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Split EPR Signals from Photosystem II Are Modified by Methanol, Reflecting S State-Dependent Binding and Alterations in the Magnetic Coupling in the CaMn 4 Cluster †
Abstract
Methanol binds to the CaMn4 cluster in photosystem II (PSII). Here we report the methanol dependence of the split EPR signals originating from the magnetic interaction between the CaMn4 cluster and the Y(Z)* radical in PSII which are induced by illumination at 5 K. We found that the magnitudes of the "split S1" and "split S3" signals induced in the S1 and S3 states of PSII centers, respectively, are diminished with an increase in the methanol concentration. The methanol concentrations at which half of the respective spectral changes had occurred ([MeOH](1/2)) were 0.12 and 0.57%, respectively. By contrast, the "split S0" signal induced in the S0 state is broadened, and its amplitude is enhanced. [MeOH](1/2) for this change was found to be 0.54%. We discuss these observations with respect to the location and nature of the methanol binding site. Furthermore, by comparing this behavior with methanol effects reported for other EPR signals in the different S states, we propose that the observed methanol-dependent changes in the split S1 and split S0 EPR signals are caused by an increase in the extent of magnetic coupling within the cluster.
... Because the substrate of the OEC is water, analysis of water interactions with the OEC is complicated, as true substrate waters must be differentiated from additional ligand and matrix waters that are not directly involved in water oxidation. To this end, the interactions of the OEC with small molecule analogues to water, such as small amines, 10,13-26 hydrazine, 25,26 hydrogen peroxide, 27-29 and several primary alcohols 19,22,[30][31][32][33][34][35][36][37][38][39][40] have been used to build a better picture of the substrate binding modes. ...
... Methanol has been shown to affect the EPR spectra of many of the oxidation states of the OEC and has been proposed to bind to the cluster by coordinating to one or more of the Mn ions. 30,[32][33][34][35][36][37] The presence of methanol changes the shape and/or intensity of almost all continuous-wave electron paramagnetic resonance (CW EPR) spectroscopic signals of the Oyala et al. ...
... OEC, including the S 0 (spin S = 1/2) multiline 33,36,[41][42][43][44][45][46][47] and S 2 (S = 1/2) multiline signal, 11,[31][32][33]48,49 the two parallel-mode signals of the S 1 state of higher plants (S = 1) 50,51 and cyanobacteria (S = 2), 52 and the split signals that result from coupling of the Y z • radical to the paramagnetic forms of S 0 , S 1 , and S 3 . 35 There appear to be differences in the concentration dependence of the effects of methanol on these signals that are S-state dependent, indicating that the binding affinity of MeOH at the Mn 4 CaO 5 cluster may change as a function of the oxidation state of the cluster. 34 While the S 2 state typically exhibits an EPR multiline (MLS) signal at g = 2 arising from an S = 1/2 species, higher spin signals at g ≥ 4.1 arising from a high-spin (S = 5/2) 53 conformation of the Mn 4 CaO 5 cluster are also observed under a variety of sample conditions. ...
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.
... In this report, we calculate solvent contact surfaces from the PSII crystal structures to identify such access channels for methanol and water molecules. In a previous study of the effects of methanol on the EPR split S 1 -, S 3 -, and S 0signals [Su et al. (2006) Biochemistry 45, 7617-7627], we proposed that methanol binds to one and the same Mn ion in all S-states. We find here that while channels of methanol dimensions were able to make contact with the CaMn 4 cluster, only 3 Mn and 4 Mn were accessible to methanol. ...
... S-state-specific EPR signals have been extensively used to study the S-cycle, and we have recently published data on the effects of methanol on each of the so-called split EPR signals then known [10], and compared those with methanol effects on other EPR signals arising from all S-states (excepting S 4 ). It was concluded that methanol binds to one and the same Mn ion of the CaMn 4 cluster for all S-states, and that the different methanol sensitivities thus reflect the structural changes in the cluster during the S-cycle, rather than different binding sites. ...
... Many small molecules are able to bind to the CaMn 4 cluster, and methanol in particular has been much studied, as it causes detectable changes in spectroscopic signals (reviewed in [10]). Pulsed EPR has been useful for studying the nature of methanol binding to the cluster. ...
Given the tightly packed environment of Photosystem II (PSII), channels are expected to exist within the protein to allow the movement of small molecules to and from the oxygen evolving centre. In this report, we calculate solvent contact surfaces from the PSII crystal structures to identify such access channels for methanol and water molecules. In a previous study of the effects of methanol on the EPR split S1-, S3-, and S0-signals [Su et al. (2006) Biochemistry 45, 7617-7627], we proposed that methanol binds to one and the same Mn ion in all S-states. We find here that while channels of methanol dimensions were able to make contact with the CaMn4 cluster, only 3Mn and 4Mn were accessible to methanol. Combining this observation with spectroscopic data in the literature, we propose that 3Mn is the ion to which methanol binds. Furthermore, by calculating solvent contact surfaces for water, we found analogous and more extensive water accessible channels within PSII. On the basis of their structure, orientation, and electrostatic properties, we propose functional assignments of these channels as passages for substrate water access to the CaMn4 cluster, and for the exit of O2 and H+ that are released during water oxidation. Finally, we discuss the possible existence of a gating mechanism for the control of substrate water access to the CaMn4 cluster, based on the observation of a gap within the channel system that is formed by Ca2+ and several mechanistically very significant residues in the vicinity of the cluster.
... In recent years, several metalloradical EPR signals (split signals) attributed to the magnetic interaction between the oxidised Y Z (Y Z ox ) and the CaMn 4 cluster in PSII have been described from each of the S 1 , S 2 , S 3 and S 0 states when samples are illuminated with visible and/or NIR light at liquid helium temperatures [27][28][29][30][31][32][33][34][35][36][37][38][39]. The split S 1 and split S 3 signals are asymmetric: the former is characterised by a low-field peak at g = 2.035 and the latter has a double trough at the high-field side of g = 2 together with a weak broad peak at the low-field side. ...
... The split S 1 and split S 0 EPR signals are induced by illumination with visible light at 5 K. This illumination also induces a narrow radical EPR signal from competing oxidation of Car or Chl via the Car-Chl Z -Cytb 559 pathway [33][34][35][37][38][39]. The time dependence of the induction of the split S 0 and split S 1 signals under our particular illumination conditions (see Materials and methods) are shown in Fig. 1A. ...
... The fraction in the S 2 state could be determined from the S 2 multiline signal as before. However, the EPR spectrum recorded after visible light illumination for 4 min at 5 K contained a mixture of the split S 0 , split S 1 and split S 3 EPR signals (Fig. 4A, spectra a-d) [37][38][39]. ...
EPR spectroscopy is very useful in studies of the oxygen evolving cycle in Photosystem II and EPR signals from the CaMn(4) cluster are known in all S states except S(4). Many signals are insufficiently understood and the S(0), S(1), and S(3) states have not yet been quantifiable through their EPR signals. Recently, split EPR signals, induced by illumination at liquid helium temperatures, have been reported in the S(0), S(1), and S(3) states. These split signals provide new spectral probes to the S state chemistry. We have studied the flash power dependence of the S state turnover in Photosystem II membranes by monitoring the split S(0), split S(1), split S(3) and S(2) state multiline EPR signals. We demonstrate that quantification of the S(1), S(3) and S(0) states, using the split EPR signals, is indeed possible in samples with mixed S state composition. The amplitudes of all three split EPR signals are linearly correlated to the concentration of the respective S state. We also show that the S(1) --> S(2) transition proceeds without misses following a saturating flash at 1 degrees C, whilst substantial misses occur in the S(2) --> S(3) transition following the second flash.
... Addition of few percent of methanol is known to produce such effects; it enhances the low spin g = 2 multiline EPR signal and fully suppresses the high spin g = 4.1 EPR signal (Deak et al. 1999). It is also known to completely eliminate or modify the so-called Split S state EPR signals (Su et al. 2006). In order to investigate the S 2 → S 3 state transition where the starting S 2 state contains both low and high spin electronic configurations of the CaMn 4 O 5 -cluster, we performed our next experiments in a sucrose containing buffer with 0% methanol. ...
... The lowering of its half-inhibition temperature by ethylene glycol is difficult to assign to the specific site and most probably is a general effect, possibly reflecting the greater ability of ethylene glycol compared to sucrose to keep protein motions and water mobility active down to lower temperatures. In contrast, methanol is a small molecule, which is known to interact with the CaMn 4 O 5 -cluster and change its spectroscopic properties (Messinger et al. 1997;Åhrling et al. 1997Force et al. 1998;Deak et al. 1999;Su et al. 2006;Oyala et al. 2014;Nagashima and Mino 2017;Yata and Noguchi 2018;Zahariou et al. 2021;Kalendra et al. 2022). It has been suggested to bind close to either Mn 1 or Mn 4 , thus disturbing the H-bonding network around the cluster Nagashima and Mino 2017;Kalendra et al. 2022). ...
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.
... Methanol is a key molecule in this respect, because it affects the electronic structure and modies the EPR signatures 75 of all states. [47][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62][63][64][65] A major effect is that it increases the energy separation between the lowest magnetic levels of the OEC, 48,51,60,62 stabilizing the S ¼ 1/2 ground spin states of the S 0 and S 2 states and the diamagnetic ground state of S 1 . Other effects relate to the amplitude enhancement of specic spectral forms, for example of the g z 2 multiline signal of the S 2 state in plants over the g $ 4 component. ...
... Methanol is a key molecule in this respect, because it affects the electronic structure and modies the EPR signatures 75 of all states. [47][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62][63][64][65] A major effect is that it increases the energy separation between the lowest magnetic levels of the OEC, 48,51,60,62 stabilizing the S ¼ 1/2 ground spin states of the S 0 and S 2 states and the diamagnetic ground state of S 1 . Other effects relate to the amplitude enhancement of specic spectral forms, for example of the g z 2 multiline signal of the S 2 state in plants over the g $ 4 component. ...
Methanol has long being used as a substrate analogue to probe access pathways and investigate water delivery at the oxygen-evolving complex (OEC) of photosystem-II. In this contribution we study the interaction of methanol with the OEC by assembling available spectroscopic data into a quantum mechanical treatment that takes into account the local channel architecture of the active site. The effect on the magnetic energy levels of the Mn4Ca cluster in the S2 state of the catalytic cycle can be explained equally well by two models that involve either methanol binding to the calcium ion of the cluster, or a second-sphere interaction in the vicinity of the "dangler" Mn4 ion. However, consideration of the latest ¹³C hyperfine interaction data shows that only one model is fully consistent with experiment. In contrast to previous hypotheses, methanol is not a direct ligand to the OEC, but is situated at the end-point of a water channel associated with the O4 bridge. Its effect on magnetic properties of plant PS-II results from disruption of hydrogen bonding between O4 and proximal channel water molecules, thus enhancing superexchange (antiferromagnetic coupling) between the Mn3 and Mn4 ions. The same interaction mode applies to the dark-stable S1 state and possibly to all other states of the complex. Comparison of protein sequences from cyanobacteria and plants reveals a channel-altering substitution (D1-Asn87 versus D1-Ala87) in the proximity of the methanol binding pocket, explaining the species-dependence of the methanol effect. The water channel established as the methanol access pathway is the same that delivers ammonia to the Mn4 ion, supporting the notion that this is the only directly solvent-accessible manganese site of the OEC. The results support the pivot mechanism for water binding at a component of the S3 state and would be consistent with partial inhibition of water delivery by methanol. Mechanistic implications for enzymatic regulation and catalytic progression are discussed.
