of the two managers.
Synthetic procedures are described that allow conversion of [MnâOâ(OAc)â(py)â(dbm)â] (1, dbmH = dibenzoylmethane) to [MnâOâX(OAc)â(dbm)â] (X = Cl, 2; X = Br, 3). Treatment of 1 with Nbuâ¿âCl in CHâClâ or hot MeCn leads to 2 in 5-8% and 35-43% yields (based on dbm), respectively. A higher yield (â¼88%) is obtained by treating 1 with 4 equiv of MeâSiCl in CHâClâ. An analogous procedure with 4 equiv of MeâSiBr in CHâBrâ gives 3 in 55% yield. Treatment of [MnâO(OAc)â(py)â](ClOâ) in MeCN with MeâSiCl followed by addition of HâO and acetic acid results in crystallization of (pyH)â[MnâOâClâ(OAc)â]·2MeCN (4) in 75% yield (based on Mn). Cyclic volammetry at 100 mV/s and differential pulse voltammetry at 5 mV/s show that both 2 and 3 support a reversible oxidation and two reductions, the first of which is reversible. The reversible processes are at 1.09/1.06 and -0.25/-0.21 V vs ferrocene and show that the [MnâOâX] core can exist at three oxidation levels spanning the 4Mn{sup III} to 2Mn{sup III}, 2Mn{sup IV} range. The combined results from 2 and 3 show that the identity of X has minimal influence on the resultant structures, magnetic properties, ¹H NMR and EPR spectral properties, or the redox behavior. Such observations are of interest with regard to the ability of Brâ» to successfully substitute for Clâ at the photosynthetic water oxidation center and thus maintain the activity of the tetranuclear Mn aggregate toward oxygen evolution.
Oxidation of water in photosynthetic organisms occurs in a chlorophyll containing enzyme, photosystem II. The catalytic manganese cluster of photosystem II cycles among five redox states called the Sn states. There are two forms of the S2 state, which give rise to different EPR signals and which differ in magnetic coupling among the manganese atoms. We have used difference infrared spectroscopy to obtain more information about the environment of the manganese cluster in these two forms of the S2 state. We present the 1600−1200 cm-1 region of the difference spectrum associated with the generation of the multiline state and the g = 4.1 S2 state. The difference spectrum associated with generation of the S2 multiline state from the dark stable S1 state shows broad spectral features in the 1500−1200 cm-1 region at 1490 (negative), 1331 (negative), 1393 (positive), and 1267 (positive) cm-1. Global 15N labeling has little impact on intensities or frequencies in this region of the spectrum. These vibrational features are not observed in manganese-depleted, EDTA-treated photosystem II. Also, these features are not observed in oxygen-evolving photosystem II upon illumination at 80 K, a temperature where the manganese cluster is not oxidized. We conclude that these lines arise from amino acid residues that are close to or ligating to the cluster. From the frequencies and the lack of sensitivity to 15N labeling, we favor the assignment of these lines to the asymmetric and symmetric stretch of one or more glutamate and/or aspartate residue(s). The spectral breadth of the lines is consistent with either an inhomogeneous or a homogeneous broadening mechanism. When illumination conditions are used that generate the g = 4.1 S2 EPR signal, these vibrational lines are not observed. These results are discussed in terms of current models for the catalytic site.
Manganese oxidation, catalyzed by chlorophyll photochemistry in photosystem II, is the key step in the pathway of redox reactions leading formation of O2 from H2O. A unique feature of Mn redox chemistry is its dependence on the presence of both Ca2+ and Cl−; the active site of H2O oxidation consists of four Mn atoms, and one atom each of Ca2+ and Cl−. The best current structural model of the inorganic ion cluster, based on polarized EXAFS experiments on crystals of the photosystem, shows Ca2+ ligated by carboxyl groups from amino acid residues (alanine, glutamate) that bridge to Mn atoms. Chloride is not resolved in the current structures. Calcium is an essential structural element of the cluster during its assembly, and is required for efficient Mn oxidation. There is also compelling evidence that Ca2+ is involved in catalysis of H2O oxidation. Chloride is also required for Mn oxidation by photosystem II, and it has been demonstrated that the anion is required for the Mn redox reactions immediately preceding oxidation of H2O to O2. Although there are no structural data on the site of Cl− binding in photosystem II, spectroscopic probing and ligand competition experiments position it in the vicinity of the Mn cluster.
The Ca2+-depleted photosystem II (PS II) was studied by pulsed EPR spectroscopy. A short (5 s) illumination of the PS II in the S′2 state resulted in formation of the doublet S′3 signal at g ≈ 2.02 with a splitting of about 15 mT concomitant with the decrease of the modified Mn multiline signal by about 40%. Further illumination up to three minutes led to the formation of another S′3 signal with a singlet-like feature at g ≈ 1.98 and to the complete disappearance of the modified Mn multiline signal. Computer simulations of the shape of the doublet S′3 signal and the dependence of its electron spin echo amplitude on the microwave field strength suggest that dipole and exchange interactions between two organic radicals are responsible for the doublet S′3 signal. The pulsed ENDOR-induced EPR measurements indicate that the ENDOR spectrum of the tyrosine radical YZ+ is associated with the doublet S′3 signal only.
