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An Oscillating Manganese Electron Paramagnetic Resonance Signal from the S 0 State of the Oxygen Evolving Complex in Photosystem II †
Abstract
Photosynthesis produces the oxygen necessary for all aerobic life. During this process, the manganese-containing oxygen evolving complex (OEC) in photosystem II (PSII), cycles through five oxidation states, S0-S4. One of these, S2, is known to be paramagnetic and gives rise to electron paramagnetic resonance (EPR) signals used to probe the catalytic structure and function of the OEC. The S0 states has long been thought to be paramagnetic. We report here a Mn EPR signal from the previously EPR invisible S0 state. The new signal oscillates with a period of four, indicating that it originates from fully active PSII centers. Although similar to the S2 state multiline signal, the new signal is wider (2200 gauss compared with 1850 gauss in samples produced by flashing), with different peak intensity and separation (82 gauss compared with 89 gauss). These characteristics are consistent with the S0 state EPR signal arising from a coupled MnII-MnIII intermediate. The new signal is more stable than the S2 state signal and its decay in tens of minutes is indicative of it originating from the S0 state. The S0 state signal will provide invaluable information toward the understanding of oxygen evolution in plants.
... In the last section, where I described a mechanistic investigation of a ruthenium water oxidation catalyst, the standard reduction potentials corresponding to the four oxidation events in the catalytic cycle were computed using a combination of density Using the explicit solvent model, we computed the standard reduction potential of nine transition metal complexes in water; this set included seven hexaaqua complexes which ISMs failed to describe plus the Co(NH 3 ) 6 and Fe(CN) 6 ions. Each computation involved averaging over hundreds of electron detachment / attachment calculations, which were performed on configurations extracted from QM/MM molecular dynamics trajectories. ...
... In the last section, where I described a mechanistic investigation of a ruthenium water oxidation catalyst, the standard reduction potentials corresponding to the four oxidation events in the catalytic cycle were computed using a combination of density Using the explicit solvent model, we computed the standard reduction potential of nine transition metal complexes in water; this set included seven hexaaqua complexes which ISMs failed to describe plus the Co(NH 3 ) 6 and Fe(CN) 6 ions. Each computation involved averaging over hundreds of electron detachment / attachment calculations, which were performed on configurations extracted from QM/MM molecular dynamics trajectories. ...
... system oxidizes water at efficiencies and rates that far exceed those of any synthetic catalyst. [242] There have been many efforts to resolve the structure of the OEC in plants using X-ray studies, [19,97,225,316] as well as experimental [6,39,122,130,184,185,189,202,237,242,286,295,314] and computational [239,253,258] efforts to understand the mechanism of water splitting within the OEC. The essential steps of the water oxidation mechanism can also be studied rigorously and comprehensively by focusing on much simpler synthetic systems, which do not have the extensive protein scaffolding that often complicates efforts to understand the natural system. ...
Solar energy conversion and water oxidation catalysis are two great scientific and engineering challenges that will play pivotal roles in a future sustainable energy economy. In this work, I apply electronic structure theory and molecular dynamics simulation methods to understand some of the detailed mechanisms behind solar energy conversion and water oxidation catalysis. I will present a detailed atomistic picture of charge separation processes at a donor-acceptor interface between two organic semiconductor materials in an organic photovoltaic device. This will be followed by an investigation of the water oxidation mechanism of a homogeneous ruthenium water-splitting catalyst and a heterogeneous cobalt phosphate water-splitting catalyst. I will also introduce several advances in theoretical methods that allow us to more accurately compute observables that are critically relevant to the operation and performance of these materials and devices.
... Despite the vast literature to understand the water oxidation process, the water oxidation mechanism is still elusive. The dynamic process of the water splitting has been vastly investigated using different methodologies; spectroscopical methods, i.e., electron paramagnetic resonance (EPR) [128][129][130][131][132][133][134][135] , Fourier transform IR spectroscopy (FTIR) 136-139 , and extended X-ray absorption fine structure (EXAFS) 92,100,[140][141][142][143][144][145][146] , and theoretical calculations [147][148][149][150][151] . Several structural changes occur during each Kok's cycle transition ( Figure 12) 82 . ...
... The PSII reaction center core consists of a heterodimer of the transmembrane subunits techniques; including X-ray crystallography 39-44, 46-50, 73, 225 , Fourier transform IR spectroscopy (FTIR) [136][137][138][139] , extended X-ray absorption fine structure (EXAFS) 92,100,[140][141][142][143][144][145][146] and electron paramagnetic resonance (EPR) [128][129][130][131][132][133][134][135] . The atomic resolution crystal structure of dPSIIcc in the dark stable S1 state from Thermosynechococcus (T.) vulcanus at 1.9 Å showed that the core of the metal cluster has the overall shape of a distorted chair 73,222 . ...
Bei der lichtinduzierten Oxidation von Wasser im Photosystem II (PSII) werden zwei wassermoleküle im katalytischen Zyklus des Metallclusters (Mn4CaO5) benötigt, und vier Protonen aus dem Cluster in den Lumen abgegeben. Daher ist es für das Verständnis des Mechanismus´ der Wasseroxidation von entscheidender Bedeutung, die Veränderung der Protonierungszustände am cluster während der Katalyse zu untersuchen. Hierbei sollten sowohl die Wasserkanäle für die Zuführung der Substratwassermoleküle als auch die Transportwege für die Freisetzung der Protonen untersucht werden. Deshalb wurde in meiner ersten Veröffentlichung ein neues Protokoll entwickelt, um einzelne große Kristalle von dPSII mit einer Länge von ~3 mm in der Längsachse zu züchten. Diese Kristalle mit einer Auflösung von ca. 8 Å gemessen. Um eine höhere Auflösung zu erzielen, ist die Verbesserung der Kristallqualität essenziell. Daher wurde in meiner zweiten Veröffentlichung die Struktur des Detergens-Protein-Komplexes von dPSII mit βDM, durch Anwendung von SANS in Kombination mit SAXS untersucht. Die Ergebnisse zeigten, dass βDM eine monomolekulare Schicht um dPSII bildet. Darüber hinaus konnten freie Mizellen von βDM in der Lösung nachgewiesen werden. Damit ist eine weitere Optimierung der βDM-Konzentration in der Proteinlösung erforderlich, um die Bildung von freien Mizellen zu minimieren. In meiner dritten Veröffentlichung wurde die strukturelle Dynamik in den Wasserkanälen, während des S2-S3 Übergangs mit Hilfe der XFEL untersucht. Ein Datensatz mit einer hohen Auflösung von 1,89 Å wurde durch die Zusammenführung von Daten gewonnen, die während des S2-S3 Übergangs gesammelt wurden. In Anbetracht der Analyse der zusammengeführten Daten und der einzelnen Zeitpunkte, die während des S2-S3 Übergangs gesammelt wurden, ist es wahrscheinlich, dass ein Substratwasser durch den O1-Kanal geliefert wird. Im Gegensatz dazu wird ein Proton aus dem Cluster durch den Cl1 Transportweg in Richtung Lumen freigesetzt.
... The active center of the OEC contains an oxo-manganese, cubane-like structure with a Ca 2+ ion occupying one of the vertices. Due to the extremely high efficiency displayed by this catalyst (with a turnover number of about 600,000 and a turn-over frequency of 30-90 s −1 according to the literature [10,11]), there have been major efforts in attempting to understand the mechanism of the overall water oxidation catalytic cycle, including the nature of the intermediates that participate in that mechanism [12][13][14][15][16]. ...
... Finally (12) and (13). The final value of the reduction potential was computed using Equation (7), where ∆G ref was 4.998 eV, which is the absolute electrode potential of the ferrocenium/ferrocene (Fc + /Fc) reference electrode [68]. ...
We present a molecular mechanics force field in AMBER format for the mixed-valence manganese vanadium oxide cluster [Mn4V4O17(OAc)3]3−—a synthetic analogue of the oxygen-evolving complex that catalyzes the water oxidation reaction in photosystem II—with parameter sets for two different oxidation states. Most force field parameters involving metal atoms have been newly parametrized and the harmonic terms refined using hybrid quantum mechanics/molecular mechanics reference simulations, although some parameters were adapted from pre-existing force fields of vanadate cages and manganese oxo dimers. The characteristic Jahn–Teller distortions of d4 MnIII ions in octahedral environments are recovered by the force field. As an application, the developed parameters have been used to calculate the redox potential of the [MnIIIMn3IV] ⇌ [Mn4IV]+e− half-reaction in acetonitrile by means of Marcus theory.