... To further check whether the weak signals at g = 2.18 and 1.85 are correlated to the S 1 Tyr Z • species, we have studied the methanol effect on these signals. It is well known that methanol can interact with the Mn-cluster and significantly modify its EPR signals in different S states (Messinger et al. 1997;Force et al. 1998;Deak et al. 1999;Su et al. 2006;Å hrling et al. 2006;Haddy 2007). Methanol can also affect the EPR signals arising from the magnetic interaction between Tyr Z • and the Mn-cluster in various S states . ...
... Figure 4 shows that both the g = 2.03 and the weak g = 2.15 and g = 1.85 EPR signals disappear after addition of 6% (v/v) methanol in the PSII core complex. The disappearance of g = 2.03 signal upon addition of methanol is consistent with the previous report Su et al. 2006). It is interesting that the weak g = 2.18 and 1.85 signals are also sensitive to the presence of methanol and disappear after addition of 6% methanol. ...
Tyrosine Z (Tyr(Z)) oxidation observed at liquid helium temperatures provides new insights into the structure and function of Tyr(Z) in active Photosystem II (PSII). However, it has not been reported in PSII core complex from higher plants. Here, we report Tyr(Z) oxidation in the S(1) and S(2) states in PSII core complex from spinach for the first time. Moreover, we identified a 500 G-wide symmetric EPR signal (peak position g = 2.18, trough position g = 1.85) together with the g = 2.03 signal induced by visible light at 10 K in the S(1) state in the PSII core complex. These two signals decay with a similar rate in the dark and both disappear in the presence of 6% methanol. We tentatively assign this new feature to the hyperfine structure of the S(1)Tyr(Z)(*) EPR signal. Furthermore, EPR signals of the S(2) state of the Mn-cluster, the oxidation of the non-heme iron, and the S(1)Tyr(Z)(*) in PSII core complexes and PSII-enriched membranes from spinach are compared, which clearly indicate that both the donor and acceptor sides of the reaction center are undisturbed after the removal of LHCII. These results suggest that the new spinach PSII core complex is suitable for the electron transfer study of PSII at cryogenic temperatures.
... This however, in striking contrast to the model proposed here, is not supported by the XFEL structural data.23 In addition it has been shown that Mn(III) is required for NIR excitation30 and the large D value of 1.523 cm -1 for the S=6 form strongly suggests the presence of Mn(III) ion in the complex. Our analysis indicates that O-O bond formation has begun between the O5 and O6 atoms in the S 3 state with the generation of the [O5O6] 3ion. ...
In this report we combine broken symmetry density functional calculations and electron paramagnetic resonance analysis to obtain the electronic structure of the penultimate S3 state of Nature’s water oxidising complex and determine the electronic pathway of O-O bond formation. Analysis of the electronic structure changes along the reaction path shows that two spin crossovers, facilitated by the geometry and magnetism of the water oxidising complex are used to provide a unique low energy pathway. The pathway is facilitated via formation and stabilisation of the [O2]3- ion. This ion is formed between ligated deprotonated substrate waters, O5 and O6, and is stabilised by antiferromagnetic interaction with the Mn ions of the complex. Combining computational, crystallographic and spectroscopic data we show that an equilibrium exists between an O5 oxo and O6 hydroxo form with an S=3 spin state and a deprotonated O6 form containing a two-centre one electron bond in [O5O6]3- which we identify as the form detected using XFEL crystallography. This form gives rise to an S=6 spin state which we demonstrate gives rise to a low intensity EPR spectrum compared with the accompanying S=3 state, making its detection via EPR difficult and overshadowed by the S=3 form. Simulations using 50% -70% of the S=6 component give rise to a superior fit to the experimental W- band EPR spectral envelope compared with an S=3 only form. The computational , crystallographic and spectroscopic data are shown to coalesce to the same picture of a predominant S=6 species containing the first one-electron oxidation product of two water molecules i.e. [O5O6]3-. Progression of this form to the two-electron oxidised peroxo and three-electron oxidised superoxo forms, leading eventually to the evolution of triplet O2 , is shown to be the pathway Nature adopts to oxidise water under ambient conditions. The study reveals the key electronic, magnetic and structural design features of Nature’s catalyst which facilitates water oxidation to O2 under ambient conditions.
... 27 This, however, in striking contrast to the model proposed here, is not supported by the XFEL structural data. 23 In addition, it has been shown that Mn(III) is required for NIR excitation, 30 and the large D value of 1.523 cm −1 for the S = 6 form strongly suggests the presence of Mn(III) ion in the complex. It should be noted that it is possible that the peroxo form, Figure 2, is also present in a low concentration, and its EPR spectrum is masked by the oxo−hydroxo form. ...
In this paper, we combine broken symmetry density functional calculations and electron paramagnetic resonance analysis to obtain the electronic structure of the penultimate S3 state of nature's water-oxidizing complex and determine the electronic pathway of O-O bond formation. Analysis of the electronic structure changes along the reaction path shows that two spin crossovers, facilitated by the geometry and magnetism of the water-oxidizing complex, are used to provide a unique low-energy pathway. The pathway is facilitated via the formation and stabilization of the [O2]3- ion. This ion is formed between ligated deprotonated substrate waters, O5 and O6, and is stabilized by antiferromagnetic interaction with the Mn ions of the complex. Combining the computational, crystallographic, and spectroscopic data, we show that an equilibrium exists between the O5 oxo and O6 hydroxo forms with an S = 3 spin state and a deprotonated O6 form containing a two-center one-electron bond in [O5O6]3- which we identify as the form detected using crystallography. This form corresponds to an S = 6 spin state which we demonstrate gives rise to a low-intensity EPR spectrum compared with the accompanying S = 3 state, making its detection via EPR difficult and overshadowed by the S = 3 form. Simulations using 70% of the S = 6 component give rise to a superior fit to the experimental W-band EPR spectral envelope compared with an S = 3 only form. Analyses of the most recent X-ray emission spectroscopy first moment changes for solution and time-resolved crystal data are also shown to support the model. The computational, crystallographic, and spectroscopic data are shown to coalesce to the same picture of a predominant S = 6 species containing the first one-electron oxidation product of two water molecules, that is, [O5O6]3-. Progression of this form to the two-electron-oxidized peroxo and three-electron-oxidized superoxo forms, leading eventually to the evolution of triplet O2, is proposed to be the pathway nature adopts to oxidize water. The study reveals the key electronic, magnetic, and structural design features of nature's catalyst which facilitates water oxidation to O2 under ambient conditions.
... It has long been known that methanol modifies the magnetic energy levels and EPR signals of the manganese cluster of the OEC, but the access pathway(s) and mode of interaction remained unknown. 167,[172][173][174][175][176][177][178] Based on the experimental determination of the 13 C hyperfine parameters of isotopically labelled methanol interacting with the S 2 state of the OEC, 167 an extensive computational screening of structural models was carried out that involved calculation of spin state energetics and 13 C isotropic and dipolar HFCs for each one. 116 This led to rejection of several possibilities such as direct binding of methanol to Ca 2+ or one of the Mn ions, supporting instead a second-sphere interaction that resulted in reorganization of the hydrogen bonding network affecting the O4 bridge and the Mn3-Mn4 exchange coupling interaction. ...
Quantum chemical approaches today are a powerful tool to study the properties and reactivity of metalloenzymes. In the field of solar fuels research these involve predominantly photosystem II and hydrogenases, which catalyze water oxidation and hydrogen evolution, as well as related biomimetic and bio-inspired models. Theoretical methods are extensively used to better comprehend the nature of catalytic intermediates, establish important structure-function and structure-property correlations, elucidate functional principles, and uncover the catalytic activity of these complex systems by unravelling the key steps of their reaction mechanism. Computations in the field of water oxidation and hydrogen evolution are used as predictive tools to elucidate structures, explain and synthesize complex experimental observations from advanced spectroscopic techniques, rationalize reactivity on the basis of atomistic models and electronic structure, and guide the design of new synthetic targets. This feature article covers recent advances in the application of quantum chemical methods for understanding the nature of catalytic intermediates and the mechanism by which photosystem II and hydrogenases achieve their function, and points at essential questions that remain only partly answered and at challenges that will have to be met by future advances and applications of quantum and computational chemistry.
... 77,78 There are, as yet, no synthetic model complexes with groups that control proton, water and oxygen movements. Styring and co-workers 79 have discussed the importance and the possible existence of a gating mechanism for controlling substrate water access to the Mn 4 Ca cluster. All the proposed functional assignments of these channels have been based on electrostatic, structural and orientational grounds. ...
The Oxygen Evolving Complex in photosystem II, which is responsible for the oxidation of water to oxygen in plants, algae and cyanobacteria, contains a cluster of one calcium and four manganese atoms. This cluster serves as a model for the splitting of water by energy obtained from sunlight. The recent published data on the mechanism and the structure of photosystem II provide a detailed architecture of the oxygen-evolving complex and the surrounding amino acids. Biomimetically, we expect to learn some strategies from this natural system to synthesize an efficient catalyst for water oxidation, that is necessary for artificial photosynthesis.
... In spinach the S 0 state multiline signal can only be detected in the presence of MeOH [49,50,58]. In contrast, the parallel mode S 1 g~4.9 [45,46] and S 3 g~8 and g~12 EPR signals [59], and the EPR "split" signals, which arise from the weak magnetic interaction between the Mn 4 O x Ca cluster and the Y Z [60,61], are no longer visible when the MeOH concentration is increased to 3-5% (v/v). Curiously, in cyanobacterial PS II the addition of MeOH does not modify the S 2 multiline signal, the addition of MeOH does not prevent the formation of high spin S 2 states under NIR illumination [62] and the S 0 state multiline is observable also in the absence of MeOH [63]. ...