The manganese complex (Mn4) which is responsible for water oxidation in photosystem II is EPR detectable in the S2-state, one of the five redox states of the enzyme cycle. The S2-state is observable at 10 K either as an EPR multiline signal (spin S = 1/2) or as a signal at g = 4.1 (spin S = 3/2 or 5/2). It has recently been shown that the state responsible for the multiline signal is converted to that responsible
for the g = 4.1 signal upon the absorption of near-infrared light [Boussac A, Girerd J-J, Rutherford AW (1996) Biochemistry 35 : 6984–6989].
It is shown here that the yield of the spin interconversion may be variable and depends on the photosystem II (PSII) preparations.
The EPR multiline signal detected after near-infrared illumination, and which originates from PSII centers not susceptible
to the near-infrared light, is shown to be different from that which originates from infrared-susceptible PSII centers. The
total S2-multiline signal results from the superposition of the two multiline signals which originate from these two PSII populations.
One S2 population gives rise to a "narrow" multiline signal characterized by strong central lines and weak outer lines. The second
population gives rise to a "broad" multiline signal in which the intensity of the outer lines, at low and high field, are
proportionally larger than those in the narrow multiline signal. The larger the relative amplitude of the outer lines at low
and high field, the higher is the proportion of the near-infrared-susceptible PSII centers and the yield of the multiline
to g = 4.1 signal conversion. This inhomogeneity of the EPR multiline signal is briefly discussed in terms of the structural properties
of the Mn4 complex.
The preparation and characterization of two new mixed-valence, trinuclear species, [Mn3O(O2CCF3)6(H2O)3]⋅CF3COOH⋅4/3H2O (1) and [Mn3O(O2CCF3)6(CH3COOH)3] (2), is reported. Compound 1 crystallizes in the triclinic space group, P¯1 (No. 2), with the parameters, a=12.3131(9) Å, b=12.4427(9) Å, c=12.965(1) Å, α=72.593(4)°, β=73.453(5)°, γ=68.345(4)°, V=1727.2(2) Å3, and Z=2. A total of 14060 reflections were collected in the range 1.68≤θ≤27.52°. The final weighted and non-weighted agreement indices, R1=0.0589 and wR2=0.1445 were based on a total of 6953 unique reflections with an R
int value of 0.0542. Compound 2 crystallizes in the monoclinic space group, P21/n (No. 14), with the parameters, a=12.876(3) Å, b=12.212(4) Å, c=17.732(4) Å, β=100.40(3)°, V=3640.4(1) Å3, and Z=4. A total of 32197 reflections were collected in the range 1.72≤θ≤27.13°. The final weighted and non-weighted agreement factors, R1=0.0647 and wR2=0.1609 were based on a total of 8018 unique reflections with an R
int
value of 0.0462. An investigation of the physical properties revealed that both compounds display an intermediate ground state of S=3/2 as a consequence of intramolecular antiferromagnetic coupling. The magnetic data for compound 1 was best fit to the parameters g=2.09, J=−5.5 cm−1, J′=−3.4 cm−1, and D
Mn(III)=−4.5 cm−1; the data for compound 2 was best fit to the parameters g=2.10, J=−2.9 cm−1, J′=−5.5 cm−1, and D
Mn(III)=−4.5 cm−1.
The multiline and g= 4.1 EPR signals from the manganese-containing water oxidation site of plant photosystem II have been studied at Q-band (35 GHz). Comparisons with X-band spectra show a significant g anisotropy in the multiline signal, which is inequivalent for the plus and minus alcohol forms. Provisional values for the plus alcohol form are g∥= 1.970, g⊥= 1.984. The Q-band 4.1 spectrum indicates that the signal arises from a quasi axial, probably spin- system, with a slight splitting of the g⊥ components into g⊥x= 4.35 and g⊥y= 4.14. Each component has a (peak-to-peak) width of ca. 30 mT, similar to that of the (unresolved) signal at X-band. The 4.1 signal from one dimensionally ordered photosystem II samples has also been studied at X-band. This shows a variation of the apparent g⊥ value with sample orientation in the magnetic field, consistent with the above limits from the powder-pattern Q-band data. Assuming the transitions around g= 4 arise from the ⊥ components of a quasi-axial spin- system, the Q- and X-band results indicate that |D| > 5 cm–1 and |E/D|≈ 0.017 for the zero field terms of the state. The oriented X-band data then show that the D(∥) axis is nearly parallel to the thylakoid membrane plane. Further, Mn hyperfine structure is resolved on the oriented X-band 4.1 signals, the first such detection in unmodified enzyme. The spacing (ca. 4 mT) is similar to that reported recently for structure on the 4.1 signal of NH3 inhibited enzyme (Kim et al., J. Am. Chem. Soc., 1990, 112, 9389), but the lines are less distinct.