... Misses are routinely used in the analysis of the S state cycle, mostly in the analysis of FIOPs, where they were rst introduced. 13,14,17,18,[22][23][24][25][26][27][28][29] However, this concept has also been used in the analysis of S state cycle intermediates studied by almost any technique, such as variable and delayed uorescence, 25,[30][31][32][33][34][35][36] transient optical, 37 EPR, [38][39][40][41][42] and EXAFS spectroscopies. [46][47][48][49][50] These analyses were done on many types of photosynthetic species with different degree of purication ranging from intact leaves and cells to PSII core complexes. ...
Photosynthesis stores solar light as chemical energy and efficiency of this process is highly important. The electrons required for CO2 reduction are extracted from water in a reaction driven by light-induced charge separations in the Photosystem II reaction center and catalyzed by the CaMn4O5-cluster. This cyclic process involves five redox intermediates known as the S0-S4 states. In this study, we quantify the flash-induced turnover efficiency of each S state by electron paramagnetic resonance spectroscopy. Measurements were performed in photosystem II membrane preparations from spinach in the presence of an exogenous electron acceptor at selected temperatures between -10 °C and +20 °C and at flash frequencies of 1.25, 5 and 10 Hz. The results show that at optimal conditions the turnover efficiencies are limited by reactions occurring in the water oxidizing complex, allowing the extraction of their S state dependence and correlating low efficiencies to structural changes and chemical events during the reaction cycle. At temperatures 10 °C and below, the highest efficiency (i.e. lowest miss parameter) was found for the S1 → S2 transition, while the S2 → S3 transition was least efficient (highest miss parameter) over the whole temperature range. These electron paramagnetic resonance results were confirmed by measurements of flash-induced oxygen release patterns in thylakoid membranes and are explained on the basis of S state dependent structural changes at the CaMn4O5-cluster that were determined recently by femtosecond X-ray crystallography. Thereby, possible "molecular errors" connected to the e - transfer, H+ transfer, H2O binding and O2 release are identified.
... However, the exact structure of the cofactor in both these states is dependent on the orientation of the Jahn-Teller axis for each of the Mn III ions of the structure (82). Interconversion between these forms represents a more subtle change to the overall geometric and electronic structure, i.e., all forms of S 0 and S 1 adopt the same low-spin state (103,104), but the spacing of the magnetic states is altered. In the higher S states (S 3 ), heterogeneity is instead correlated with the stepwise process of water molecule insertion, which is discussed in the next section. ...
The investigation of water oxidation in photosynthesis has remained a central topic in biochemical research for the last few decades due to the importance of this catalytic process for technological applications. Significant progress has been made following the 2011 report of a high-resolution X-ray crystallographic structure resolving the site of catalysis, a protein-bound Mn 4 CaO x complex, which passes through ≥5 intermediate states in the water-splitting cycle. Spectroscopic techniques complemented by quantum chemical calculations aided in understanding the electronic structure of the cofactor in all (detectable) states of the enzymatic process. Together with isotope labeling, these techniques also revealed the binding of the two substrate water molecules to the cluster. These results are described in the context of recent progress using X-ray crystallography with free-electron lasers on these intermediates. The data are instrumental for developing a model for the biological water oxidation cycle.
Expected final online publication date for the Annual Review of Biochemistry, Volume 89 is June 22, 2020. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... The studies of the groups of Ono and Kusunoki on oriented PS II membranes and theoretical investigations (Hasegawa et al. 1998(Hasegawa et al. , 1999 and subsequent advanced EPR studies from the Britt (UC Davis) (Britt et al. , 2004Peloquin et al. 2000) and Lubitz (MPI Mülheim) laboratories Kulik et al. 2005aKulik et al. , b, 2007Lohmiller et al. 2012Lohmiller et al. , 2014Su et al. 2011) further constrained all four 55 Mn hyperfine tensors in the S 2 state and allowed the spin coupling in the tetranuclear manganese cluster to be interrogated. These data together with results collected on the S 0 state, which also resolves a multiline signal (Ahrling et al. 1997;Kulik et al. 2005bKulik et al. , 2007Lohmiller et al. 2017;Messinger et al. 1997), and density functional theory calculations allowed the local oxidation states and set of coupling pathways to be determined (Ames et al. 2011;Krewald et al. 2013Krewald et al. , 2015Krewald et al. , 2016Pantazis et al. 2012Pantazis et al. , 2009). The oxidation state assignment for the S 2 state, which comes from this analysis, is shown in Fig. 5 (top), i.e., Mn 4 (III, IV, IV, IV). ...
Biological water oxidation, performed by a single enzyme, photosystem II, is a central research topic not only in understanding the photosynthetic apparatus but also for the development of water splitting catalysts for technological applications. Great progress has been made in this endeavor following the report of a high-resolution X-ray crystallographic structure in 2011 resolving the cofactor site (Umena et al. in Nature 473:55–60, 2011), a tetra-manganese calcium complex. The electronic properties of the protein-bound water oxidizing Mn4OxCa complex are crucial to understand its catalytic activity. These properties include: its redox state(s) which are tuned by the protein matrix, the distribution of the manganese valence and spin states and the complex interactions that exist between the four manganese ions. In this short review we describe how magnetic resonance techniques, particularly EPR, complemented by quantum chemical calculations, have played an important role in understanding the electronic structure of the cofactor. Together with isotope labeling, these techniques have also been instrumental in deciphering the binding of the two substrate water molecules to the cluster. These results are briefly described in the context of the history of biological water oxidation with special emphasis on recent work using time resolved X-ray diffraction with free electron lasers. It is shown that these data are instrumental for developing a model of the biological water oxidation cycle.
... The oxidized P 680 + is reduced by a nearby redox active tyrosine residue, which in turn oxidizes a Mn 4 CaO 5 cluster for [10,11,14,[29][30][31][32][33][34][35][36][37][38]. Spectroscopic study and DFT calculations strongly suggesting that the S0 state is the most reduced state in OEC, containing three Mn(III) and a Mn(IV) ion in the Mn 4 CaO 5 cluster and has a ground spin state of ½ [40][41][42]. Initially, after the electron transfer cycle the nearby tyrosine radical oxidise one of the Mn(III) to Mn(IV) in the Mn 4 CaO 5 cluster with concomitant proton transfer and the S1 state contains the Mn oxidation state pattern III, IV, IV, III with spin state of 0 and is diamagnetic [43]. ...
Manganese plays multiple role in many biological redox reactions in which it exists in different oxidation states from Mn(II) to Mn(IV). Among them the high-valent manganese-oxo intermediate plays important role in the activity of certain enzymes and lessons from the natural system provide inspiration for new developments of artificial systems for a sustainable energy supply and various organic conversions. This review describes recent advances and key lessons learned from the nature on high-valent Mn-oxo intermediates. Also we focus on the elemental science developed from the natural system, how the novel strategies are realised in nano particles and molecular sites at heterogeneous and homogeneous reaction conditions respectively. Finally, perspectives on the utilisation of the high-valent manganese-oxo species towards other organic reactions are proposed.
... Elucidating how the electronic and geometric structures change in the OEC during catalysis at each S state will pave the way for a better understanding of the mechanism of water splitting and the formation of the O-O bond. The occurrence of structural changes during water oxidation have been demonstrated by several biochemical and biophysical techniques; including X-ray crystallography 10,11,[15][16][17][18][19][20][21][22][23][24][25] , Fourier transform IR spectroscopy (FTIR) [26][27][28][29] , extended X-ray absorption fine structure (EXAFS) 4, 30-37 and electron paramagnetic resonance (EPR) [38][39][40][41][42][43][44][45] . The atomic resolution crystal structure of dPSIIcc in the dark stable S1 state from Thermosynechococcus (T.) vulcanus at 1.9 Å showed that the core of the metal cluster has the overall shape of a distorted chair 9,11 . ...
Photosystem II (PSII) catalyzes the photo-oxidation of water to molecular oxygen and protons. The water splitting reaction occurs inside the oxygen-evolving complex (OEC) via a Mn4CaO5 cluster. To elucidate the reaction mechanism, detailed structural information for each intermediate state of the OEC is required. Despite the current high-resolution crystal structure of PSII at 1.85 Å and other efforts to follow the structural changes of the Mn4CaO5 cluster using X-ray free electron laser (XFEL) crystallography in addition to spectroscopic methods, many details about the reaction mechanism and conformational changes in the catalytic site during water oxidation still remain elusive. In this study, we present a rarely found successful application of the conventional macroseeding method to a large membrane protein like the dimeric PSII core complex (dPSIIcc). Combining microseeding with macroseeding crystallization techniques allowed us to reproducibly grow large dPSIIcc crystals with a size of ~ 3 mm. These large crystals will help improve the data collected from spectroscopic methods like polarized extended X-ray absorption fine structure (EXAFS) and single crystal electron paramagnetic resonance (EPR) techniques and are a prerequisite for determining a 3D structure using neutron diffraction.