The electronic properties of the Mn4OxCa cluster in the S2 state of the oxygen-evolving complex (OEC) were studied using X- and Q-band EPR and Q-band 55Mn-ENDOR using photosystem II preparations isolated from the thermophilic cyanobacterium T. elongatus and higher plants (spinach). The data presented here show that there is very little difference between the two species. Specifically it is shown that: (i) only small changes are seen in the fitted isotropic hyperfine values, suggesting that there is no significant difference in the overall spin distribution (electronic coupling scheme) between the two species; (ii) the inferred fine-structure tensor of the only MnIII ion in the cluster is of the same magnitude and geometry for both species types, suggesting that the MnIII ion has the same coordination sphere in both sample preparations; and (iii) the data from both species are consistent with only one structural model available in the literature, namely the Siegbahn structure [Siegbahn, P. E. M. Accounts Chem. Res. 2009, 42, 1871-1880, Pantazis, D. A. et al., Phys. Chem. Chem. Phys. 2009, 11, 6788-6798]. These measurements were made in the presence of methanol because it confers favorable magnetic relaxation properties to the cluster that facilitate pulse-EPR techniques. In the absence of methanol the separation of the ground state and the first excited state of the spin system is smaller. For cyanobacteria this effect is minor but in plant PS II it leads to a break-down of the ST = ½ spin model of the S2 state. This suggests that the methanol-OEC interaction is species dependent. It is proposed that the effect of small organic solvents on the electronic structure of the cluster is to change the coupling between the outer Mn (MnA) and the other three Mn ions that form the trimeric part of the cluster (MnB, MnC, MnD), by perturbing the linking bis-μ-oxo bridge. The flexibility of this bridging unit is discussed with regard to the mechanism of O-O bond formation.
... Fig. 6C displays the Split S 3 signals induced by visible light (black line) and 830 nm light (grey line) at 5 K in Arabidopsis. Both signals are very similar to the corresponding signals reported from spinach [107,109,113] and cyanobacteria [114]. The signal has a double trough at g = 1.95 and 1.93 position and a peak (for the near infrared induced signal) at g = 2.06 (Table 2). ...
Arabidopsis thaliana is widely used as a model organism in plant biology as its genome has been sequenced and transformation is known to be efficient. A large number of mutant lines and genomic resources are available for Arabidopsis. All this makes Arabidopsis a useful tool for studies of photosynthetic reactions in higher plants. In this study, photosystem II (PSII) enriched membranes were successfully isolated from thylakoids of Arabidopsis plants and for the first time the electron transfer cofactors in PSII were systematically studied using electron paramagnetic resonance (EPR) spectroscopy. EPR signals from both of the donor and acceptor sides of PSII, as well as from auxiliary electron donors were recorded. From the acceptor side of PSII, EPR signals from Q(A)- Fe²(+) and Phe- Q(A)- Fe²(+) as well as from the free Phe- radical were observed. The multiline EPR signals from the S₀- and S₂-states of CaMn₄O(x)-cluster in the water oxidation complex were characterized. Moreover, split EPR signals, the interaction signals from Y(Z) and CaMn₄O(x)-cluster in the S₀-, S₁-, S₂-, and the S₃-state were induced by illumination of the PSII membranes at 5K and characterized. In addition, EPR signals from auxiliary donors Y(D), Chl(+) and cytochrome b₅₅₉ were observed. In total, we were able to detect about 20 different EPR signals covering all electron transfer components in PSII. Use of this spectroscopic platform opens a possibility to study PSII reactions in the library of mutants available in Arabidopsis.
... • signal [59,60], but to leave the Q A -• EPR signal unchanged [55]. Indeed, we find that the peaks at g = 2.03, 2.18 and 1.85 due to the S 1 Tyr Z ...
The correlation between the reduction of Q(A) and the oxidation of Tyr(Z) or Car/Chl(Z)/Cyt(b559) in spinach PSII enriched membranes induced by visible light at 10 K is studied by using electron paramagnetic resonance spectroscopy. Similar g=1.95-1.86 Q(A)(-*)EPR signals are observed in both Mn-depleted and intact samples, and both signals are long lived at low temperatures. The presence of PPBQ significantly diminished the light induced EPR signals from Q(A)(-*), Car(+*)/Chl(+*) and oxidized Cyt(b559), while enhancing the amplitude of the S(1)Tyr(Z)* EPR signal in the intact PSII sample. The quantification and stability of the g=1.95-1.86 EPR signal and signals arising from the oxidized Tyr(Z) and the side-path electron donors, respectively, indicate that the EPR-detectable g=1.95-1.86 Q(A)(-*) signal is only correlated to reaction centers undergoing oxidation of the side-path electron donors (Car/Chl(Z)/Cyt(b559)), but not of Tyr(Z). These results imply that two types of Q(A)(-*) probably exist in the intact PSII sample. The structural difference and possible function of the two types of Q(A) are discussed.
In this report we combine broken symmetry density functional calculations and electron paramagnetic resonance analysis to obtain the electronic structure of the penultimate S3 state of Nature’s water oxidising complex and determine the electronic pathway of O-O bond formation. Analysis of the electronic structure changes along the reaction path shows that two spin crossovers, facilitated by the geometry and magnetism of the water oxidising complex are used to provide a unique low energy pathway. The pathway is facilitated via formation and stabilisation of the [O2]3- ion. This ion is formed between ligated deprotonated substrate waters, O5 and O6, and is stabilised by antiferromagnetic interaction with the Mn ions of the complex. Combining computational, crystallographic and spectroscopic data we show that an equilibrium exists between an O5 oxo and O6 hydroxo form with an S=3 spin state and a deprotonated O6 form containing a two-centre one electron bond in [O5O6]3- which we identify as the form detected using crystallography. This form corresponds to an S=6 spin state which we demonstrate gives rise to a low intensity EPR spectrum compared with the accompanying S=3 state, making its detection via EPR difficult and overshadowed by the S=3 form. Simulations using 70% of the S=6 component give rise to a superior fit to the experimental W- band EPR spectral envelope compared with an S=3 only form. Analysis of the most recent X-ray emission spectroscopy (XES) first moment changes for solution and time resolved crystal data are also shown to support the model. The computational, crystallographic and spectroscopic data are shown to coalesce to the same picture of a predominant S=6 species containing the first one-electron oxidation product of two water molecules i.e. [O5O6]3-. Progression of this form to the two-electron oxidised peroxo and three-electron oxidised superoxo forms, leading eventually to the evolution of triplet O2, is proposed to be the pathway Nature adopts to oxidise water. The study reveals the key electronic, magnetic and structural design features of Nature’s catalyst which facilitates water oxidation to O2 under ambient conditions.
The solar water-splitting protein complex, photosystem II (PSII), catalyzes one of the most energetically demanding reactions in nature by using light energy to drive a catalyst capable of oxidizing water. The water oxidation reaction is catalyzed at the Mn4Ca-oxo cluster in the oxygen-evolving complex (OEC), which cycles through five light-driven S-state intermediates (S0-S4). A detailed mechanism of the reaction remains elusive as it requires knowledge of the delivery and binding of substrate water in the higher S-state intermediates. In this study, we use two-dimensional (2D) hyperfine sublevel correlation spectroscopy, in conjunction with quantum mechanics/molecular mechanics (QM/MM) and density functional theory (DFT), to probe the binding of the substrate analog, methanol, in the S2 state of the D1-N87A variant of PSII from Synechocystis sp. PCC 6803. The results indicate that the size and specificity of the "narrow" channel is altered in D1-N87A PSII, allowing for the binding of deprotonated 13C-labeled methanol at the Mn4(IV) ion of the catalytic cluster in the S2 state. This has important implications on the mechanistic models for water oxidation in PSII.
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 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.
A broken symmetry density functional theory (BS-DFT) magnetic analysis of the S2, S2YZ● and S3 states of Nature’s oxygen evolving complex is performed for both the native Ca and Sr substituted forms. Good agreement with experiment is observed between the tyrosyl calculated g-tensor and 1H hyperfine couplings for the native Ca form. Changes in the hydrogen bonding environment of the tyrosyl radical in S2YZ• caused by Sr substitution leads to notable changes in the calculated g-tensor of the tyrosyl radical. Comparison of calculated and experimental 55Mn hyperfine couplings for the S3 state presently favours an open cubane form of the complex with an additional OH ligand coordinating to MnD. In Ca models this additional ligation can arise by closed-cubane form deprotonation of the Ca ligand W3 in the S2YZ• state accompanied by spontaneous movement to the vacant Mn coordination site or by addition of an external OH group. For the Sr form, no spontaneous movement of W3 to the vacant Mn coordination site is observed in contrast to the native Ca form; a difference which may lead to the reduced catalytic activity of the Sr substituted form. Preliminary BS-DFT studies on peroxide models of S3 as indicated by a recent X-ray free electron laser (XFEL) crystallography study give rise to an S = 3 ground state in agreement with EPR studies.
Proton matrix electron nuclear double resonance (ENDOR) spectroscopy was performed to specify the location of the methanol molecule near the manganese cluster in photosystem II. Comparison of the ENDOR spectra in the presence of CH3OH and CD3OH revealed two pairs of hyperfine couplings, 1.2 MHz for A(perpendicular to) and 2.5 MHz for A(//), arising from the methyl group in methanol. On the basis of the crystal structure, the possible location of methanol close to the manganese cluster was discussed.
The EPR "split signals" represent key intermediates of the S-state cycle where the redox active D1-Tyr161 (YZ) has been oxidized by the reaction center of the photosystem II enzyme to its tyrosyl radical form, but the successive oxidation of the Mn4CaO5 cluster has not yet occurred (SiYZ˙). Here we focus on the S2YZ˙ state, which is formed en route to the final metastable state of the catalyst, the S3 state, the state which immediately precedes O-O bond formation. Quantum chemical calculations demonstrate that both isomeric forms of the S2 state, the open and closed cubane isomers, can form states with an oxidized YZ˙ residue without prior deprotonation of the Mn4CaO5 cluster. The two forms are expected to lie close in energy and retain the electronic structure and magnetic topology of the corresponding S2 state of the inorganic core. As expected, tyrosine oxidation results in a proton shift towards His190. Analysis of the electronic rearrangements that occur upon formation of the tyrosyl radical suggests that a likely next step in the catalytic cycle is the deprotonation of a terminal water ligand (W1) of the Mn4CaO5 cluster. Diamagnetic metal ion substitution is used in our calculations to obtain the molecular g-tensor of YZ˙. It is known that the gx value is a sensitive probe not only of the extent of the proton shift between the tyrosine-histidine pair, but also of the polarization environment of the tyrosine, especially about the phenolic oxygen. It is shown for PSII that this environment is determined by the Ca(2+) ion, which locates two water molecules about the phenoxyl oxygen, indirectly modulating the oxidation potential of YZ.