The complex Mn-2(H2O)(OAc)(4)(tmeda)(2) (tmeda = N,N,N',N'-tetramethylethylenediamine) is a model for the active site of hydrolase enzymes containing acetate-bridged dimanganese cores. The two high-spin Mn(II) ions are antiferromagnetically coupled, as determined by previous magnetic susceptibility studies (Yu, S.-B; Lippard, S. J.; Shweky, I; Bino, A. Inorg. Chem. 1992, 31, 3502-3504) to yield a spin "ladder" with total spin S = 0, 1, 2,..., 5 in increasing energy. In this study, the complex was characterized by Q-band and X-band EPR spectroscopy in frozen solution. Analysis of the temperature dependence of these EPR spectra indicates that the primary spectral contribution is from the S = 2 manifold. The EPR spectra were simulated using a full spin Hamiltonian for this manifold of a coupled spin system, which provided the fit parameters J = -2.9 cm(-1), g = 2.00, and D-2 = -0.060 +/- 0.003 cm(-1). An additional multiline EPR signal is observed which is proposed to arise from the total spin S = 5/2 ground state of a Mn(II) trimer of the type Mn-3(OAc)(6)(tmeda)(2).
A new type of 90–240 G wide EPR signal from the modified S3 state of Ca2+-depleted photosystem II (PSII) is concluded to arise from a partially oxidized water radical with spin S = 12 interacting with the S = 12 S2-state manganese tetramer (‘Mn4’). This is based exclusively on the fact that the average g value of the radical is ≈ 2.010–2.012, a value close to that of OH radical (2.011) and significantly larger than either one of an oxidized imidazole (2.00226) or an oxidized tyrosine (2.0046), indicating that the radical may be (HOOH)−, the most probable intermediate produced by abstracting two protons and one electron from a bound water dimer. The effective interactions between the Mn4 and radical spins (S1 and S2, respectively) of the form Hint = J12S1 · S2 + S1 · D12 · S12 have been thoroughly investigated to find which Mn4-radical complex can reasonably make both J12andD12 as small as ≈ 100 G in magnitude and can, simultaneously, yield an X-ray absorption Mn K-edge energy 0.7 ± 0.3 eV higher than that in the modified S2 state. As the most probable model, we propose that the radical must form the third bridging ligand between di-μ-oxo or μ2-oxo-(μ3-oxo) bridged Mna(III) and Mnb(IV) ions on the opposite side of mono-μ2-oxo bridged Mnc and Mnd ions.
Publisher Summary
This chapter discusses properties, structures, and reactivity of manganese redox enzymes and model systems. Manganese is one of several first-row transition elements that are employed by biological systems to assist in varied metabolic and structural roles. Manganese is used to give structural support to proteins and is a cofactor in chemical transformations that include hydrolytic and redox reactions. Perhaps the best-known function, and the one of great importance to aerobic life, is in the oxygen-evolving complex, which oxidizes water to dioxygen during photosynthesis. The discussions of data gathering on enzyme systems and model chemistry, both structural and functional, presented in this chapter provides a foundation for exploring current and as yet undiscovered manganese enzymes. The biological applications of manganese are numerous and quite varied. The manganese-containing redox enzymes are unique. Most of these deal with dioxygen metabolism in one form or another.
An ESEEM (electron spin−echo envelope modulation) spectroscopic study employing a series of 2H-labeled alcohols provides direct evidence that small alcohols (methanol and ethanol) ligate to the Mn cluster of the oxygen evolving complex (OEC) of Photosystem II in the S2-state of the Kok cycle. A numerical method for calculating the through-space hyperfine interactions for exchange-coupled tetranuclear Mn clusters is described. This method is used to calculate hyperfine interaction tensors for protons [deuterons] in the vicinity of two different arrangements of Mn ions in a tetranuclear cluster: a symmetric cubane model and the EXAFS-based Berkeley “dimer-of-dimers” model. The Mn−H distances derived from the spectroscopically observed coupling constants for methanol and ethanol protons [deuterons] and interpreted with these cluster models are consistent with the direct ligation of these small alcohols to the OEC Mn cluster. Specifically, for methanol we can simulate the three-pulse ESEEM time domain pattern with three dipolar hyperfine interactions of 2.92, 1.33, and 1.15 MHz, corresponding to a range of maximal Mn−H distances in the models of 3.7−5.6 Å (dimer-of-dimers) and 3.6−4.9 Å (symmetric cubane). We also find evidence for limited access of n-propanol, but no evidence for 2-propanol or DMSO access. Implications for substrate accessibility to the OEC are discussed.