... To test the stability of the S 0 state over longer time periods, we monitored the EPR signal associated with S 0 [66,67] before and after 2-days dark-incubation on ice and even after 3-days at 20°C. No significant change in the amplitude of the S 0 signal was observed, showing that no unwanted redox agent interacts with S 0 on a long time scale (see Text S2 with Fig. S5 in the Supplementary Material). ...
Photosynthetic water oxidation to molecular oxygen is carried out by photosystem II (PSII) over a reaction cycle involving four photochemical steps that drive the oxygen-evolving complex through five redox states Si (i=0,.., 4). For understanding the catalytic strategy of biological water oxidation it is important to elucidate the energetic landscape of PSII and in particular that of the final S4→S0 transition. In this short-lived chemical step the four oxidizing equivalents accumulated in the preceding photochemical events are used up to form molecular oxygen, two protons are released and at least one substrate water molecule binds to the Mn4CaO5 cluster. In this study we probed the probability to form S4 from S0 and O2 by incubating YD-less PSII in the S0 state for 2-3days in the presence of (18)O2 and H2(16)O. The absence of any measurable (16,18)O2 formation by water-exchange in the S4 state suggests that the S4 state is hardly ever populated. On the basis of a detailed analysis we determined that the equilibrium constant K of the S4→S0 transition is larger than 1.0×10(7) so that this step is highly exergonic. We argue that this finding is consistent with current knowledge of the energetics of the S0 to S4 reactions, and that the high exergonicity is required for the kinetic efficiency of PSII.
... The molecular oxygen is formed from S 3 through unstable S 4 by releasing an electron and two protons. The oxidation states of the four Mn atoms at the S 0 state have been suggested as Mn 4 (II, III, IV, IV) or Mn 4 (III, III, III, IV) [10][11][12][13][14][15][16]. Even if the oxidation states of four manganese atoms at S 0 are different, the oxidation states change as Mn 4 (III, III, IV, IV) at the S 1 state → Mn 4 (III, IV, IV, IV) at S 2 → Mn 4 (III, IV, IV, IV) at S 3 → Mn 4 (III, IV, IV, IV) at S 4 . ...
Electronic and molecular structures of [(X)mMn(μ-oxo)2Mn(Y)n]q+ (X, Y = H2O, OH and O), which are Mn cluster models at catalytic sites of OEC, were studied by broken-symmetry unrestricted B3LYP method. Two paths from the S0 to S3 states of Kok cycle were investigated. One is a path starting from [Mn(II) (μ-oxo)2Mn(III)] at the S0 state, and another is from [Mn(III) (μ-oxo)2Mn(III)] at the S0. Results found in this study are summarized as, 1) In [Mn(II), Mn(III)], it is not possible that H2O molecules coordinate to the Mn atoms with retaining the octahedral configuration. 2) The OHˉ anion selectively coordinates to Mn(IV) rather than Mn(III). 3) When the oxo atom directly bind to the Mn atom, the Mn atom must be a Mn(IV). From these results, the catalytic mechanism for four-electron oxidation of two H2O molecules in OEC is proposed. 1) The Mn4(II, III, IV, IV) at S0 is ruled out. 2) For Mn4(III, III, IV, IV) at S1, the Mn atom coordinated by OHˉ anion is a Mn(IV) not Mn(III). 3) Only Mn(III) ion which is coordinated by a H2O molecule at S0 plays crucial roles for the oxidation.
... 31,161 An alternative to the (III) 3 (IV) oxidation state assignment, i.e. (II)(III)(IV) 2 , arises if the S 0 -S 1 transition involves a Mn(II)-Mn(III) rather than a Mn(III)-Mn(IV) oxidation. This possibility was le open in early EPR and XANES work, 26,36,38,176 but excluded in subsequent 55 Mn-ENDOR studies. 31,161,177 In the present models, a Mn(II) ion is found in the Mn4 position of S 0 H-3c (see Fig. S4 †) but in line with previous reports, 82,178,179 it is strongly disfavored energetically by more than 23 kcal mol À1 over its redox isomer S 0 H-3a. ...
A central question in biological water splitting concerns the oxidation states of the manganese ions that comprise the oxygen-evolving complex of photosystem II. Understanding the nature and order of oxidation events that occur during the catalytic cycle of five S
i
states (i = 0-4) is of fundamental importance both for the natural system and for artificial water oxidation catalysts. Despite the widespread adoption of the so-called "high-valent scheme"-where, for example, the Mn oxidation states in the S2 state are assigned as III, IV, IV, IV-the competing "low-valent scheme" that differs by a total of two metal unpaired electrons (i.e. III, III, III, IV in the S2 state) is favored by several recent studies for the biological catalyst. The question of the correct oxidation state assignment is addressed here by a detailed computational comparison of the two schemes using a common structural platform and theoretical approach. Models based on crystallographic constraints were constructed for all conceivable oxidation state assignments in the four (semi)stable S states of the oxygen evolving complex, sampling various protonation levels and patterns to ensure comprehensive coverage. The models are evaluated with respect to their geometric, energetic, electronic, and spectroscopic properties against available experimental EXAFS, XFEL-XRD, EPR, ENDOR and Mn K pre-edge XANES data. New 2.5 K 55Mn ENDOR data of the S2 state are also reported. Our results conclusively show that the entire S state phenomenology can only be accommodated within the high-valent scheme by adopting a single motif and protonation pattern that progresses smoothly from S0 (III, III, III, IV) to S3 (IV, IV, IV, IV), satisfying all experimental constraints and reproducing all observables. By contrast, it was impossible to construct a consistent cycle based on the low-valent scheme for all S states. Instead, the low-valent models developed here may provide new insight into the over-reduced S states and the states involved in the assembly of the catalytically active water oxidizing cluster.
In Photosystem II, light-induced water splitting occurs via the S state cycle of the CaMn4O5-cluster. To understand the role of various possible conformations of the CaMn4O5-cluster in this process, the temperature dependence of the S1 → S2 and S2 → S3 state transitions, induced by saturating laser flashes, was studied in spinach photosystem II membrane preparations under different conditions. The S1 → S2 transition temperature dependence was shown to be much dependent on the type of the cryoprotectant and presence of 3.5% methanol, resulting in the variation of transition half-inhibition temperature by 50 K. No similar effect was observed for the S2 → S3 state transition, for which we also show that both the low spin g = 2.0 multiline and high spin g = 4.1 EPR configurations of the S2 state advance with similar efficiency to the S3 state, both showing a transition half-inhibition temperature of 240 K. This was further confirmed by following the appearance of the Split S3 EPR signal. The results are discussed in relevance to the functional and structural heterogeneity of the water oxidizing complex intermediates in photosystem II.
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.
Virtually all life on our planet is powered by the sun through the process of photosynthesis. Understanding the design and operating principles of natural photosynthesis is a central challenge for fundamental research because biology presents a unique paradigm and possible blueprint for the realization of artificial alternatives. All processes of natural photosynthesis, including light harvesting, charge separation and accumulation, water oxidation, and carbon fixation, embody “solutions” optimized through billions of years of evolution to challenges currently faced by scientists developing components for artificial photosynthetic devices. The present chapter provides an overview of the most important aspects of natural photosynthesis and discusses the ways in which they motivate biomimetic and bioinspired research into artificial photosynthesis.
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.
Domain-based local pair natural orbital (DLPNO) coupled cluster single and double (CCSD) with triple perturbation (T) correction methods were performed to elucidate the relative stabilities of ten different intermediate structures of the CaMn4Ox cluster in the S0 state of the oxygen evolving complex (OEC) of photosystem II (PSII). Full geometry optimizations of all the S0 intermediates were performed by the UB3LYP-D3/Def2-TZVP methods, providing the assumed geometrical structures and starting natural orbitals (UNO) for DLPNO-CCSD(T)/Def2TZVP calculations. The effective exchange integrals (J) for the spin Hamiltonian models for the ten intermediates were obtained by the UB3LYP/Def2-TZVP calculations followed by the general spin projections. DLPNO-CCSD(T) calculations followed by the CBS extrapolation procedure elucidated that the (II, III, IV, IV) and (III, III, III, IV) valence states in the CaMn4O5 cluster of the OEC of the PS II were nearly degenerated in energy in the S0 state, indicating an important role of dynamical electron correlation effects for the valence and spin fluctuations in strongly correlated electron systems (SCESs) consisting of 3d transition metals.
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.
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.