This article provides an overview of the application of electron paramagnetic resonance (EPR) and related hyperfine spectroscopies for the investigation of the electronic and geometric structure of the tetramanganese cluster that constitutes the oxygen-evolving complex (OEC) of photosystem II. Starting from the spin physics of Mn ions, a quantum-mechanical description of exchange-coupled oligonuclear Mn systems is given. Then, the focus shifts to the characterization of the OEC with emphasis on the two half-integer spin states of its catalytic cycle, namely the S0 and S2. The development of electronic models based on EPR and related spectroscopies, such as electron nuclear double resonance (ENDOR), is described in detail. The role of broken-symmetry density functional theory (BS-DFT) is also outlined. The characterization of Mn ligands and bound substrate waters via electron nuclear magnetic interactions is illustrated. Implications for the mechanism of water oxidation catalysis are discussed.
Illuminating a photosystem II sample at low temperatures (here 5-10 K) yields so called split signals detectable with CW-EPR. These signals reflect the oxidized, deprotonated radical of D1-Tyr161 (YZ(•)) in a magnetic interaction with the CaMn4 cluster in a particular S state. The intensity of the split EPR signals are affected by the addition of the water substrate analogue methanol. This was previously shown by the induction of split EPR signals from the S1, S3 and S0 states [Su, J-H. et al. (2006) Biochemistry 45, 7617-7627.]. Here, we use two split EPR signals induced from photosystem II trapped in the S2 state to further probe the binding of methanol in an S state dependent manner. The signals are induced with either visible or near-infrared light illumination provided at 5-10 K where methanol cannot bind or un-bind from its site. The results imply that the binding of methanol not only changes the magnetic properties of the CaMn4 cluster but also the hydrogen bond network in the OEC, thereby affecting the relative charge of the S2 state. The induction mechanisms for the two split signals are different resulting in two different redox states, S2YZ(•) and S1YZ(•) respectively. The two states show different methanol dependence for their induction. This indicates the existence of two binding sites for methanol in the CaMn4 cluster. It is proposed that methanol binds to MnA with high affinity and to MnD with lower affinity. The molecular nature and S-state dependence of the methanol binding to each respective site is discussed.
The redox-active tyrosine residue (YZ) plays a crucial role in the mechanism of the water oxidation. Metalloradical electron paramagnetic resonance (EPR) signals
reflecting the light-induced YZ· in magnetic interaction with the CaMn4-cluster in the particular S-state, YZ·SX intermediates, have been found in intact photosystem II. These so-called split EPR signals are induced by illumination at
cryogenic temperatures and provide means to both study the otherwise transient YZ· and to probe the S-states with EPR spectroscopy. The illumination used for signal induction grouped the observed split EPR signals in two categories:
(i) YZ in the lower S-states was oxidized by P680+ formed via charge separation, while (ii) YZ in the higher S-states was oxidized by an excited, highly oxidizing Mn species. Applied mechanistic studies of the YZ·SX intermediates in the different S-states are reviewed and compared to investigations in photosystem II at physiological temperature. Addition of methanol induced
S-state characteristic changes in the split signals’ formation which reflect changes in the magnetic coupling within the CaMn4-cluster due to methanol binding. The pH titration of the split EPR signals, on the other hand, could probe the proton-coupled
electron transfer properties of the YZ oxidation. The apparent pK
as found for decreased split signal induction were interpreted in the fate of the phenol proton.
Water oxidation in photosystem II is catalyzed by the CaMn(4) cluster. The electrons extracted from the CaMn(4) cluster are transferred to P(680)(+) via the redox-active tyrosine residue D1-Tyr161 (Y(Z)). The oxidation of Y(Z) is coupled to a deprotonation creating the neutral radical Y(Z)(*). Light-induced oxidation of Y(Z) is possible down to extreme temperatures. This can be observed as a split EPR signal from Y(Z)(*) in a magnetic interaction with the CaMn(4) cluster, offering a way to probe for Y(Z) oxidation in active PSII. Here we have used the split S(0) EPR signal to study the mechanism of Y(Z) oxidation at 5 K in the S(0) state. The state of the hydrogen bond between Y(Z) and its proposed hydrogen bond partner D1-His190 is investigated by varying the pH. The split S(0) EPR signal was induced by illumination at 5 K between pH 3.9 and pH 9.0. Maximum signal intensity was observed between pH 6 and pH 7. On both the acidic and alkaline sides the signal intensity decreased with the apparent pK(a)s (pK(app)) approximately 4.8 and approximately 7.9, respectively. The illumination protocol used to induce the split S(0) EPR signal also induces a mixed radical signal in the g approximately 2 region. One part of this signal decays with similar kinetics as the split S(0) EPR signal ( approximately 3 min, at 5 K) and is easily distinguished from a stable radical originating from Car/Chl. We suggest that this fast-decaying radical originates from Y(Z)(*). The pH dependence of the light-induced fast-decaying radical was measured in the same pH range as for the split S(0) EPR signal. The pK(app) for the light-induced fast-decaying radical was identical at acidic pH ( approximately 4.8). At alkaline pH the behavior was more complex. Between pH 6.6 and pH 7.7 the signal decreased with pK(app) approximately 7.2. However, above pH 7.7 the induction of the radical species was pH independent. We compare our results with the pH dependence of the split S(1) EPR signal induced at 5 K and the S(0) --> S(1) and S(1) --> S(2) transitions at room temperature. The result allows mechanistic conclusions concerning differences between the hydrogen bond pattern around Y(Z) in the S(0) and S(1) states.
Genome sequence of Arabidopsis thaliana (Arabidopsis) revealed two psbO genes (At5g66570 and At3g50820) which encode two distinct PsbO isoforms: PsbO1 and PsbO2, respectively. To get insights into the function of the PsbO1 and PsbO2 isoforms in Arabidopsis we have performed systematic and comprehensive investigations of the whole photosynthetic electron transfer chain in the T-DNA insertion mutant lines, psbo1 and psbo2. The absence of the PsbO1 isoform and presence of only the PsbO2 isoform in the psbo1 mutant results in (i) malfunction of both the donor and acceptor sides of Photosystem (PS) II and (ii) high sensitivity of PSII centers to photodamage, thus implying the importance of the PsbO1 isoform for proper structure and function of PSII. The presence of only the PsbO2 isoform in the PSII centers has consequences not only to the function of PSII but also to the PSI/PSII ratio in thylakoids. These results in modification of the whole electron transfer chain with higher rate of cyclic electron transfer around PSI, faster induction of NPQ and a larger size of the PQ-pool compared to WT, being in line with apparently increased chlororespiration in the psbo1 mutant plants. The presence of only the PsbO1 isoform in the psbo2 mutant did not induce any significant differences in the performance of PSII under standard growth conditions as compared to WT. Nevertheless, under high light illumination, it seems that the presence of also the PsbO2 isoform becomes favourable for efficient repair of the PSII complex.
Detailed optical and EPR analyses of states induced in dark-adapted PS II membranes by cryogenic illumination permit characterization and quantification of all pigment derived donors and acceptors, as well as optically silent (in the visible, near infrared) species which are EPR active. Near complete turnover formation of Q(A)((-)) is seen in all centers, but with variable efficiency, depending on the donor species. In minimally detergent-exposed PS II membranes, negligible (<5%) oxidation of chlorophyll or carotenoid centers occurs for illumination temperatures 5-20 K. An optically silent electron donor to P680(+) is observed with the same decay kinetics as the S(1) split signal. Cryogenic donors to P680(+) seen are: (i) transient (t(1/2) approximately 150 s) tyrosine related species, including 'split signals' ( approximately 15% total centers), (ii) reduced cytochrome b(559) ( approximately 30-50% centers), and (iii) an organic donor, possibly an amino acid side chain, ( approximately 30% centers).
To understand the wear-splitting chemistry of enzyme photosystem II (PSII), researchers have sought insights from a great number of different sorts of experiments all directed both at the protein and at model metal complexes. Sophisticated spectroscopic techniques such as EPR (electron paramagnetic resonance), FTIR (Fourier transform infrared) and XAS (X-ray absorption spectroscopy) as well as time-resolved mass spectrometry have been deployed against the OEC (oxygen evolving complex), yielding for the first time detailed information in a variety of S-states. The recent crystallographic studies have made it possible to propose and to test more detailed mechanistic proposals than have previously been tenable.
The S2 state of the oxygen-evolving complex (OEC) of photosystem II is heterogeneous, exhibiting two main EPR spectral forms, the multiline and the g = 4.1 signal. It is not clearly established whether this heterogeneity develops during the S1 to S2 transition or is already present in the precursor states. We have compared the spectra of the S1YZ* intermediate, obtained by visible light excitation (induction of charge separation) of the S1 state at liquid He temperatures, (S1YZ*)vis, or by near-infrared (NIR) light excitation of the S2 state (utilization of the unusual property of the Mn cluster to act as an oxidant of Yz when excited by NIR), (S1YZ*)NIR. The decay kinetics of the (S1YZ*)vis spectrum at 11 K was also studied by the application of rapid-scan EPR. The two spectra share in common a signal with a characteristic feature at g = 2.035, but the (S1YZ*)vis spectrum contains in addition a fast decaying component 26 G wide. The analysis of the surface of the rapid-scan spectra yielded 270 +/- 35 and 90 +/- 15 s for the respective half-times of the two components of the (S1YZ*)vis spectrum at 11 K. (S1YZ*)vis advances efficiently to S2 when annealed at 200 K; notably the g = 2.035 signal advances to the multiline while the 26 G component advances to the g = 4.1 conformation. The "26 G" component is absent or very small, respectively, in thermophilic cyanobacteria or glycerol-containing spinach samples, in correlation to vanishing or very small amounts of the g = 4.1 component in the S2 spectrum. The results validate the assignment of S1YZ* to a true S1 to S2 intermediate and imply that the heterogeneity observed in S2 is already present in S1. Tentative valences are assigned to the individual Mn ions of the OEC in the two heterogeneous conformations of S1.
The interaction EPR split signals from photosystem II (PSII) have been reported from the S0, S1, and S3 states. The signals are induced by illumination at cryogenic temperatures and are proposed to reflect the magnetic interaction between YZ* and the Mn4Ca cluster. We have investigated the formation spectra of these split EPR signals induced in PSII enriched membranes at 5 K using monochromatic laser light from 400 to 900 nm. We found that the formation spectra of the split S0, split S1, and split S3 EPR signals were quite similar, but not identical, between 400 and 690 nm, with maximum formation at 550 nm. The major deviations were found between 440 and 480 nm and between 580 and 680 nm. In the regions around 460 and 680 nm the amplitudes of the formation spectra were 25-50% of that at 550 nm. A similar formation spectrum was found for the S2-state multiline EPR signal induced at 0 degrees C. In general, the formation spectra of these signals in the visible region resemble the reciprocal of the absorption spectra of our PSII membranes. This reflects the high chlorophyll concentration necessary for the EPR measurements which mask the spectral properties of other absorbing species. No split signal formation was found by the application of infrared laser illumination between 730 and 900 nm from PSII in the S0 and S1 states. However, when such illumination was applied to PSII membranes poised in the S3 state, formation of the split S3 EPR signal was observed with maximum formation at 740 nm. The quantum yield was much less than in the visible region, but the application of intensive illumination at 830 nm resulted in accumulation of the signal to an amplitude comparable to that obtained with illumination with visible light. The split S3 EPR signal induced by NIR light was much more stable at 5 K (no observable decay within 60 min) than the split S3 signal induced by visible light (50% of the signal decayed within 30 min). The split S3 signals induced by each of these light regimes showed the same EPR spectral features and microwave power saturation properties, indicating that illumination of PSII in the S3 state by visible light or by NIR light produces a similar configuration of YZ* and the Mn4Ca cluster.