The two forms of the g ≈ 4.1 signal recently identified in photosystem II (Smith, P. J.; Pace, R. J. Biochim. Biophys. Acta 1996, 1275, 213) have been simulated at several frequencies as near-axial spin 3/2 centers. In both cases, an explicit spin coupling model is assumed, involving two magnetically isolated Mn pairs, one for each signal type. For that pair assumed to give rise to the spin 1/2 multiline signal as the ground state, the modeling of the first-excited-state 4.1 signal gives estimates of the fine structure parameters for the individual Mn centers and the exchange coupling constant for the pair. The fine structure terms suggest that one Mn ion is a conventional MnIII ion in a highly axially distorted environment. The other Mn center, which is formally spin 3/2, is unlikely to be a conventional MnIV ion, but rather a MnIII−radical ligand pair, strongly antiferromagnetically coupled to give a net spin 3/2 state. The coupling between this Mn−radical center and the other MnIII is weak (J = −2.3 cm-1) in the absence of alcohol in the buffer medium, as determined earlier (Smith and Pace). The model is shown to be quantitatively consistent with the behavior of other signals proposed to arise from this coupled dimer. Comparison of our own data with those of others (Haddy, A.; et al. Biochim. Biophys. Acta 1992, 1099, 25−34) on one-dimensionally ordered photosystem II samples shows a generally consistent orientation of the molecular axis system for the dimer in the membrane plane. The second 4.1 signal, which exhibits ground-state behavior, may be simulated at X- and Q-band frequencies as an isolated system with D = +1.1 cm-1 and E/D = 0.037. The spin center is suggested to arise from a radical-bridged Mn homodimer, and the modeling parameters have been interpreted within this framework. The resulting proposal, involving two isolated dimers for the Mn organization within the oxygen evolving center, is critically examined in the light of recent work from other groups.
The Mn4 complex which is involved in water oxidation in photosystem II is known to exhibit three types of EPR signals in the S2 state, one of the five redox states of the enzyme cycle: a multiline signal (spin 1/2), signals at g > 5 (spin 5/2), and a signal at g = 4.1 (spin value 3/2 or 5/2). The multiline and g = 4.1 signals are those the most readily observed. The relative proportions of the g = 4.1 signal and of the multiline signal are affected by many biochemical treatments including the substitution of Ca2+and Cl- which are two essential cofactors for O2 evolution. The state responsible for the multiline signal can also be converted, reversibly, to that responsible for the g = 4.1 signal upon the absorption of near-IR light at around 150 K. These infrared-induced effects are confined to the Mn4 cluster, and no other redox change occurs in the enzyme. Here, we have used the IR-induced photochemistry of the Mn4 cluster to measure the changes in magnetization occurring upon interconversion of the state responsible for the spin 1/2 state and the g = 4.1 state. Measurements were performed with a SQUID magnetometer below 20 K and at magnetic fields ≤5.5 T. Simulations of experimental data provide strong indication that the spin value of the state responsible for the g = 4.1 state is 5/2. Results are discussed in terms of a model implying an IR-triggered spin conversion of the MnIII (from the spin 2 to spin 1) of the Mn4 cluster.
We have performed continuous-wave electron paramagnetic resonance (CW-EPR) and electron spin echo electron nuclear double resonance (ESE-ENDOR) experiments on the multiline form of the S2-state of untreated, MeOH-treated, and ammonia-treated spinach photosystem II (PS II) centers. Through simultaneously constrained simulations of the CW-EPR and ESE-ENDOR data, we conclude that four effective 55Mn hyperfine tensors (AX, AY, AZ) are required to properly simulate the experimental data [untreated and MeOH-treated PS II centers (MHz): −232, −232, −270; 200, 200, 250; −311, −311, −270; 180, 180, 240; ammonia-treated PS II centers (MHz): 208, 208, 158; −150, −150, −112; 222, 222, 172; −295, −315, −390]. We further show that these effective hyperfine tensors are best supported by a trimer/monomer arrangement of three Mn(IV) ions and one Mn(III) ion. In this topology, MnA, MnB, and MnC form a strongly exchange coupled core (JAB and JBC < −100 cm-1) while MnD is weakly exchange coupled (JCD) to one end of the trinuclear core. For untreated and MeOH-treated PS II centers, the Mn(III) ion is either MnA or MnC, with a zero-field-splitting of D = −1.25 to −2.25 cm-1. For ammonia-treated PS II centers, the Mn(III) ion is MnD, with a zero-field-splitting of D = +0.75 to +1.75 cm-1. The binding of the ammonia ligand results in a shift of the Mn(III) ion from the trinuclear core to the monomer Mn ion. This structural model can also account for the higher spin of the g = 4.1 signal and the magnetic properties of the S0-state.