The sunlight-powered oxidation of water by photosystem II (PSII) of algae, plants, and cyanobacteria underpins the energy conversion processes that sustain most of life on our planet. Understanding the structure and function of the “engine of life”, the oxygen-evolving complex (OEC) in the active site of PSII, has been one of the great and persistent challenges of modern science. Immense progress has been achieved in recent years through combined contributions of diverse disciplines and research approaches, yet the challenge remains. The improved understanding of the tetramanganese-calcium cluster of the OEC for the experimentally accessible catalytic states often creates a more complex picture of the system than previously imagined, while the various strands of evidence cannot always be unified into a coherent model. This review focuses on selected current problems that relate to structural–electronic features of the OEC, emphasizing conceptual aspects and highlighting topics of structure and function that remain uncertain or controversial. The Mn4CaOx cluster of the OEC cycles through five redox states (S0–S4) to store the oxidizing equivalents required for the final step of dioxygen evolution in the spontaneously decaying S4 state. Remarkably, even the dark-stable state of the OEC, the S1 state, is still incompletely understood because the available structural models do not fully explain the complexity revealed by spectroscopic investigations. In addition to the nature of the dioxygen-evolving S4 state and the precise mechanism of O–O bond formation, major current open questions include the type and role of structural heterogeneity in various intermediate states of the OEC, the sequence of events in the highly complex S2–S3 transition, the heterogeneous nature of the S3 state, the accessibility of substrate or substrate analogues, the identification of substrate oxygen atoms, and the role of the protein matrix in mediating proton removal and substrate delivery. These open questions and their implications for understanding the principles of catalytic control in the OEC must be convincingly addressed before biological water oxidation can be understood in its full complexity on both the atomic and the systemic levels.
Nature’s water splitting catalyst, an oxygen-bridged tetra-manganese calcium (Mn4O5Ca) complex, sequentially activates two substrate water molecules generating molecular O2. Its reaction cycle is composed of five intermediate (Si) states, where the index i indicates the number of oxidizing equivalents stored by the cofactor. After formation of the S4 state, the product dioxygen is released and the cofactor returns to its lowest oxidation state, S0. Membrane-inlet mass spectrometry measurements suggest that at least one substrate is bound throughout the catalytic cycle, as the rate of ¹⁸O-labeled water incorporation into the product O2 is slow, on a millisecond to second timescale depending on the S state. Here, we demonstrate that the Mn4O5Ca complex poised in the S0 state contains an exchangeable hydroxo bridge. Based on a combination of magnetic multiresonance (EPR) spectroscopies, comparison to biochemical models and theoretical calculations we assign this bridge to O5, the same bridge identified in the S2 state as an exchangeable fully deprotonated oxo bridge [Pérez Navarro et al. Proc. Natl. Acad. Sci. U.S.A. 2013 110, 15561]. This oxygen species is the most probable candidate for the slowly-exchanging substrate water in the S0 state. Additional measurements provide new information on the Mn ions that constitute the catalyst. A structural model for the S0 state is proposed that is consistent with available experimental data and explains the observed evolution of water exchange kinetics in the first three states of the catalytic cycle.
The PsbO protein of photosystem II stabilizes the active-site manganese cluster and is thought to act as a proton antenna. To enable neutron diffraction studies, crystals of the β-barrel core of PsbO were grown in capillaries. The crystals were optimized by screening additives in a counter-diffusion setup in which the protein and reservoir solutions were separated by a 1% agarose plug. Crystals were cross-linked with glutaraldehyde. Initial neutron diffraction data were collected from a 0.25 mm ³ crystal at room temperature using the MaNDi single-crystal diffractometer at the Spallation Neutron Source, Oak Ridge National Laboratory.
Photosynthetic water oxidation involves cycling of the Oxygen Evolving Complex (OEC) of Photosystem II (PSII) through 5 intermediate states (S0 to S4 with S1 being the dark-stable state) [1]. It is commonly assumed that the catalytic center of the water-splitting reaction consists of a tetranuclear multivalent Mn complex. The reaction mechanism of water oxidation, however, is at present insufficiently understood.
Significant progress has been made recently in probing the lower S-states by EPR spectroscopy. The S1 state previously characterised by a broad integer spin EPR signal at g= 4.8 [1, 2] has been shown more recently to exhibit an alternative multiline signal at g=12. The latter signal is detected in Synechocystis preparations but also, after removal of the 23 and 17 kDa extrinsic proteins, in spinach preparations [3]. A half integer spin signal has been recently detected in the S0 state produced either by chemical reduction [4] or after the 3rd flash in a flash sequence [5, 6]. The presence of 0.5–3% methanol is required for the observation of the weak hyperfine structure of the signal, but the signal is also observed as a broad derivative in the absence of methanol [6].
Molecular oxygen - essential for all living organisms — is produced by the photosynthetic oxidation of water in photosystem II (PSII) of higher plants, algae and cyanobacteria. The water-oxidizing complex of PSII contains a cluster of 4 Mn atoms, and turns over through five different redox states, denoted by S0-S4, during its function.
Photosystem II (PSII) catalyses the light-driven oxidation of water to molecular oxygen. The site for this process, the oxygen evolving complex (OEC), contains four Mn atoms, and cycles through the five oxidation states S0 - S4. EPR spectroscopy has been used ex-tensively to investigate the chemistry of this cycle, as well as the structure of the catalytic site. The S0 and S2 states are paramagnetic, each giving rise to a broad, fine-structured Mn EPR signal centered around g=2. The S2 multiline signal has been extensively characterized, while the S0 signal was discovered only last year [1, 2]
Photosystem II (PS II) catalyzes the light driven oxidation of water to molecular oxygen and the reduction of plastoquinone to plastohydroquinone. Water oxidation occurs in the oxygen evolving complex (OEC) of PS II that is known to cycle through five different redox states, referred to as the S states (S0,..,S4). A cluster of four Mn, one Ca and Cl⁻ is thought to form the central unit of the OEC which stores most of the oxidizing equivalents and binds the substrate water.
The function of oxygen evolution is known to be catalyzed by a manganese cluster located on the donor side of PS II. Successive abstraction of four electrons from oxygen evolving center was carried out by four times absorption of light by P680 and results in evolution of molecular oxygen. The oxidizing center cycles through a series of the five oxidation states, called S-states with Sn (n = 0 to 4), n denoting the number of stored oxidizing equivalents. The S4-state is a transient state because it rapidly decays to the S0-state by releasing molecular oxygen. The S1-state is most stable of five oxidation states in dark at room temperature. The S2- and S3-states are unstable and decay back to the S1-state. (1, 2)
The evolution of oxygen as a result of light-driven water oxidation is catalyzed by photosystem II (PS2) in which a cluster of four manganese ions acts both as a charge accumulating device and as the active site. During the enzyme cycle, the oxidizing side of PS2 goes through five different redox states that are denoted Sn, n varying from 0 to 4. Oxygen is released during the S3 to S0 transition in which S4 is a transient state (1,2). The Mn4 is known to exhibit in the S2-state a multiline EPR signal (spin 1/2) (1–7, and references therein), a signal at g = 4.1 or g = 4.25 (1–7) (spin = 5/2, (8–12)) and signals at g 5 (spin 5/2) (8–12). A different multiline signal has also been observed in the S0-state (13-15). The detection of an S0 signal by cw-EPR required the presence of methanol in the sample (13–15). In the present study we report experiments on the nature of the interaction of methanol with the Mn4-cluster, on the number of spins contributing in the S0-multiline signal and on the effects of infrared light.
The source of molecular oxygen on Earth is the oxidation of water by Photosystem II in oxygenic photosynthesis. The water oxidising complex is composed of a Mn-cluster ligated to a protein environment and Cl⁻ and Ca²⁺ ions. During water oxidation the Mn-cluster cycles through five different oxidation states (S0-S4).
The oxygen evolving centre (OEC) of photosystem II (PSII) is capable of storing, in a cyclic manner, four oxidising equivalents before it oxidises water to molecular oxygen. The intermediate oxidation states of the OEC are denoted S0...S4, where the suffix refers to the number of electron holes stored (1).