The heart of the oxygen-evolving complex (OEC) of photosystem II is a Mn4OxCa cluster that cycles through five different oxidation states (S0 to S4) during the light-driven water-splitting reaction cycle. In this study we interpret the recently obtained 55Mn hyperfine coupling constants of the S0 and S2 states of the OEC [Kulik et al. J. Am. Chem. Soc. 2005, 127, 2392-2393] on the basis of Y-shaped spin-coupling schemes with up to four nonzero exchange coupling constants, J. This analysis rules out the presence of one or more Mn(II) ions in S0 in methanol (3%) containing samples and thereby establishes that the oxidation states of the manganese ions in S0 and S2 are, at 4 K, Mn4(III, III, III, IV) and Mn4(III, IV, IV, IV), respectively. By applying a "structure filter" that is based on the recently reported single-crystal EXAFS data on the Mn4OxCa cluster [Yano et al. Science 2006, 314, 821-825] we (i) show that this new structural model is fully consistent with EPR and 55Mn-ENDOR data, (ii) assign the Mn oxidation states to the individual Mn ions, and (iii) propose that the known shortening of one 2.85 A Mn-Mn distance in S0 to 2.75 A in S1 [Robblee et al. J. Am. Chem. Soc. 2002, 124, 7459-7471] corresponds to a deprotonation of a mu-hydroxo bridge between MnA and MnB, i.e., between the outer Mn and its neighboring Mn of the mu3-oxo bridged moiety of the cluster. We summarize our results in a molecular model for the S0 --> S1 and S1 --> S2 transitions.
Tyr Z of photosystem II mediates electron transfer from the water splitting site, a Mn4Ca cluster, to the specialized chlorophyll assembly P680. Due to its proton-limited redox properties and the proximity to the Mn cluster, it is thought to play a critical role in the proton-coupled electron transfer reactions that constitute the four-step oxidation mechanism (so-called S-state transitions) of water to molecular oxygen. Spectroscopic evidence for the Tyr Z radical has been scarce in intact preparations (it is difficult to probe it optically, and too short-lived for EPR characterization) until recently. Advances in recent years have allowed the trapping at liquid helium temperatures and EPR characterization of metalloradical intermediates, attributed to tyrosyl Z* magnetically interacting with the Mn cluster. We have extended these studies and examined the evolution of the spectra of five intermediates: S0YZ*, S0YZ* (with 5% MeOH), S1YZ*, S2YZ*, and S2YZ* (with 5% MeOH) in the temperature range of 11-230 K. A rapid-scan EPR method has been applied at elevated temperatures. The tyrosyl radical decouples progressively from Mn, as the Mn relaxation rate increases with an increase in temperature. Above approximately 100 K, the spectra collapse to the unperturbed spectrum of Tyr Z*, which is found to be somewhat broader than that of the stable Tyr D* radical. This study provides a simple means for recording the spectrum of Tyr Z* and extends earlier observations that link the photochemistry at liquid helium temperatures to the photochemistry at temperatures that support S-state transitions.
The O2-evolving complex of photosystem II, Mn 4Ca, cycles through five oxidation states, S0,..., S4, during its catalytic function, which involves the gradual abstraction of four electrons and four protons from two bound water molecules. The direct oxidant of the complex is the tyrosine neutral radical, YZ(*), which is transiently produced by the highly oxidizing power of the photoexcited chlorophyll species P680. EPR characterization of YZ(*) has been limited, until recently, to inhibited (non-oxygen-evolving) preparations. A number of relatively recent papers have demonstrated the trapping of YZ(*) in O2-evolving preparations at liquid helium temperatures as an intermediate of the S0 to S1, S1 to S2, and S2 to S3 transitions. The respective EPR spectra are broadened and split at g approximately 2 by the magnetic interaction with the Mn cluster, but this interaction collapses at temperatures higher than about 100K [Zahariou et al. (2007) Biochemistry 46, 14335 -14341]. We have conducted a study of the Tyr Z(*) transient in the temperature range 77-240 K by employing rapid or slow EPR scans. The results reveal for the first time high-resolution X-band spectra of Tyr Z(*) in the functional system and at temperatures close to the onset of the S-state transitions. We have simulated the S 2Y Z(*) spectrum using the simulation algorithm of Svistunenko and Cooper [(2004) Biophys. J. 87, 582 -595]. The small g(x) = 2.00689 value inferred from the analysis suggests either a H-bonding of Tyr Z (*) (presumably with His190) that is stronger than what has been assumed from studies of Tyr D(*) or Tyr Z(*) in Mn-depleted preparations or a more electropositive environment around Tyr Z(*). The study has also yielded for the first time direct information on the temperature variation of the YZ(*)/QA(-) recombination reaction in the various S states. The reaction follows biphasic kinetics with the slow phase dominating at low temperatures and the fast phase dominating at high temperatures. It is tentatively proposed that the slow phase represents the action of the YZ(*)/YZ(-) redox couple while the fast phase represents that of the YZ(*)/YZH couple; it is inferred that Tyr Z at elevated temperatures is protonated at rest. It is also proposed that YZ(*)/YZH is the couple that oxidizes the Mn cluster during the S1-S2 and S2-S3 transitions. A simple mechanism ensuring a rapid (concerted) protonation of Tyr Z upon oxidation of the Mn cluster is discussed, and also, a structure-based molecular model suggesting the participation of His190 into two hydrogen bonds is proposed.
We have investigated the electron transfer from reduced tyrosine Y D (YDred) and cytochrome b559 to the S2 and S3 states of the water oxidizing complex (WOC) in Photosystem II. The EPR signal of oxidized cyt b559, the S 2 state multiline EPR signal and the EPR signal from Y D· were measured to follow the electron transfer to the S2 and S3 states at 245 and 275 K. The majority of the S2 centers was reduced directly from YDred but at 245 K we observed oxidation of cyt b559 in about 20% of the centers. Incubation of the YDredS3 state resulted in biphasic changes of the S2 multiline signal. The signal first increased due to reduction of the S3 state. Thereafter, the signal decreased due to decay of the S2 state. In contrast, the YD· signal increased with a monophasic kinetics at both temperatures. Again, we observed oxidation of cyt b559 in about 20% of the PSII centers at 245 K. This oxidation correlated with the decay of the S2 state. The complex changes can be explained by the conversion of YDredS3 centers (present initially) to YD·S1 centers, via the intermediate YD·S2 state. The early increase of the S2 state multiline signal involves electron transfer from Y Dred to the S3 state resulting in formation of YD·S2. This state is reduced by cyt b559 resulting in a single exponential oxidation of cyt b 559. Taken together, these results indicate that the electron donor to S2 is cyt b559 while cyt b559 is unable to compete with YDred in the reduction of the S3 state in the pre-reduced samples. We also followed the decay of the S 2 and S3 states and the oxidation of cyt b559 in samples where YD was oxidized from the start. In this case cyt b559 was able to reduce both the S2 and the S3 states suggesting that different pathways exist for the electron transfer from cyt b559 to the WOC. The activation energies for the Y DredS2→YD·S1 and YDredS 3→YD·S2 transformations are 0.57 and 0.67 eV, respectively, and the reason for these large activation energies is discussed.
Oxygenic photosynthesis is the principal energy converter on earth. It is driven by photosystems I and II, two large protein-cofactor complexes located in the thylakoid membrane and acting in series. In photosystem II, water is oxidized; this event provides the overall process with the necessary electrons and protons, and the atmosphere with oxygen. To date, structural information on the architecture of the complex has been provided by electron microscopy of intact, active photosystem II at 15-30 A resolution, and by electron crystallography on two-dimensional crystals of D1-D2-CP47 photosystem II fragments without water oxidizing activity at 8 A resolution. Here we describe the X-ray structure of photosystem II on the basis of crystals fully active in water oxidation. The structure shows how protein subunits and cofactors are spatially organized. The larger subunits are assigned and the locations and orientations of the cofactors are defined. We also provide new information on the position, size and shape of the manganese cluster, which catalyzes water oxidation.
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.
We report the detection of a “split” electron paramagnetic resonance (EPR) signal during illumination of dark-adapted (S1 state) oxygen-evolving photosystem II (PSII) membranes at <20 K. The characteristics of this signal indicate that it arises from an interaction between an organic radical and the Mn cluster of PSII. The broad radical signal decays in the dark following illumination either by back-reaction with Qa•- or by forward electron transfer from the Mn cluster. The forward electron transfer (either from illumination at 11 K followed by incubation in the dark at 77 K or by illumination at 77 K) results in the formation of a multiline signal similar to, but distinct from, other well-characterized multiline forms found in the S0 and S2 states. The relative yield of the “S1 split signal”, which we provisionally assign to S1X•, where X could be YZ• or Car•+, and that of the 77 K multiline signal indicate a relationship between the two states. An approximate quantitation of the yield of these signals indicates that up to 40−50% of PSII centers can form the S1 split signal. Ethanol addition removes the ability to observe the S1 split signal, but the multiline signal is still formed at 77 K. The multiline forms with <700 nm light and is not affected by near-infrared (IR) light, showing that we are detecting electron transfer in centers not responsive to IR illumination. The results provide important new information about the mechanism of electron abstraction from the water oxidizing complex (WOC).
The oxygen evolving complex (OEC) of photosystem II (PSII) gives rise to manganese-derived electron paramagnetic resonance (EPR) signals in the S-0 and S-2 oxidation states. These signals exhibit different microwave power saturation behavior between 4 and 10 K. Below 8 K, the S-0 state EPR signal is a faster relaxer than the S-2 multiline signal, but above 8 K, the So signal is the slower relaxer of the two. The different temperature dependencies of the relaxation of the S-0 and S-2 ground-state Mn signals are due to differences in the spin-lattice relaxation process, The dominating spin-lattice relaxation mechanism is concluded to be a Raman mechanism in the So slate, with a T-4.1 temperature dependence of the relaxation rate. It is proposed that the relaxation of the St State arises from a Raman mechanism as well, with a T-6.8 temperature dependence of the relaxation rate, although the data also fit an Orbach process. If both signals relax through a Raman mechanism, the different exponents are proposed to reflect structural differences in the proteins surrounding the Mn cluster between the S-0 and S-2 states. The saturation of SIIslow from the Y-D(OX) radical on the D2 protein was also studied, and found to vary between the S-0 and the S-2 states of the enzyme in a manner similar to the EPR signals from the OEC, Furthermore, we found that the S-2 multiline signal in the second turnover of the enzyme is significantly more difficult to saturate than in the first turnover. This suggests differences in the OEC between the first and second cycles of the enzyme. The increased relaxation rate may be caused by the appearance of a relaxation enhancer, or it may be due to subtle structural changes as the OEC is brought into an active state.