Electron paramagnetic resonance of spinach chloroplasts given a series of laser flashes, n = 0, 1,..., 6, at room temperature and rapidly cooled to -140 degrees C reveals a signal possessing at least 16 and possibly 21 or more hyperfine lines when observed below 35 K. The spectrum is consistent with a pair of antiferromagnetically coupled Mn ions, or possibly a tetramer of Mn ions, in which Mn(III) and Mn(IV) oxidation states are present. The intensity of this signal peaks on the first and fifth flashes, suggesting a cyclic change in oxidation state of period 4. The multiline signal produced on the first flash is not affected by the electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea but is abolished by agents that influence the state of bound manganese, such as incubation with alkaline Tris, or dithionite, and by extraction with cholate detergent in the presence of ammonium sulfate. These results indicate that the paramagnetic signal is monitoring oxidation state changes in the enzyme involved in oxidation of water.
The EPR spectra of Mn2+ were studied in the As�S�I and As�Te�I glass systems over a range of iodine concentration. For all samples the main spectral line was centered at g=2.00. Exchange narrowing was sometimes present with the higher manganese concentrations. Some of the As�S�I glasses exhibited partially resolved hyperfine structure. In the case of the As�Te�I glasses, under certain conditions an additional line was observed at g=4.3. This line was explained as arising from a particular zero field splitting, and it was found that ∣E∣ ∼ ∣D/3∣.
Previous research (Sandusky, P.O. and Yocum, C.F. (1983) FEBS Lett. 162, 339–343 and (1984) Biochim. Biophys. Acta 766, 603–611) has documented a competition between chloride and ammonia or Tris for a binding site within the oxygen-evolving complex of Photosystem II. This competition is in fact a general property of inhibitory amines which is related to their nucleophilicity; this in turn suggests that the binding site is associated with a metal. Only ammonia, of all amines tested, is able to occupy a second binding site which is unrelated to the site of chloride binding; this sterically hindered site may be identical to the site already described for binding of hydroxylamine, hydrazine, and certain of their derivatives (Radmer, R. and Ollinger, O. (1983) FEBS Lett. 152, 39–43). When the interaction between amines, chloride and the inhibitory halide fluoride was examined, steady-state kinetic plotting procedures revealed that amines and fluoride compete for the chloride binding site; binding of one inhibitor precludes the binding of the other. It was also observed that the intensity of inhibitor binding to the oxygen-evolving complex was influenced by the electron acceptor present during assays; stronger inhibition was observed with a PS II-specific electron acceptor (2,5-dichloro-p-benzoquinone) than with an acceptor (ferricyanide) which requires electron transport to the reducing terminus of Photosystem I. These results are interpreted in terms of a model which proposes that the binding site for chloride on the oxidizing side of Photosystem II resides within the pool of functional manganese associated with the oxygen-evolving complex of Photosystem II.
Hyperfine structure observed at g′≃9.4 and g′≃4.3 in the EPR spectrum of Mn2+ in several glasses is analyzed on the basis of the theory of Griscom and Griscom [J. Chem. Phys. 47, 2711 (1967)] and Aasa [J. Chem. Phys. 52, 3919 (1970)]. The complex hyperfine structure at g′=4.3 is found to be a superposition of three sextets, but at g′=9.4 only a single set of six hyperfine lines is observed.
Integrated intensities of EPR lines and the simulation of powder shapes in the presence of large anisotropy are discussed for field-sweep spectra. It is pointed out that the shape function, normalized by integration over the magnetic field, must be multiplied by a factor which in the case equals the inverse of the g-value. This factor seemingly has been omitted in previous calculations of intensities and shapes. As a consequence, the integrated intensity of an isotropic line is proportional to its g-value and not to g2. The total intensity of a powder spectrum is calculated, and examples of simulations of such spectra are given. A method for the determination of total intensities from the area under an “absorption” peak in a first derivative powder spectrum is also given.
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.
In this contribution, the authors provide a more complete interpretation of earlier EPR data and include new experimental data on the magnetic properties of the g = 4.1 Sâ state EPR signal and the Sâ state multiline EPR signal produced by 245 K illumination in active state samples. An interpretation of the temperature-dependence data of the Sâ state EPR signal from NHâCl-treated PSII membranes was presented. These data can be best explained by a model of the Sâ state which consists of a mixed-valence manganese tetramer.