The active site of photosynthetic water oxidation is the oxygen-evolving complex (OEC) in the photosystem II (PSII) reaction center. The OEC is a Mn4CaO5 cluster embedded in the PSII protein matrix, and it cycles through redox intermediates known as Si states (i = 0-4). Significant progress has been made in understanding the inorganic and physical chemistry of states S0-S3 through experiment and theory. The chemical steps from S3 to S0 are more poorly understood, however, because the identity of the substrate water molecules and the mechanism of O-O bond formation are not well established. In this review, we highlight both the consensuses and the remaining challenges of PSII research. Expected final online publication date for the Annual Review of Physical Chemistry Volume 68 is April 20, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Plants, algae and cyanobacteria capture sunlight, extracting electrons from H2O to reduce CO2 into sugars, while releasing O2 in the oxygenic photosynthetic phenomenon. Because of the important role of water oxidation for artificial photosynthesis and many solar fuel systems, understanding the structure and function of this unique biological catalyst forms a requisite research field. Herein the structure of the water-oxidizing complex and its ligand environment are described with reference to the 1.9 Å resolution X-ray-derived crystallographic model of the water-oxidizing complex from the cyanobacterium Thermosynechococcus vulcanus. Proposed mechanisms for water oxidation by Photosystem II and nanosized manganese oxides are also reviewed and discussed in the paper.
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.
Photosynthetic water oxidation is one of the most important biochemical processes on Earth. Oxygen-evolving photosynthetic organisms contain two types of photosynthetic reaction center, called photosystem I (PS I) and photosystem II (PS II). The PS II reaction center uses water as a source of electrons, light energy being used to oxidize water to provide protons and oxygen. How water is oxidized is a difficult problem that has already taken many years of work to understand, however, both the structure of oxygen evolving complex(OEC) and the mechanism of water oxidation still remain to be understood. In this review, we collected the current research results on this area and it will hopefully stimulate further work on the structure of oxygen evolving complex and the mechanism of water oxidation.
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.
A frequent challenge when dealing with multinuclear transition metal clusters in biology is to determine the absolute oxidation states of the individual metal ions and to identify how they evolve during catalytic turnover. The oxygen-evolving complex of biological photosynthesis, an active site that harbors an oxo-bridged Mn4Ca cluster as the water-oxidizing species, offers a prime example of such a challenge that withstood satisfactory resolution for decades. A multitude of experimental studies have approached this question and have offered insights from different angles, but they were also accompanied by incomplete or inconclusive interpretations. Only very recently, through a combination of experiment and theory, has a definitive assignment of the individual Mn oxidation states been achieved for all observable catalytic states of the complex. Here we review the information obtained by structural and spectroscopic methods, describe the interpretation and synthesis achieved through quantum chemistry, and summarize our current understanding of the electronic structure of nature’s water splitting catalyst.
Water splitting is considered as a method to storage of renewable energies to hydrogen. The water-oxidation reaction in water splitting is an efficiency-limiting process for water splitting and, thus, there has been notable progress to find highly efficient water-oxidizing catalysts made from cost-effective and earth-abundant elements. In addition to efficiency, the stability of the water-oxidizing compounds is very important. Herein we focus on self-healing in manganese-based water-oxidizing catalysts in artificial photosynthetic systems.
Photosystem II (PSII) uses light energy to oxidise water and reduce quinone. The water oxidation site is a Mn4Ca cluster located on the luminal side of the membrane protein complex, while the quinone reduction site is made up of two quinones (QA and QB) and a non-heme Fe2+ located on the stromal side of the membrane protein. In this thesis I worked on both oxidation and reduction functions of the enzyme. QA•- and QB•- are magnetically couple to the Fe2+ giving weak and complex EPR signals. The distorted octahedral Fe2+ has four histidines ligands and an exchangeable (bi)carbonate ligand. Formate can displace the exchangeable (bi)carbonate ligand, slowing electron transfer out of the PSII reaction centre. Here I report the formate-modified QB•- Fe2+ EPR signal, and this shows marked spectral changes and has a greatly enhanced intensity. I also discovered a second new EPR signal from formate-treated PSII that is attributed to formate-modified QA•- Fe2+ in the presence of a 2-electron reduced form of QB. In addition, I found that the native QA•- Fe2+ and QB•- Fe2+ EPR signals have a strong feature that had been previously missed because of overlapping signals (mainly the stable tyrosyl radical TyrD•). These previously unreported EPR signals should allow for the redox potential of this cofactor to be directly determined for the first time. I also observed that when QB•-Fe was formed; it was able to oxidise the iron slowly in the dark. This occurred in samples pumped to remove O2. This observation implies that at least in some centres, the QB•-/QBH2 couple has a higher potential then is often assumed and thus that the protein-bound semiquinone is thermodynamically less stable expected. It has yet to be determined if this represents a situation occurring in the majority of centres. Treatment of the system with dithionite generated a modified form of QA•-Fe2+ state and a change in the association of the proteins on gels. This indicates a redox induced modification of the protein, possibly structurally important cysteine bridge in PSII.On the water oxidation side of the enzyme, I studied the first step in the assembly of the Mn4Ca cluster looking at Mn2+ oxidation using kinetic EPR and high field EPR. Conditions were found for stabilising the first oxidised state and some discrepancies with the literature were observed. I also found that dithionite could be used to reduce the Mn4Ca, forming states that are formally equivalent to those that exist during the assembly of the enzyme.
Nature relies on a unique and intricate biochemical setup to achieve sunlight-driven water splitting. Combined experimental and computational efforts have produced significant insights into the structural and functional principles governing the operation of the water-oxidizing enzyme Photosystem II in general, and of the oxygen-evolving manganese-calcium cluster at its active site in particular. Here we review the most important aspects of biological water oxidation, emphasizing current knowledge on the organization of the enzyme, the geometric and electronic structure of the catalyst, and the role of calcium and chloride cofactors. The combination of recent experimental work on the identification of possible substrate sites with computational modeling have considerably limited the possible mechanistic pathways for the critical O-O bond formation step. Taken together, the key features and principles of natural photosynthesis may serve as inspiration for the design, development, and implementation of artificial systems.
Syntheses and magnetic functionalities of exchange-coupled magnetic systems in a controlled fashion of molecular basis have been the focus of the current topics in chemistry and materials science; particularly extremely large spins in molecular frames and molecular high-spin clusters have attracted much attention among the diverse topics of molecule-based magnetics and high spin chemistry. Magnetic characterizations of molecule-based exchange-coupled high-spin clusters are described in terms of conventional as well as high-field/high-frequency ESR spectroscopy. Off-principal-axis extra lines as a salient feature of fine-structure ESR spectroscopy in non-oriented media are emphasized in the spectral analyses. Pulse-ESR-based two-dimensional electron spin transient nutation spectroscopy applied to molecular high-spin clusters is also dealt with, briefly. Solution-phase fine-structure ESR spectroscopy is reviewed in terms of molecular magnetics. In addition to finite molecular high-spin clusters, salient features of molecule-based low-dimensional magnetic materials are dealt with. Throughout the chapter, electron spin resonance for high-spin systems is treated in a general manner in terms of theory. Hybrid eigenfield method is formulated in terms of direct products, and is described as a powerful and facile approach to the exact numerical calculation of resonance fields and transition probabilities for molecular high spin systems. Exact analytical expressions for resonance fields of high spin systems in their principal orientations are for the first time given.
Electron transfer and structure of plant photosystem II were studied by advanced EPR techniques. Pulsed EPR, pulsed electron electron double resonance (PELDOR), and spin polarized radical pair ESEEM were applied to determine the distance between radical pairs of electron transfer components. These methods can detect accurately the dipolar interaction between a pair of radicals, from which the distance is derived. The determined distances and their orientations were compared with recently observed X-ray data. EPR of the manganese cluster in water oxidizing complexes in photosystem II were discussed with respect of their functions.
The results from a multifrequency high-field EPR study of five di-μ-oxo bridged mixed-valence binuclear Mn(III)Mn(IV) complexes are reported. Spectra were obtained at 9, 95, and 285 GHz. The g anisotropy was unambiguously observable at 285 GHz. Hyperfine and g tensor values were estimated using spectral simulation procedures that cyclically and simultaneously fit the multifrequency data. In all five cases, the g tensors of the mixed-valence complexes were found to be rhombic. The g tensors were analyzed using the vector projection model. Most, but not all, of the g anisotropy originates from the Mn(III) center. The rhombic g tensors result from the low symmetry of the manganese centers. The size of the effective g anisotropy for a given complex was found to be a linear function of the average bond distance between the manganese and axial nitrogens. This relationship can be understood in terms of the influence of tetragonal distortion on the electronic levels of the Mn(III) center. The frequency-dependent line broadening observed in these mixed-valence complexes is explained in terms of the relationship between g anisotropy and structure.