The Mn-4-cluster of photosystem II (PSII) from Synechococcus elongatus was studied by electron paramagnetic resonance (EPR) spectroscopy after a series of saturating laser flashes given in the presence of either methanol or ethanol. Results were compared to those obtained in similar experiments done on PSII isolated from plants. The flash-dependent changes in amplitude of the EPR multiline signals were virtually identical in all samples. In agreement with earlier work [Messinger, J., Nugent, J, H. A., and Evans, M. C. W. (1997) Biochemistry, 36, 11055-11060; Ahrling, K. A., Peterson, S., and Styring, S, (1997) Biochemistry 36, 13148-13152], detection of an EPR multiline signal from the So state in PSII from plants was only possible with methanol present. In PSII from S. elongatus, it is shown that the S-0 state exhibits an EPR multiline signal in the absence of methanol (however, ethanol was present as a solvent for the artificial electron acceptor). The hyperfine lines are better resolved when methanol is present. The S-0 multiline signals detected in plant PSII and in S, elongatus were similar but not identical. Unlike the situation seen in plant PSII, the S-2 state in S. elongatus is not affected by the addition of methanol in that (i) the S-2 multiline EPR signal is not modified by methanol and (ii) the spin state of the S-2 state is affected by infrared light when methanol is present. It is also shown that the magnetic relaxation properties of an oxidized low-spin heme, attributed to cytochrome c(550), vary with the S states. This heme then is in the magnetic environment of the Mn-4 cluster.
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.
The whereabouts of the Ca2+ site in Photosystem II (PSII) was investigated by experiments in which Mn2+ was substituted for Ca2+. When stoichiometric amounts of Mn2+ ions were added to Ca2+-depleted PSII, the Mn2+ was not detected by EPR. The titration of Ca2+ back into Ca2+-depleted/Mn2+-containing PSII resulted in the simultaneous release of the Mn2+ and the loss of the two EPR signals which are characteristic of the Ca2+-depleted enzyme (i.e., the stable, modified S2 multiline signal arising from the intrinsic Mn cluster and the split S3 signal from an organic radical interacting with the Mn cluster). These results indicate that the Mn2+ occupies the functional Ca2+ site. The S2 and S3 EPR signal characteristic of this kind of Ca2+-depleted preparation were unaffected by the binding of the Mn2+ Since, from earlier results, it seems likely that the modification and stability of S2 multiline signal in these PSII preparations is due to binding of chelator to or close to the Mn cluster, the present results indicate that the Ca2+ site (at least when occupied by Mn2+) does not overlap with the chelator binding site. Since Mn2+ binding does not effect the S2 EPR signal from the Mn cluster, it can be concluded that the Mn2+ is not involved in detectable magnetic interactions with the cluster. This result indicates that the Mn2+-occupied Ca2+ binding site is outside the first co-ordination sphere of the Mn cluster. The relaxation properties of TyrD. were enhanced by the presence of the Mn2+ when the Mn cluster was in the S1 state.
An X-ray structure of photosystem II refined to 3.5 Å resolution has revealed details of the metal cluster and protein environment of the oxygen-evolving centre and provides a framework for developing a molecular mechanism for the water splitting reaction.
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.
Oxygen evolution by the mangano-enzyme of photosystem II is inhibited by Ca2+ depletion induced by NaCl washing and restored by Ca2+ addition. The effectiveness of NaCl treatment in inhibiting oxygen evolution in photosystem II was studied after a series of preilluminating flashes. The susceptibility of the enzyme to NaCl treatment varied with the number of preilluminating flashes and this variation showed an oscillation pattern with a period of four. This pattern is characteristic of cycling through the four long-lived intermediate states in the enzyme cycle (i.e. the states, S0, S1, S2, S3). The relative extent of inhibition corresponding to each of the S states was as follows: S3 > S0≈S2 > S1. From these results it is concluded that Ca2+ binding is dependent on the S states and that Ca2+ probably plays a fundamental role in the mechanism of water splitting. The results also help to explain the conflicting reports of the extent of inhibition induced by NaCl washing and the controversy over which electron transfer step is inhibited by Ca2+ depletion.
— 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).
The light-driven oxidation of water to dioxygen is catalyzed by the enzyme photosystem II. A four-manganese ion cluster and a tyrosine, YZ, are present in the catalytic site. In preparations inhibited by addition of acetate or removal of the calcium cofactor, it is possible to trap the tyrosyl radical in interaction with the metal cluster. The coupled species is characterized by a broad split EPR signal at 9 GHz. In this work, high-field EPR has been used for further characterization of the coupling. The 285, 190 and 95 GHz EPR spectra of the interacting system are reported. Analysis of these spectra yielded exchange and dipolar couplings of the same magnitude as those found with 9 GHz EPR. However, the high-field spectra show that the coupling between the radical and the manganese cluster has opposite sign in acetate-treated compared to calcium-depleted samples. The sign difference indicates differences in the electronic structure of the radical−metal center pair. Comparisons are made between photosystem II and other enzymes containing radicals interacting with metal centers. Possible explanations for the difference in sign are proposed. The difficulty in obtaining reliable structural information for the spin coupled system is addressed.
Upon room-temperature illumination, acetate-inhibited photosystem II membranes are known to exhibit a 240 G wide X-band (9.5 GHz) electron paramagnetic resonance (EPR) signal at 10 K. This EPR signal arises from an interaction between the S = 1/2 multiline S2 state of the tetranuclear manganese cluster and an oxidized tyrosine residue, YZ•. In the present study, the exchange and dipolar interactions between the two paramagnetic species are simulated at X- and Q-band (33 GHz) frequencies utilizing second-order perturbation theory. The positions and relative intensities of the hyperfine lines in the S = 1/2 S2 state multiline EPR signal of the noninteracting Mn4 cluster are accurately simulated by including g anisotropy and four sets of axially symmetric 55Mn hyperfine tensors. These parameters are then used to simulate the dipolar and exchange interactions giving rise to the interacting S2YZ• (formerly referred to as S3) EPR signal. Relative intensities of components of the S2YZ• EPR spectrum, at both X- and Q-band frequencies, are best reproduced with a dipolar coupling corresponding to an interspin distance of 7.7 Å and an exchange coupling (J) of −280 × 10-4 cm-1.
The photosynthetic oxidation of water to oxygen occurs in photosystem II (PSII) at an active site composed of a tetranuclear cluster of manganese ions, a redox active tyrosine, YZ, and two essential cofactors, calcium and chloride. Recently, several experimental observations have led to the proposal of a metalloradical catalytic cycle in which water oxidation occurs via hydrogen-atom abstraction by the tyrosyl radical from water bound to the manganese cluster. This model predicts a close proximity between the radical tyrosine, Yz•, and the Mn cluster and the involvement of the radical in a bifurcated hydrogen bond. Magnetic resonance techniques have been used in this work to probe the interaction of the tyrosyl radical with its environment in PSII samples in which the catalytic cycle is blocked by acetate treatment and the enzyme is trapped in a paramagnetic S2Yz• state. Radical interaction with the metal cluster has been studied via simulations of the EPR spectra obtained for this state. The simulations were based on a radical-pair model and included terms for both electron−electron dipolar and exchange interactions. The results show a dominant exchange interaction between the radical and the manganese cluster in these preparations and led to an estimate of 8−9 Å for the spin−spin distance. ESEEM spectroscopy and 1H2O/2H2O exchange were used to study interactions of the S2Yz• state with exchangeable hydrogen nuclei in the site. Two-pulse ESEEM data show features expected for a radical-pair species, in support of our analysis of the continuous-wave EPR spectrum. An ESEEM analysis based on an electron spin 1/2, nuclear spin 1 model shows that both two- and three-pulse ESEEM data are consistent with four deuterons that exhibit an electron−nuclear dipole−dipole coupling of 0.42 MHz. The validity of this analysis and its implications for the oxygen-evolving apparatus are discussed.
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 evaluation of Mn X-ray absorption fine structure (EXAFS) studies on the oxygen-evolving complex (OEC) from photosystem II is described for preparations from both spinach and the cyanobacterium Synechococcus sp. poised in the S[sub 1] and S[sub 2] states. In addition to reproducing previous results suggesting the presence of bis([mu]-oxo)-bridged Mn centers in the OEC, a Fourier transform peak due to scatterers at an average distance of > 3 [angstrom] is detected in both types of preparation. In addition, subtle but reproducible changes are found in the relative amplitudes of the Fourier transform peaks due to mainly O ([approximately]1.8 [angstrom]) and Mn ([approximately] 2.7 [angstrom]) neighbors upon cryogenic advance from the S[sub 1] to the S[sub 2] state. Analysis of the peak due to scatterers at [approximately] 3 [angstrom] favors assignment to (per 4 Mn in the OEC) 1-2 heavy atom (Mn, Ca) scatterers at an average distance of 3.3-3.4 [angstrom]. The EXAFS data of several multinuclear Mn model compounds containing such scattering interactions are analyzed and compared with the data for the OEC. Structural models for the OEC are evaluated on the basis of these results. 40 refs., 9 figs., 5 tabs.
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.
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.
EPR was used to study the binding of NH3 to the photosynthetic O2-evolving center, NH3-treated, Ca2+-depleted Photosystem II (PS II) membranes exposed to continuous light at 250 K showed a 10 mT-wide asymmetric EPR signal, centered around g = 2. When dark-adapted material was illuminated with a sequence of laser flashes the same signal appeared after the second flash, indicating that the g = 2 signal arises from a modified S3 state. The signal is different from the 15–16.5 mT-wide EPR signal at g = 2 ascribed to the S3′ state. Illumination of native NH3-treated PS II membranes with continuous light results in the appearance of an EPR signal at g = 2 with a width similar to that in Ca2+-depleted. NH3-treated membranes. The conditions for the formation of the signal and its properties suggest that it also arises from a perturbed S3 state with NH3 in close association with the manganese.