The Mn-containing catalytic site for photosynthetic water oxidation undergoes changes in oxidation states during the catalytic cycle. One of these intermediates, the S2 state, can be studied directly by e.s.r. at liquid-helium temperatures. Two distinct e.s.r. signals from the S2 state are produced when dark-adapted Photosystem II membranes are illuminated in the 130–200 K range: a g= 4.1 signal or a signal centred at g= 2.0 with many hyperfine lines, referred to as the multiline e.s.r. signal. The yields and magnetic properties of these e.s.r. signals are found to depend on the temperature at which the S2 state is formed and the choice of cryoprotectant (ethylene glycol or sucrose). The intensity of the g= 4.1 e.s.r. signal obeys the Curie law in the 4.0–20.0 K temperature range. The S2-state multiline e.s.r. signal exhibits an intensity maximum at 7.0 K which is independent of microwave powers below 2 mW, if the samples contain 30 % ethylene glycol. This non-Curie behaviour is not observed in samples containing 0.4 mol dm–3 sucrose. A model is presented in which the S2 state e.s.r. signals arise from an exchange-coupled Mn tetramer, where both ferromagnetic and antiferromagnetic exchange occur. According to our model, the multiline e.s.r. signal observed in samples suspended in 30 % ethylene glycol originates from the thermally populated first excited s= 1/2 state of the exchange-coupled Mn tetramer, whereas the g= 4.1 e.s.r. signal arises from the ground s= 3/2 state of the Mn tetramer in a configuration that makes the s= 1/2 state thermally inaccessible. The different behaviour of the S2-state multiline e.s.r. signal in samples containing sucrose can be explained by a small conformational change of the Mn complex which alters the exchange couplings. In support of our assignment of the multiline e.s.r. signal, we present spectral simulations at S-, X- and Q-bands. The fits to the experimental spectra at X- and Q-bands are improved if a small degree of anisotropy is introduced in the g tensor of the Mn complex.
New evidence on the chloride requirement for photosynthetic O 2 evolution has indicated that Cl - facilitates oxidation of the manganese cluster by the photosystem II (PSII) Tyr-Z + radical. Illumination above 250K of spinach PSII centers which are inhibited in O 2 evolution bu either Cl - depletion of F - substitution produces a new EPR signal which has magnetic characteristics similar to one recently discovered in samples inhibited by depletion of Ca 2+ only.
The reaction of ammonia with the oxygen-evolving system was investigated using EPR. Two sites with distinct binding properties were found. One site, previously known to be responsible for the modification by ammonia of the multiline EPR signal from the S2 state and believed to be accessible in this state only, was found to bind ammonia also in the S1 state although weaker. The second binding site, identified by the effect of bound ammonia on the shape and position of the g = 4.1 EPR signal, was also found to be accessible in both the S1 and S2 states. The apparent dissociation constants for ammonia at the two sites in the S1 and S2 states were determined. In neither state did the binding the ammonia account for the observed inhibition of oxygen evolution, suggesting that binding to other S states plays an important role in the inhibition. Chloride, which is known to interfere with ammonia-induced inhibition of oxygen evolution, was found to compete with ammonia at the site associated with the modification of the g = 4.1 EPR signal. The broadening of the hyperfine lines of the multiline EPR signal, seen in the presence of 17O-labeled water, was still observed after the modification of the signal by ammonia. This indicates that ammonia has not completely displaced water bound to the catalytic site in the S2 state. The results of the binding studies are interpreted in terms of a two state — two site model, where the two states are identified by their EPR signals, the multiline and the g = 4.1 signal, respectively, and the two sites identified by the effects of ammonia on these signals and where the equilibrium between the two states is regulated by the binding of ligands to the sites.
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.
— 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.
The positions of powder lines in the electron paramagnetic spectra of high‐spin ferric systems (d5,S = ) have been calculated by solving the spin Hamiltonian = gβB⋅S + ⅓D[3Sz2 − S(S + 1)] + E(Sx2 − Sy2) for a broad range of parameters. Powder lines are obtained for every transition when the magnetic field points along the principal axes of the fine structure tensor. However, it was found that for most transitions extra powder lines are often found when the field lies in any of the principal planes but not along the axes. Particular attention is directed to the transition responsible for the g′ ≈ 4.2 absorption in nearly rhombic (E / D ∼ ⅓) ferric complexes. The calculations show that, depending on the value of the ratio between the microwave quantum and the parameter D, this transition may consist of 3–6 powder lines near g′ = 4.2. The g′ values for all these powder lines were also obtained from a third‐order perturbation calculation which assumes nearly rhombic symmetry and D > gβB. The 9.2‐ and 34‐GHz spectra of Fe(III)–EDTA diluted in the corresponding diamagnetic Co(III) compound and the 2.7‐, 9.2‐, and 34‐GHz spectra of native human serum transferrin have been analyzed by the aid of the calculations. It was determined that for FeEDTA∣ D ∣ = 0.83cm−1 and ∣ E / D ∣ = 0.31, while for transferrin ∣ D ∣ = 0.27cm−1 and ∣ E / D ∣ = 0.31–0.32.
The title compound 1 is a model for the Mn 3IIIMnIV center near the photosystem II. It follows from the dependence of the susceptibility of 1 on temperature and field strength that its magnetic properties are determined both by ferromagnetic as well as antiferromagnetic interactions and that the ground state has a spin of 9/2. The first excited states should be more than 200 cm⁻¹ energy‐richer than the ground state. (Figure Presented.)