In addition to the reaction-center chlorophyll, at least two other organic cofactors are involved in the photosynthetic oxygen-evolution process. One of these cofactors, called "Z," transfers electrons from the site of water oxidation to the reaction center of photosystem II. The other species, "D," has an uncertain function but gives rise to the stable EPR signal known as signal II. Z+. and D+. have identical EPR spectra and are generally assumed to arise from species with the same chemical structure. Results from a variety of experiments have suggested that Z and D are plastoquinones or plastoquinone derivatives. In general, however, the evidence to support this assignment is indirect. To address this situation, we have developed more direct methods to assign the structure of the Z+./D+. radicals. By selective in vivo deuteration of the methyl groups of plastoquinone in cyanobacteria, we show that hyperfine couplings from the methyl protons cannot be responsible for the partially resolved structure seen in the D+. EPR spectrum. That is, we verify by extraction and mass spectrometry that quinones are labeled in algae fed deuterated methionine, but no change is observed in the line shape of signal II. Considering the spectral properties of the D+. radical, a tyrosine origin is a reasonable alternative. In a second series of experiments, we have found that deuteration of tyrosine does indeed narrow the D+. signal. Extraction and mass spectral analysis of the quinones in these cultures show that they are not labeled by tyrosine. These results eliminate a plastoquinone origin for D+.; we conclude instead that D+., and most likely Z+., are tyrosine radicals.
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 authors report the detection of a new electron paramagnetic resonance (EPR) signal that demonstrates the presence of a paramagnetic intermediate in the resting (S{sub 1}) state of the photosynthetic oxygen-evolving complex. The signal was detected using the method of parallel polarization EPR, which is sensitive to {Delta}m = 0 transitions in high spin systems. The properties of the parallel polarization EPR signal in the S{sub 1} state are consistent with an S=1 spin state of and exchange-coupled manganese center that corresponds to the reduced form of the species giving rise to the multiline EPR signal in the light-induced S{sub 2} state. The implications for the electronic structure of the oxygen-evolving complex are discussed. 36 refs., 2 fig., 1 tab.
The decay kinetics for the S2 and S3 states of the oxygen-evolving complex in Photosystem II have been measured in the presence of an external electron acceptor. The S2- and S3-states decay monophasically with half-decay times at 18°C of 3–3.5 min and 3.5–4 min, respectively. The results also show that S3 decays via S2 under these circumstances. The temperature dependence of the individual S-state transitions has been measured in single flash experiments in which the multiline EPR signal originating from the S2 state has been used as spectroscopic probe. The half-inhibition temperatures are for S0 to S1 220–225 K, for S1 to S2 135–140 K, for S2 to S3 230 K and for the S3-to-S0 transition 235 K.
Microwave power saturation studies have been performed over the range 4–20 K on EPR signals photogenerated in PS II particles by low-temperature illumination (180–240 K). In the presence of 3% methanol (+ MeOH), with no g 4.1 signal present, the multiline signal intensity (extrapolated to zero power) shows strict Curie law behaviour over the 4–20 K range. With no MeOH present in the suspension buffer (−MeOH), both the multiline and 4.1 signals show complementary deviations from Curie law behaviour. These are consistent with the signals arising respectively from the ground, S = 1/2, and first excited S = 3/2, states of a total spin = 7/2 multiplet, such as could occur in an MnIV-MnIII antiferromagnetically coupled pair. The deduced height of the 3/2 state above the ½ state is 9.0 K. An inferential estimate, from relaxation data, of this height for the +MeOH case is about 40 K. A broad, featureless component around g = 2 appears to underlie the multiline pattern in the presence of the 4.1 signal, and has a similar temperature behaviour to the latter. A possible exchange coupling model, involving four Mn centres, is presented to accommodate these and other findings on the S2 state signals.
— 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).
Interactions of water and methanol with a mixed valence Mn(III)Mn(IV) complex are explored with 1H electron spin echo (ESE)-electron nuclear double resonance (ENDOR) and 1H and 2H ESE envelope modulation (ESEEM). Derivatives of the (2-OH-3,5-Cl2-SALPN)2 Mn(III)Mn(IV) complex are ideal for structural and spectroscopic modeling of water binding to multinuclear Mn complexes in metalloproteins, specifically photosystem II (PSII) and manganese catalase (MnCat). Using ESE-ENDOR and ESEEM techniques, 1H hyperfine parameters are determined for both water and methanol ligated to the Mn(III) ion of the complex. The protons of water directly bound to Mn(III) are inequivalent and exhibit roughly axial dipolar hyperfine interactions (Tdip = 8.4 MHz and Tdip = 7.4 MHz), permitting orientations and radial distances to be determined using a model where the proton experiences a point dipole interaction with each Mn ion. General equations are given for the components of the rhombic dipolar hyperfine interaction between a proton and a spin coupled dinuclear metal cluster. The observed ENDOR pattern is from water protons 2.65 and 2.74 Å from the Mn(III) which make an Mn(IV)−Mn(III)−H angle of 160°. For the alcohol proton in the analogous methanol bound complex, a 2.65 Å Mn(III)−H distance is observed. Three pulse 2H ESEEM gives best fit Mn(III)−2H(1H) radial distances of 3.0, 3.5, and 4.0 Å for the three methyl deuterons in this complex.
Binding of NH3 to the S2 state of the O2-evolving complex of photosystem II (PSII) causes a structural change in the Mn site that is detectable with low-temperature electron paramagnetic resonance (EPR) spectroscopy. Untreated spinach PSII membranes at pH 7.5 produce a S2 state multiline EPR spectrum when illuminated at either 210 K or at 0°C in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) having an average hyperfine line spacing of 87.5 G. The temperature dependence of the S2 state multiline EPR signal observed from untreated samples deviates from the Curie law above 5 K, with a maximum signal intensity at 6.9 K as has been previously observed. In contrast, 100 mM NH4Cl-treated PSII membranes at pH 7.5 exhibit a new S2 state EPR spectrum when illuminated at 0°C in the presence of DCMU. The novel S2 state EPR spectrum from NH4Cl-treated PSII membranes has an average hyperfine line spacing of 67.5 G and a temperature dependence obeying the Curie law except for small deviations at low temperature. We assign the new S2 state EPR signal from NH4Cl-treated PSII membranes to a form of the S2 state having one or more NH3 molecules directly coordinated to the Mn site. NH3 does not bind to Mn in the dark-stable S1 state present before illumination, since generation of the S2 state in NH4Cl-treated PSII membranes by illumination at 210 K does not yield the new S2 state EPR spectrum. Since inhibition of O2 evolution activity in the presence of NH4Cl probably occurs through binding of NH3 to the O2-evolving complex in competition with substrate H2O molecules, these results indicate that the EPR-detectable Mn site functions as the substrate-binding site of the O2-evolving complex.
Analysis of Mn-55 electron spin echo-electron nuclear double resonance (ESE-ENDOR) spectra obtained on a dinuclear mixed valence Mn(III)Mn(IV) complex [di-mu-oxotetrakis(2,2'-bipyridine)dimanganese(III,IV)] (1) reveals the hyperfine and nuclear quadrupolar parameters for the spin I = 5/2 Mn-55 nucleus of both Mn(III) and Mn(IV) ions. The following parameters are obtained: for Mn(IV), A(perpendicular to) = +212 MHz, A(parallel to) = +231 MHz, P-parallel to 3e(2)Qq/40 = +2.0 MHz, and eta = 0.3, and for Mn(III), A perpendicular to = -480 MHz, A(parallel to) -360 MHz, P-parallel to = -4.5 MHz, and eta = 0.1. The Mn-55 ESE-ENDOR data obtained on the g = 2 Mn multiline EPR signal of the S-2 state of the photosystem II oxygen-evolving complex demonstrate that this EPR signal cannot arise from a dinuclear Mn(III)Mn(IV) center. The ENDOR spectra are consistent with a tetranuclear Mn cluster origin for the photosystem II multiline EPR signal.