The effect of trypsin treatment on Photosystem-II particles has been investigated by measurements of oxygen evolution, 2,6-dichlorophenolindophenol (DCIP)-reduction and Mn-abundance and by analyzing the peptide pattern. The following results were obtained. (1) Trypsin modifies both the acceptor and donor side of PS II, but striking differences are observed for the pH dependence: whereas the acceptor side is severely attacked between pH 5.5 and 9.0, the destruction of the donor side (oxygen-evolving capacity) by trypsin becomes significant only at pH values higher than 7.25. (2) The pH-dependence of the susceptibility of oxygen evolution to trypsin closely resembles that observed in inside-out thylakoids (Renger, G., Völker, M. and Weiss, W. (1984) Biochim. Biophys. Acta 766, 582–591). (3) The effect of trypsin on the functional integrity of water oxidation cannot be due to an attack on the surface exposed 16 kDa, 24 kDa and 33 kDa polypeptides, because they are digested rapidly even at pH 6.5, where the oxygen-evolving capacity remains almost unaffected. (4) Trypsination of PS-II particles as well as of the isolated 33 kDa protein leads to a 15 kDa fragment. In trypsinized PS-II particles this fragment remains membrane-bound. The amount of the 15 kDa fragment and Mn content are correlated with the oxygen-evolving capcity. These results indicate pH-dependent structural modifications at the donor side of System II which make target proteins accessible to trypsin. The 33 kDa protein is inferred to play a regulatory role in photosynthetic oxygen evolution and this function is realized by only a part of the protein, i.e., the 15 kDa fragment, that remains resistant to mild trypsination.
Using EPR and EXAFS spectroscopies we show that high concentrations of ammonium cations at alkaline pH are required for (1) inhibition of oxygen evolution: (2) an alteration of the EPR properties of the oxygen evolving complex: (3) the ability to detect Y.Z; and (4) the slow reduction of the Mn complex leading to the appearance of EPR detectable Mn2+. The inhibition of S state cycling, slowing of Y.Z reduction, appearance of Mn2+ and the yield of a Hpp < 10 mT S3 type EPR signal are decreased by calcium addition. This indicates that these effects were probably associated with calcium depletion arising from the high concentration of ammonium cation. The ammonia-induced changes to the S2 multiline EPR signal are not affected by calcium addition. The appearance of Mn2+ is shown to be reversible on illumination, suggesting that the Mn reduced from the native state is located at or near the native site. Simulations of the interaction which gives rise to the S3 EPR signal are also presented and discussed. These indicate that lineshape differences occur through small changes in the exchange component of the interaction between the manganese complex and organic radical, probably through minor structural changes between the variously treated samples.
Visible light illumination at liquid He temperatures of photosystem II (PSII) membranes poised in the S1-state, results in the production of a metalloradical signal with resonances at g
= 2.035 and g
2.0 at X-band (J. H. A. Nugent, I. P. Muhiuddin, and M. C. W. Evans, Biochemistry, 2002, 41, 4117–4126). A similar signal has been obtained by near IR excitation of samples poised in the S2 state (D. Koulougliotis, J.-R. Shen, N. Ioannidis, and V. Petrouleas, Biochemistry, 2003, 42, 3045–3053). The signal has been attributed to the magnetic interaction of the tyrosyl Z radical with the Mn cluster in the S1 state. In an effort to obtain further information about the interactions of tyrosine Z with the Mn cluster, and about the integer-spin S1 state we have employed EPR spectroscopy at two frequencies, X and W-band. The spectrum at W band is characterized by novel resonances at g
= 2.019, g
2.00 and g
= 1.987. For the analysis of the spectra at the two microwave frequency bands a spin Hamiltonian has been applied under the following basic assumptions: The S1 state of the Mn cluster is characterized by two low lying spin states Sa
= 0 and 1. The major features of the spectra are attributed to the interaction of the Sa
= 1 state with the spin Sb
= 1/2 of the tyrosyl radical. Potential contributions from the Sa
= 0 state are suppressed under the present experimental conditions. A satisfactory fit reproducing all features of the spectra is achieved with the same set of fitting parameters for the signals at both bands. An anisotropic ferromagnetic exchange interaction results from the fit with the coupling value being of the same order of magnitude with the value of the zero field splitting term of the Mn cluster (S
= 1).
Thesis (doctoral)--Rijksuniversiteit te Leiden, 1976.
THE evolution of oxygen as a result of light-driven water oxidation
occurs in plants and is catalysed by photosystem-II (PS-II). A
manganese-cluster probably acts both as the active site and as a
charge-accumulating device (for a review, see ref. 1). The enzyme cycle
involves five redox states which are denoted as
S0-S4, depend-ing on the number of positive
equivalents stored2. Oxygen is released after formation of
the transient S4 state. Ca2+ is an obligatory
cofactor in this process and its depletion inhibits the enzyme cycle at
the step before water oxidation, that is, after formation of the
S3 state3. In chelator-treated,
Ca2+-depleted PS-II, a new electron paramagnetic resonance
(EPR) signal arising from a formal S3 state has been
reported4. It was suggested that the S3 EPR signal
could originate from the oxidation of an amino acid, interacting
magnetically with the manganese cluster4. There are only a
few examples of amino-acid oxidation in enzyme chemistry and these are
limited to tyrosine5 and tryptophan6. Here we
report evidence that the S2 to S3 transition
occurring in Ca2+-depleted PS-II corresponds to the oxidation
of histidine.
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 g = 4 and g = 2 multiline EPR signals arising from the Mn cluster of the photosynthetic oxygen-evolving complex (OEC) in the S2 state were studied in preparations of oriented photosystem II (PSII) membranes. The ammonia-modified forms of these two signals were also examined. The g = 4 signal obtained in oriented PSII membranes treated with NH4Cl at pH 7.5 displays at least 16 partially resolved Mn hyperfine transitions with a regular spacing of 36 G [Kim, D.H., Britt, R.D., Klein, M.P., & Sauer, K. (1990) J. Am. Chem. Soc. 112, 9389-9391]. The observation of this g = 4 "multiline signal" provides strong spectral evidence for a tetranuclear Mn origin for the g = 4 signal and is strongly suggestive of a model in which different spin state configurations of a single exchange-coupled Mn cluster give rise to the g = 4 and g = 2 multiline signals. A simulation shows the observed spectrum to be consistent with an S = 3/2 or S = 5/2 state of a tetranuclear Mn complex. The resolution of hyperfine structure on the NH3-modified g = 4 signal is strongly dependent on sample orientation, with no resolved hyperfine structure when the membrane normal is oriented perpendicular to the applied magnetic field. The dramatic NH3-induced changes in the g = 4 signal resolved in the spectra of oriented samples are suggestive that NH3 binding at the Cl- site of the OEC may represent direct coordination of NH3 to the Mn cluster.(ABSTRACT TRUNCATED AT 250 WORDS)
This guide is intended to aid in the detection and identification of paramagnetic species in Photosystem II membranes, by electron paramagnetic resonance spectroscopy. The spectral features and occurrence of each of the electron paramagnetic resonance signals from Photosystem II are discussed, in relation to the nature of the moiety giving rise to the signal and the role of that species in photosynthetic electron transport. Examples of most of the signals discussed are shown. The electron paramagnetic resonance signals produced by the cytochrome b6f and Photosystem I complexes, as well as the signals from other common contaminants, are also reviewed. Furthermore, references to seminal experiments on bacterial reaction centers are included. By reviewing both the spectroscopic and biochemical bases for the electron paramagnetic resonance signals of the cofactors that mediate photosynthetic electron transport, this paper provides an introduction to the use and interpretation of electron paramagnetic resonance spectroscopy in the study of Photosystem II.
The effects of NH3 on the oxygen evolving enzyme have been investigated with EPR and steady-state O2 evolution. The following results were obtained. At low light intensity O2 evolution occurs in all centers even though ammonia is bound. This binding occurs in the S2 state and results in a modification of the multiline signal as reported earlier. However, the oscillations with flash number of the amplitude of the EPR signal are virtually unaffected, indicating that NH3 binding does not prevent S-state advancement. Inhibition of O2 evolution by NH3 measured at light intensities that are nearly saturating for untreated photosystem II is interpreted as being due to a slow down in the rate of S-state cycling. At very high light intensities NH3 is not able to inhibit oxygen evolution presumably because NH3 binding is S state dependent and the susceptible S state (S2) is turned over too quickly. NH3 binding resulting in the modified multiline signal does not occur in S1. When S1 is formed from fully NH3 modified S2 by deactivation or by three further flashes, the S1 state does not have NH3 bound. NH3 thus dissociates easily from S1. Earlier reports of NH3 binding in S1 may be explained by the observation that NH3 binding can occur upon incubation of samples in S2 at temperatures as low as 198 K. Evidence is obtained for an NH3 binding occurring slowly (30 s) in S3. This binding results in a block in S-state advancement as suggested earlier [Velthuys, B. R. (1975) Thesis, University of Leiden]. The results are interpreted in two possible models: (1) NH3 binding in S2 occurs in a substrate site, but it is rapidly exchanged by water upon S4 formation. (2) NH3 binding in S2 is not in a substrate site but instead in a structural site and remains bound while water is oxidized. Inherent in this model is that other NH3 binding sites, i.e., the Cl- site, and the slow NH3 binding site in S3 could be the true substrate sites. Some mechanistic implications are discussed.
The electron spin-lattice relaxation rate (1/T1) of the g = 2 "multiline" manganese electron paramagnetic resonance (EPR) signal arising from the photosystem II oxygen-evolving complex poised in the S2 state has been directly measured over the temperature range of 4.2-11 K via the inversion-recovery pulsed EPR technique. The electron spin echo amplitude of the g = 2 "multiline" signal varies inversely with temperature over this range, indicating a ground spin state Curie law behavior in agreement with our previously reported work [Britt et al. (1992) Biochim. Biophys. Acta 1140, 95-101]. Results of a plot of the natural log of the electron spin-lattice relaxation rate versus reciprocal temperature are consistent with an Orbach mechanism serving as the dominant relaxation pathway for the "multiline" signal in this temperature range. The slope of the plot indicates that an excited spin state manifold exists 36.5 cm-1 above the ground-state manifold that gives rise to the "multiline" signal.
Experiments are described which allow the detection and characterization of new EPR signals in photosystem II (PSII). PSII has been extensively studied with the water oxidising complex (WOC) poised in the S1 and S2 states. Other stages in the cycle of water oxidation lack characteristic EPR signals for use as probes. In this study, experiments use multiple turnovers of PSII from an initial S1 state to allow new states of PSII to be studied. The first EPR signal detected, centered at g = 4.85 and termed the g = 5 signal, is suggested to be a new form of S2 probably formed by decay of S3 at cryogenic temperatures, but a novel form of oxidized non-heme iron cannot be fully excluded at present. The second signal is split around g = 2 and shows characteristics of signals formed by spin-spin interaction between two paramagnetic species. The split g = 2 signal is reversibly formed by illumination at <30 K of a sample containing the g = 5 signal. The g = 2 signal may be a form of the "S3" EPR signal previously only found in a variety of PSII preparations where oxygen evolution has been inhibited. Those "S3" signals are thought to arise from the interaction of an oxidized amino acid radical and the S2 state, i.e., S2X+. Illumination at higher temperatures or illumination at <30 K, followed by dark-adaptation at 77 K, removes the g = 5 signal and prevents subsequent detection of the g = 2 signal on illumination at <30 K. The most likely explanation of our data is that illumination at <30 K of centers containing the g = 5 species allows accumulation of an oxidized intermediate and that at higher temperatures electron transfer proceeds to re-form an EPR-silent S state equivalent to that initially trapped during sample preparation. Study of these signals should provide an important new insight into the WOC and PSII.