This chapter discusses the manganese (Mn) binding sites and presumed manganese proteins in chloroplasts. At least three “classes” of Mn binding sites are identifiable in well-washed, O2 evolving type II chloroplasts. One “class” is nonfunctional in electron transport, whereas the other two appear to be intimately involved within reactions of system II. Equilibration of type II spinach chloroplasts in iso-osmotic media with Mn2+ results in extensive binding of Mn2+ to chloroplasts. Many proteins, nucleic acids, and anionic lipids bind Mn2+, thus this apparent discrepancy in dissociation constants values may reflect differences in macromolecular composition of chloroplasts as isolated by different workers. In contrast to the nonfunctional Mn, the functional bound Mn is not removed from chloroplasts by washes with grinding media or with chelating agents such as EDTA even though this reagent readily penetrates thylakoid membranes and chelates any free Mn2+ within the thylakoids.
The process of water oxidation and dioxygen evolution by the photosystem II (PSII) component of plant photosynthesis is cyclic, with intermediate states of the oxygen-evolving complex (OEC) designated S{sub 0} through S{sub 4}. Two electron paramagnetic resonance (EPR) signals have been assigned to the S{sub 2} state of the complex. A multiline' EPR signal centered at the g = 2 region of the spectrum shows 16 or more partially resolved Mn hyperfine transitions and arises from a cluster with a minimum of two exchange-coupled mixed-valence Mn atoms. The other S{sub 2} EPR signal occurs in the g = 4.1 region of the spectrum. The lack of resolved Mn hyperfine couplings has prevented conclusive assignment of the g = 4.1 EPR signal to a Mn center. However, a shift of the Mn X-ray K edge to higher energies is correlated with the appearance of the g = 4.1 signal in PSII membranes illuminated at 140 K. A considerable body of experimental work, including measurements of the temperature dependence of the EPR signals and observations of the interconversion between the multiline and the g = 4.1 signals, has given rise to two different models involving S = 3/2 Mn origins for themore » g = 4.1 signal. In this communication, the authors present direct spectral evidence of a multinuclear Mn origin for the S{sub 2} g = 4.1 signal.« less
The S2-state multiline EPR signal observed in photosynthetic membrane preparations has been previously well characterized at X-band frequencies (9.1-9.5 GHz). These studies have indicated that the signal, centered at g = 2, arises from a multinuclear mixed-valence Mn center of the O2-evolving complex. In the present study, the multiline EPR signal from spinach photosystem II enriched membranes is characterized at an S-band frequency (3.9 GHz). At this lower frequency, the resolution and complexity of the signal increase markedly compared with its appearance in the X-band. While the multiline signal covers similar magnetic field ranges at the two frequencies, the S-band signal has a greater number of lines, narrower line widths, and a different overall appearance. Replacement of Cl- with Br- or 1H2O with 2H2O in the buffer shows that neither exchangeable Cl- nor protons cause superhyperfine structure in the S-band multiline signal. Membrane preparations oriented on mylar sheets show dependence of the S-band signal on the angle between the mylar sheet normal and the magnetic field direction, indicating that the multiplicity of lines is in part due to signal anisotropy. The results, combined with previous work at X-band, indicate that a minimal working model for the species responsible for the multiline signal is a mixed-valence binuclear Mn complex with an anisotropic hyperfine interaction that includes second-order contributions.
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 prospects are shrinking rapidly for a future for society based on liquid hydrocarbons as a major source of energy. Among the wide array of alternative sources that are currently undergoing scrutiny, much attention is attracted to the photolysis of water to produce hydrogen and oxygen gases. Water, the starting material, does not suffer from lack of abundance, and there is every likelihood that the environmental consequences of water splitting will be negligible. Solar radiation is the obvious candiate for the ultimate energy source, but of course water cannot be photolyzed directly by the relatively low-energy wave-lengths, greater than 300 nm, that penetrate the earth's atmosphere. Nevertheless, the photolysis of water to produce O and reduced substances, with reduction potentials equivalent to that of H, is accomplished efficiently using sunlight by higher plant photosynthesis. There are even organisms that, under special conditions, will evolve H gas photosynthetically, but not efficiently when coupled with O production. To produce a molecule of O from water requires the removal of four electrons from two HO molecules.
Abstract— This contribution provides an analysis of the basic coordination tendencies of manganese with specific emphasis on the biological chemistry of this element. The review is broken into four parts. First, a discussion of the basic coordination chemistry of manganese is mononuclear and multinuclear environments is presented. Second, the biophysical data essential to the development of models for the active center of the photosystem is examined. Third, recently reported mononuclear and cluster manganese compounds are profiled with an emphasis on relating the physical parameters of the models to the structure and function of the enzymatic system. Finally, a comparison is made between the OEC and other known manganoenzymes which metabolize the O2n-moiety.