Catalysts which functionally mimic the bacterial dimanganese catalase enzymes have been synthesized and their structure, electrochemical, redox, and EPR spectra have been compared to the enzyme. These compounds are formulated as [LMn(2)(II,II)X]Y-2,mu-X=CH3CO2, ClCH2CO2; Y=ClO4, BPh(4), CH3CO2, possessing a bridging mu-alkoxide from the ligand, HL = N,N,N'N'-tetrakis(2-methylenebenzimidazole)-1,3-diaminopropan-2-ol. An X-ray diffraction structure of [LMn(2)(CH3CO2)(butanol)](ClO4)(2).H2O, in the monoclinic space group P2(1)/c, confirmed the anticipated N6O septadentate coordination of the HL ligand, the bridging mu-acetate, and revealed both five- and six-coordinate Mn ions; the latter arising from a butanol solvent molecule. This contrasts with the six-coordinate Mn ions observed for the mu-Cl and mu-OH derivatives, LMn(2)Cl(3) and LMn(2)(OH)Br-2 (Mathur et al. J. Am. Chem. Soc. 1987, 109, 5227-5232). Like the enzyme, three electrons can be removed from these complexes to form four oxidation states ranging from Mn-2(II,II) to Mn-2(III,IV). Three of these have been characterized by EPR and found to possess electronic ground states, Mn-III electron orbital configurations, Mn-55 hyperfine parameters, and Heisenberg exchange interactions analogous to those observed in the enzyme. For the mu-carboxylate derivatives electrochemistry reveals the initial oxidation process involves loss of two electrons at 0.81-0.86 V, forming Mn-2(III,III), followed by dismutation to yield a Mn-2(II,III) and Mn-2(III,IV) species. By contrast, the mu-Cl and mu-OH derivatives oxidize by an initial one-electron process (0.49-0.54 V). For the mu-carboxylate derivatives chemical oxidation with Pb(OAc)(4) also reveals an initial two-electron oxidation to a Mn-2(III,III) species, which dismutates to form both Mn-2(II,III) and Mn-2(III,IV) species. The two Mn-2(II,III) species formed by these methods exhibit Mn-55 hyperfine fields differing in magnitude by 9% (150 G), implying different Mn coordination environments induced by the electrolyte. The different ligand coordination observed in the enzyme (predominantly oxo and carboxylato) appears to be responsible for stabilization of the MnCat(III,III) oxidation state as the resting state.
A general approach for simulation of EPR spectra of mixed-valence dimanganese complexes and proteins is presented, based on the theory of Sage et al. (J. Am. Chem,Soc. 1989, 111, 7239-7247),which overcomes limitations inherent in the theory of strongly coupled ions. This enables explanation of ''anomalous'' spectral parameters and extraction of accurate g tensors and Mn-55 magnetic hyperfine tensors from which the spatial distribution of the unpaired spin density, the electronic configuration, and ligand field parameters have been obtained, This is used to analyze highly accurate simulations of the EPR spectra, obtained by least-squares fits of two mixed valence oxidation states, from a series of dimanganese(II,III) and dimanganese(III,IV) complexes and from the dimanganese catalase enzyme, MnCat(II,III) and MnCat(III,IV), from Thermus thermophilus. The sign of the Mn-55 dipolar hyperfine anisotropy (Delta a) reveals that the valence orbital configuration of the Mn(III) ion in MnCat(III,IV) and all dimanganese(III,IV) complexes possessing sterically unconstrained bis(mu-oxo) bridges is d(pi)(3)(d(22))(1) with the antibonding d(22) electron oriented perpendicular to the plane of the Mn-2(mu-O)(2) rhombus. This accounts for the strong Mn-O bonding and slow ligand exchange kinetics widely observed. The asymmetry of the spin density of Mn(III) increases substantially from Delta a/a(iso) = 0.27 in MnCat(III,IV) to 0.46 in MnCat(II,III), reflecting a change in manganese coordination. Comparison with model complexes suggest this may be due to protonation and opening of the (mu-O)(2) bridge upon reduction to yield a single mu-OH bridge. The presence of strong Mn-O bonding in an unreactive (mu-O)(2) core of MnCat(III,IV) offers a plausible explanation for the 10(12) slower kinetics of peroxide dismutation compared to what is observed for the physiologically important oxidation state MnCat(II,II). For the dimanganese(II,III) oxidation state, the theory also provides the first explanation for the anomalously large (similar to 30%) Mn-55(II) hyperfine anisotropy in terms of admiring of the S = 3/2 excited state into the ground state (S = 1/2) via the zero-freld splitting interaction of Mn(III). This ''transferred'' anisotropy obscures the otherwise typical isotropic high-spin 3d(5) orbital configuration of Mn(II). An estimate of the ratio of the zero-field splitting to the Heisenberg exchange interaction (D/J) is obtained. The theory also explains the unusuarl 12-line EPR spectrum for a weakly coupled dimanganese(III,IV) complex (Larson et al. J. Am. Chem. Sec. 1992, 114, 6263-6265), in contrast to the typical 16-line ''multiline'' spectra seen in strongly coupled dimanganese(III,IV) complexes. The theory shows this is due to a weak J = -10 cm(-1) which results in a D/J ratio approaching unity and not to unusual intrinsic magnetic hyperfine parameters of the Mn ions.
The microwave power for half-saturation (P1/2) for the radical in photosystem II giving rise to signal IIslow (SIIs) has been measured by EPR in samples illuminated by a series of flashes. The charge storage state of the oxygen-evolving complex (S0-S4) was monitored by measuring the multiline EPR signal arising from the S2 state. The following results were obtained: (1) SIIs becomes easier to saturate after tris(hydroxymethyl)aminomethane (Tris) washing, a treatment that partially removes the Mn cluster. (2) P1/2 for SIIs oscillates with the flash number. P1/2 is lower in S1 (in dark-adapted material and after four flashes) than in S2, S3, or S0. (3) P1/2(S2) = P1/2(S3). (4) At 8 K P1/2(S2) > P1/2(S2), but at 20 K P1/2(S0) < P1/2(S2). (5) P1/2 for SIIs increases with temperature (8-70 K) in the S1 state. SHs is more difficult to saturate in S2, S3, and S0 than in S1 over the investigated temperature range. In addition, the increase in P1/2 is complex around 20-30 K in S2, S3, and S0. (6) In S0, P1/2 for SIIs decreases with time (decay half-time 30-60 s) to a stable level significantly above the dark level. The data are explained in terms of cross relaxation between the radical giving rise to SIIs and an efficient relaxer, which is suggested to be the Mn cluster. This relaxes more slowly in S1 than in the other S states. Since it is known that a mixed-valence Mn cluster is present in S2, and because P1/2 of SIIs in S3 and S0 is comparable to that in S2, it is suggested that mixed-valence Mn clusters are present in the S3 and S0 states also. Different models with these features can be proposed, the simplest of which is the following: S0 [Mn(H)-Mn(III)], S1 [Mn(III)-Mn(III)], S2 [Mn(III)-Mn(IV)], and S3 [Mn(III)-Mn(IV)].
A study of electron paramagnetic resonance (EPR) signals from components on the electron donor side of photosystem II has been performed. By measurement of EPR signal IIslow (D+) it is shown that, after three flashes, D+ decays slowly in the dark at room temperature in the fraction of the centers that was in the S0 state (t1/2 of 20 min in thylakoid membranes and 50 min in photosystem II enriched membranes). This reaction is accompanied by a conversion of S0 to S1. The concentration of S1 was estimated from the amplitude of the S2-state multiline EPR signal that could be generated by illumination at 200 K. These observations indicate that D+ accepts an electron from S0 in a dark reaction in which D and S1 are formed. In addition, the reactions by which D donates an electron to S2 or S3 have been directly measured by monitoring both signal IIslow and the multiline signal. The redox interactions between the D/D+ couple and the S states are explained in terms of a model in which D/D+ has a midpoint potential between those of S0/S1, and S1/S2. In addition, this model provides explanations for a number of previously unrelated phenomena, and the proposal is put forward that the reaction between D+ and Mn2+ is involved in the so-called photoactivation process.
The effect of protonation events on the charge equilibrium between tyrosine-D and the water-oxidizing complex in photosystem II has been studied by time-resolved measurements of the EPR signal IIslow at room temperature. The flash-induced oxidation of YD by the water-oxidizing complex in the S2 state is a monophasic process above pH 6.5 and biphasic at lower pHs, showing a slow and a fast phase. The half-time of the slow phase increases from about 1 s at pH 8.0 to about 20 s at pH 5.0, whereas the half-time of the fast phase is pH independent (0.4-1 s). The dark reduction of YD+ was followed by measuring the decay of signal IIslow at room temperature. YD+ decays in a biphasic way on the tens of minutes to hours time scale. The minutes phase is due to the electron transfer to YD+ from the S0 state of the water-oxidizing complex. The half-time of this process increases from about 5 min at pH 8.0 to 40 min at pH 4.5. The hours phase of YD+ has a constant half-time of about 500 min between pH 4.7 and 7.2, which abruptly decreases above pH 7.2 and below pH 4.7. This phase reflects the reduction of YD+ either from the medium or by an unidentified redox component of PSII in those centers that are in the S1 state. The titration curve of the half-times for the oxidation of YD reveals a proton binding with a pK around 7.3-7.5 that retards the electron transfer from YD to the water-oxidizing complex. We propose that this monoprotic event reflects the protonation of an amino acid residue, probably histidine-190 on the D2 protein, to which YD is hydrogen bonded. The titration curves for the oxidation of YD and for the reduction of YD+ show a second proton binding with pK approximately 5.8-6.0 that accelerates the electron transfer from YD to the water-oxidizing complex and retards the process in the opposite direction. This protonation most probably affects the water-oxidizing complex. From the measured kinetic parameters, the lowest limits for the equilibrium constants between the S0YD+ and the S1YD as well as between the S1YD+ and S2YD states were estimated to be 5 and 750-1000, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
Photosystem II enriched membranes were depleted of Ca2+ and the 17- and 23-kDa polypeptides by treatment with NaCl and EGTA. The 17- and 23-kDa polypeptides were then reconstituted. This preparation was incapable of O2 evolution until Ca2+ was added. An EPR study revealed the presence of two new EPR signals. One of these is a modified S2 multiline signal with an isotropic g value of 1.96 with at least 26 hyperfine peaks (average spacing 55 G) distributed over approximately 1600 G. The other is a near-Gaussian signal with an isotropic g value of 2.004, which is attributed to a formal S3 state. Experiments involving the interconversion of these signals and the effect of Ca2+ and Sr2+ rebinding provide evidence for these assignments. From these results the following conclusions are drawn: (1) These results are consistent with our earlier demonstration that charge accumulation is blocked after formation of S3 when Ca2+ is deficient. (2) Binding of the 17- and 23-kDa polypeptides to photosystem II in the absence of Ca2+ results in the perturbation of the Mn cluster. This is taken as a further indication that the Ca2+-binding site is close to or even an integral part of the Mn cluster. (3) The S3 signal may arise from an organic free radical interacting magnetically with the Mn cluster. However, other possible origins for this signal, including the Mn cluster itself, must also be considered.
The light-driven water-splitting/oxygen-evolving enzyme remains one of the great enigmas of plant biology. However, due to the recent expansion of research efforts on this enzyme, it is grudgingly giving up some of its secrets.
The application of magnetic resonance techniques to biological systems has permitted a detailed study of the nature of the active sites of many proteins that had not been possible previously. Among these is the whole class of iron—sulphur proteins which have been implicated as electron transport proteins in a variety of fundamental processes: photosynthesis, hydroxylation and nitrogen fixation to name but a few.
The single-iron proteins in this class, the rubredoxins, have been studied extensively by chemical, spectroscopic and X-ray crystallographic techniques (Lovenberg, 1973), and the active site is composed of a single iron atom bound in a distorted tetrahedron of cysteine sulphur ligands. The iron is high-spin ferric in the oxidized state and high-spin ferrous in the reduced state. This structure is shown in Fig. I (α).
The S2 state electron paramagnetic resonance (EPR) multiline signal of Photosystem II has been simulated at Q-band (35 Ghz), X-band (9 GHz) and S-band (4 GHz) frequencies. The model used for the simulation assumes that the signal arises from an essentially magnetically isolated MnIII-MnIV dimer, with a ground state electronic spin ST = 1/2. The spectra are generated from exact numerical solution of a general spin Hamiltonian containing anisotropic hyperfine and quadrupolar interactions at both Mn nuclei. The features that distinguish the multiline from the EPR spectra of model manganese dimer complexes (additional width of the spectrum (195 mT), additional peaks (22), internal "superhyperfine" structure) are plausibly explained assuming an unusual ligand geometry at both Mn nuclei, giving rise to normally forbidden transitions from quadrupole interactions as well as hyperfine anisotropy. The fitted parameters indicate that the hyperfine and quadrupole interactions arise from Mn ions in low symmetry environments, corresponding approximately to the removal of one ligand from an octahedral geometry in both cases. For a quadrupole interaction of the magnitude indicated here to be present, the MnIII ion must be 5-coordinate and the MnIV 5-coordinate or possibly have a sixth, weakly bound ligand. The hyperfine parameters indicate a quasi-axial anisotropy at MnIII, which while consistent with Jahn-Teller distortion as expected for a d4 ion, corresponds here to the unpaired spin being in the ligand deficient, z direction of the molecular reference axis. The fitted parameters for MnIV are very unusual, showing a high degree of anisotropy not expected in a d3 ion. This degree of anisotropy could be qualitatively accounted for by a histidine ligand providing pi backbonding into the metal dxy orbital, together with a weakly bound or absent ligand in the x direction.
In the photosynthetic evolution of oxygen, water oxidation occurs at a catalytic site that includes four manganese atoms together
with the essential cofactors, the calcium and chlorine ions. A structural model and a determination of the manganese oxidation
states based on x-ray absorption spectroscopy are presented. The salient features, in both higher plants and cyanobacteria,
are a pair of di-mu-oxo bridged manganese binuclear clusters linked by a mono-mu-oxo bridge, one proximal calcium atom, and
one halide. In dark-adapted samples, manganese occurs in oxidation states (III) and (IV). Data from oriented membranes display
distinct dichroism, precluding highly symmetrical structures for the manganese complex.
Magnetic properties of the S1-state manganese cluster in the oxygen-evolving photosystem II were studied by parallel polarization electron paramagnetic resonance spectroscopy. Dark minus light spectra gave rise to a broad S1-state signal with a g value of about 4.9 [Dexheimer, S. L., Klein, M. P. (1992) J. Am. Chem. Soc. 114, 2821-2826]. Temperature variation of the signal intensity between 1.9 and 10 K observed in PS II with a sucrose buffer indicates that the signal originates from an excited state with a spin S of 1 with separation from the ground state (S = 0) of about 2.5 K. The S1-state signal was also observed in the sucrose buffer supplemented by 50% glycerol. However, no S1-state signal was detected by addition of 3% methanol or 30% ethylene glycol in the sucrose buffer, although illumination at 200 K in the presence of these alcohols induced the normal multiline S2 signal. Furthermore, modification of the Mn cluster by Cl- or Ca2+ depletion from PS II membranes failed to produce a detectable S1-state signal. A possible magnetic structure of the Mn cluster responsible for the generation of the S1-state signal is discussed on the basis of these observations.
The S0* state was generated by incubation of dark-adapted (S1 state) photosystem II membranes either with the exogenous two electron reductant hydrazine and subsequent 273 K illumination in the presence of DCMU or by dark incubation with low amounts of the one electron reductant hydroxylamine. In agreement with earlier reports, the S1 and S-1 states were found to be electron paramagnetic resonance (EPR) silent. However, in the presence of 0.5-1.5% methanol, a weak EPR multiline signal centered around g = 2.0 was observed at 7 K for the S0* states generated by both procedures. This signal has a similar average line splitting to the well-characterized S2 state multiline EPR signal, but can be clearly distinguished from that and other modified S2 multiline signals by differences in line position and intensities. In addition, at 4 K it can be seen that the S0* multiline has a greater spectral breadth than the S2 multilines and is composed of up to 26 peaks. The S0* signal is not seen in the absence of methanol and is not affected by 1 mM EDTA in the buffer medium. We assign the S0* multiline signal to the manganese cluster of the oxygen evolving complex in a mixed valence state of the form MnIIMnIIIMnIIIMnIII,MnIIMnIIIMnIVMnIV, or MnIIIMnIIIMnIIIMnIV. Addition of methanol may be helpful in future to find an EPR signal originating form the natural S0 state.
The essential involvement of manganese in photosynthetic water oxidation was implicit in the observation by Pirson in 1937 that plants and algae deprived of Mn in their growth medium lost the ability to evolve Oâ. Addition of this essential element to the growth medium resulted in the restoration of water oxidation within 30 min. There is increased interest in the study of Mn in biological chemistry and dioxygen metabolism in the last two decades with the discovery of several Mn redox enzymes. The list of enzymes where Mn is required for redox activity includes a Mn superoxide dismutase, a binuclear Mn-containing catalase, a binuclear Mn-containing ribonucleotide reductase, a proposed binuclear Mn site in thiosulfate oxidase, a Mn peroxidase that is capable of oxidative degradation of lignin, and perhaps the most complex and important, the tetranuclear Mn-containing oxygen-evolving complex in photosystem II (Mn-OEC). Mn is well suited for the redox role with accessible oxidation states of II, III, and IV, and possibly V: oxidation states that have all been proposed to explain the mechanisms of the Mn redox enzymes.
X-ray absorption near-edge structure spectra of the manganese (Mn) cluster in physiologically native intermediate states of photosynthetic water oxidation induced by short laser flash were measured with a compact heat-insulated chamber equipped with an x-ray detector near the sample surface. The half-height energy of the Mn Kedge showed a period-four oscillation dependent on cycling of the Joliot-Kok's oxygen clock. The flash number-dependent shift in the Mn K-edge suggests that the Mn cluster is oxidized by one electron upon the S(0)-to-S(1), S(1)-to-S(2), and S(2)-to-S(3) transitions and then reduced upon the S(3)-to-S(0) transition that releases molecular oxygen.