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.
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.
In the presence of Cl−, the severity of ammonia-induced inhibition of photosynthetic oxygen evolution is attenuated in spinach thylakoid membranes (Sandusky, P.O. and Yocum, C.F. (1983) FEBS Lett. 162, 339–343). A further examination of this phenomenon using steady-state kinetic analysis suggests that there are two sites of ammonia attack, only one of which is protected by the presence of Cl−. In the case of Tris-induced inhibition of oxygen evolution only the Cl− protected site is evident. In both cases the mechanism of Cl− protection involves the binding of Cl− in competition with the inhibitory amine. Anions (Br− and NO−3) known to reactive oxygen evolution in Cl−-depleted membranes also protect against Tris-induced inhibition, and reactivation of Cl−-depleted membranes by Cl− is competitively inhibited by ammonia. Inactivation of the oxygen-evolving complex by NH2OH is impeded by Cl−, whereas Cl− does not affect the inhibition induced by so-called ADRY reagents. We propose that Cl− functions in the oxygen-evolving complex as a ligand bridging manganese atoms to mediate electron transfer. This model accounts both for the well known Cl− requirement of oxygen evolution, and for the inhibitory effects of amines on this reaction.
An oxygen-evolving Photosystem II reaction center complex was characterized by using both biophysical and biochemical techniques. A low-temperature EPR study of this preparation has revealed that cytochrome b-559 has been converted to its low-potential form(s); although in the presence of Ca2+ and Cl− the PS II reaction center complex shows high rates of oxygen-evolution activity, cytochrome b-559 is not converted to its high-potential form. The same study also demonstrated that Ca2+ and Cl−, not the 17 and 23 kDa proteins, are the cofactors required for the generation of the multiline signal which is associated with the S2 state. Further solubilization of the PS II reaction center complex, followed by gel filtration chromotography, resulted in the isolation of a purified oxygen-evolving PS II reaction center core and a 28 kDa Chl-a-binding protein. The purified oxygen-evolving preparation contains polypeptides with molecular masses of 47, 43, 32, 30 and 9 kDa as well as the extrinsic 33 kDa polpeptide. These proteins, along with manganese, chloride and calcium, appear to form the simplest structure thus far reported to retain the enzymatic activity necessary for oxidation of water to molecular oxygen.
Continuous illumination at 200 K of photosystem (PS) II-enriched membranes generates two electron paramagnetic resonance (EPR) signals that both are connected with the S(2) state: a multiline signal at g 2 and a single line at g = 4.1. From measurements at three different X-band frequencies and at 34 GHz, the g tensor of the multiline species was found to be isotropic with g = 1.982. It has an excited spin multiplet at approximately 30 cm(-1), inferred from the temperature-dependence of the linewidth. The intensity ratio of the g = 4.1 signal to the multiline signal was found to be almost constant from 5 to 23 K. Based on these findings and on spin quantitation of the two signals in samples with and without 4% ethanol, it is concluded that they arise from the ground doublets of paramagnetic species in different PS II centers. It is suggested that the two signals originate from separate PS II electron donors that are in a redox equilibrium with each other in the S(2) state and that the g = 4.1 signal arises from monomeric Mn(IV).
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)
X-ray absorption spectroscopy at the Mn K-edge has been utilized to study the origin of the g = 4.1 EPR signal associated with the Mn-containing photosynthetic O2-evolving complex. Formation of the g = 4.1 signal by illumination of Photosystem II preparations at 140 K is associated with a shift of the Mn edge inflection point to higher energy. This shift is similar to that observed upon formation of the S2 multiline EPR signal by 190 K illumination. The g = 4.1 signal is assigned to the Mn complex in the S2 state.
Properties of the S2 state formed in photosystem II membranes in which Cl- had been replaced by various anions were investigated by means of thermoluminescence measurements and low temperature EPR spectroscopy. The Br--substituted membranes showed the normal thermoluminescence B-band arising from S2Q-B charge recombination, whereas the SO2-4-, F--, CH3COO--, and NO-3-substituted membranes showed modified B-bands with variously upshifted peak temperatures. The extent of the peak temperature upshift varied in parallel with the extent of inhibition of O2 evolution depending on the anion species. A normal EPR S2 multiline signal was induced in Br--substituted membranes, but its amplitude was reduced to less than 10% in F--, NO-3-, CH3COO--, and SO2-4-substituted membranes, In contrast, the g = 4.1 signal from S2 was markedly enhanced in F-- and NO-3-substituted membranes, not much affected in CH3COO-- and SO2-4-substituted membranes, and decreased to 70% in Br--substituted membranes. Based on these data, the effect of various types of S2 modification on the O2-evolving activity was discussed. It was suggested that anions have an important role in regulating the interaction between the Mn atoms, and thereby adjust the redox properties of the S2 state to enable further transitions beyond S2.
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