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.
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.
During oxygen evolution, the Mn cluster in Photosystem II cycles through five oxidation states, S0-S4. S0 and S2 are paramagnetic, and can be monitored by electron paramagnetic resonance (EPR). Recently a new EPR signal from the S0 state was discovered [Ahrling et al. (1997) Biochemistry 36, 13148-13152, Messinger et al. (1997) J. Am. Chem. Soc. 119, 11349-11350]. Here, we present a well-resolved S0 spectrum, taken at high power and low temperature. The spectrum is wider and more resolved than previously thought, with structure over more than 2500 G, and appears to have at least 20 reproducible peaks on each side of g = 2. We also present the temperature dependence of the unsaturated S0 signal amplitude. A linear relationship was found between signal intensity and reciprocal temperature (1/T) in the region 5-25 K, clearly extrapolating to 0. This obeys the Curie law, indicating that the S0 state is a ground S = 1/2 state with no thermally accessible excited state. The data are consistent with a minimum energy gap of 30 cm-1 between the ground and first excited states.
The light-induced new EPR signals at g = 12 and 8 were observed in photosystem II (PS II) membranes by parallel polarization EPR. The signals were generated after two flashes of illumination at room temperature, and the signal intensity had four flashes period oscillation, indicating that the signal origin could be ascribed to the S3-state. Successful simulations were obtained assuming S = 1 spin for the values of the zero-field parameters, D = +/-0.435 +/- 0. 005 cm-1 and E/D = -0.317 +/- 0.002. Orientation dependence of the g =12 and 8 signal intensities shows that the axial direction of the zero-field interaction of the manganese cluster is nearly parallel to the membrane normal.
The oxidation of carotenoid upon illumination at low temperature has been studied in Mn-depleted photosystem II (PSII) using EPR and electronic absorption spectroscopy. Illumination of PSII at 20 K results in carotenoid cation radical (Car+*) formation in essentially all of the centers. When a sample which was preilluminated at 20 K was warmed in darkness to 120 K, Car+* was replaced by a chlorophyll cation radical. This suggests that carotenoid functions as an electron carrier between P680, the photooxidizable chlorophyll in PSII, and ChlZ, the monomeric chlorophyll which acts as a secondary electron donor under some conditions. By correlating with the absorption spectra at different temperatures, specific EPR signals from Car+* and ChlZ+* are distinguished in terms of their g-values and widths. When cytochrome b559 (Cyt b559) is prereduced, illumination at 20 K results in the oxidation of Cyt b559 without the prior formation of a stable Car+*. Although these results can be reconciled with a linear pathway, they are more straightforwardly explained in terms of a branched electron-transfer pathway, where Car is a direct electron donor to P680(+), while Cyt b559 and ChlZ are both capable of donating electrons to Car+*, and where the ChlZ donates electrons when Cyt b559 is oxidized prior to illumination. These results have significant repercussions on the current thinking concerning the protective role of the Cyt b559/ChlZ electron-transfer pathways and on structural models of PSII.
The Mn-derived electron paramagnetic resonance (EPR) multiline signal from the S(0) state of the water-oxidizing complex is observable only in the presence methanol. In the present study, we have characterized the effect of methanol on the EPR signals from the S(0) and S(2) states as well as on the EPR Signal II(slow) originating from the Tyrosine(D)(ox) radical. The amplitudes of the S(0) and S(2) multiline signals increase with the methanol concentration in a similar way, whereas the S(2) g=4.1 excited state signal amplitude shows a concomitant decrease. The methanol concentration at which half of the spectral change has occurred is approximately 0.2% and the effect is saturating around 5%. Methanol has an effect on the microwave power saturation of the S(2) multiline signal, as well. The microwave power at half saturation (P(1/2)) is 85 mW in the presence of methanol, whereas the signal relaxes much slower (P(1/2) approximately 27 mW) without. The relaxation of Signal II(slow) in the presence of methanol has also been investigated. The P(1/2) value of Signal II(slow) oscillates with the S cycle in a similar way as without methanol, but the P(1/2) values are consistently lower in the methanol-containing samples. From the results, we conclude that methanol modifies the magnetic properties of the S(0) and S(2) states in a similar way. The possible site and nature of methanol binding is discussed.
The oxygen evolving complex (OEC) of photosystem II (PSII) gives rise to manganese-derived electron paramagnetic resonance (EPR) signals in the S0 and S2 oxidation states. These signals exhibit different microwave power saturation behavior between 4 and 10 K. Below 8 K, the S0 state EPR signal is a faster relaxer than the S2 multiline signal, but above 8 K, the S0 signal is the slower relaxer of the two. The different temperature dependencies of the relaxation of the S0 and S2 ground-state Mn signals are due to differences in the spin-lattice relaxation process. The dominating spin-lattice relaxation mechanism is concluded to be a Raman mechanism in the S0 state, with a T(4.1) temperature dependence of the relaxation rate. It is proposed that the relaxation of the S2 state arises from a Raman mechanism as well, with a T(6.8) temperature dependence of the relaxation rate, although the data also fit an Orbach process. If both signals relax through a Raman mechanism, the different exponents are proposed to reflect structural differences in the proteins surrounding the Mn cluster between the S0 and S2 states. The saturation of SII(slow) from the Y(D)(ox) radical on the D2 protein was also studied, and found to vary between the S0 and the S2 states of the enzyme in a manner similar to the EPR signals from the OEC. Furthermore, we found that the S2 multiline signal in the second turnover of the enzyme is significantly more difficult to saturate than in the first turnover. This suggests differences in the OEC between the first and second cycles of the enzyme. The increased relaxation rate may be caused by the appearance of a relaxation enhancer, or it may be due to subtle structural changes as the OEC is brought into an active state.
The tetranuclear manganese cluster responsible for the oxidation of water in photosystem II cycles through five redox states denoted S(i)() (i = 0, 1, 2, 3, 4). Progress has been made recently in the detection of weak low-field EPR absorptions in both the perpendicular and parallel modes, associated with the integer spin state S(3) [Matsukawa, T., Mino, H., Yoneda, D., and Kawamori, A. (1999) Biochemistry 38, 4072-4077]. We confirm observation of these signals and have obtained them in high yield by illumination of photosystem II membranes, in which the non-heme iron was chemically preoxidized. It is shown that a split g = 4 signal accompanies the S(3) state signals. The signals diminish in the presence of ethanol and vanish in the presence of methanol. This effect is similar to that exerted by these alcohols to the high-spin component (g = 4.1) of the S(2) state and suggests that the latter spin configuration is the precursor of the S(3) state low-field signals. The S(3) state shows similar sensitivity to infrared illumination as has been observed previously in the S(2) state [Boussac, A., Un, S., Horner, O., and Rutherford, A. W. (1998) Biochemistry 37, 4001-4007]. Illumination of the S(3) state with near-infrared light (700-900 nm), at temperatures around 50 K, results in the modification of the low-field signals and most notably to the appearance of a broad (DeltaH approximately 200 G) radical-type signal centered at g = 2. The signal is tentatively assigned to the interaction of the Mn cluster in a modified S(2) state with a radical.
Photosystem II uses visible light to drive the oxidation of water, resulting in bioactivated electrons and protons, with the production of molecular oxygen as a byproduct. This water-splitting reaction is carried out by a manganese cluster/tyrosine radial ensemble, the oxygen -evolving complex. Although conventional continuous-wave, perpendicular -polarization electron paramagnetic resonance (EPR) spectroscopy has significantly advanced our knowledge of the structure and function of the oxygen-evolving complex, significant additional information can be obtained with the application of additional EPR methodologies. Specifically, parallel-polarization EPR spectroscopy can be use to obtain highly resolved EPR spectra of integer spin Mn species, and pulsed EPR spectroscopy with electron spin echo-based sequences, such as electron spin echo envelope modulation and electron spin echo-electron nuclear double resonance, can be used to measure weak interactions obscured in continuous-wave spectroscopy by inhomogeneous broadening.
Electron paramagnetic resonance (EPR) spectroscopy has often played a crucial role in characterizing the various cofactors and processes of photosynthesis, and photosystem II and its oxygen evolving chemistry is no exception. Until recently, the application of EPR spectroscopy to the characterization of the oxygen evolving complex (OEC) has been limited to the S2-state of the Kok cycle. However, in the past few years, continuous wave-EPR signals have been obtained for both the S0- and S1-state as well as for the S2 (radical)(Z)-state of a number of inhibited systems. Furthermore, the pulsed EPR technique of electron spin echo electron nuclear double resonance spectroscopy has been used to directly probe the 55Mn nuclei of the manganese cluster. In this review, we discuss how the EPR data obtained from each of these states of the OEC Kok cycle are being used to provide insight into the physical and electronic structure of the manganese cluster and its interaction with the key tyrosine, Y(Z).
Electron paramagnetic resonance (EPR) spectroscopy is one of the major techniques used to analyse the structure and function of the water oxidising complex (WOC) in Photosystem II. The discovery of an EPR signal from the S0 state has opened the way for new experiments, aiming to characterise the S0 state and elucidate the differences between the different S states. We present a review of the biochemical and biophysical characterisation of the S0 state multiline signal that has evolved since its discovery, and compare these results to previous and recent data from the S2 multiline signal. We also present some new data from the S2 state reached on the second turnover of the enzyme.
The mechanism by which the Mn-containing oxygen evolving complex (OEC) produces oxygen from water has been of great interest for over 40 years. This review focuses on how X-ray spectroscopy has provided important information about the structure of this Mn complex and its intermediates, or S-states, in the water oxidation cycle. X-ray absorption near-edge structure spectroscopy and high-resolution Mn Kbeta X-ray emission spectroscopy experiments have identified the oxidation states of the Mn in the OEC in each of the intermediate S-states, while extended X-ray absorption fine structure experiments have shown that 2.7 A Mn-Mn di-mu-oxo and 3.3 A Mn-Mn mono-mu-oxo motifs are present in the OEC. X-ray spectroscopy has also been used to probe the two essential cofactors in the OEC, Ca2+ and Cl-, and has shown that Ca2+ is an integral component of the OEC and is proximal to Mn. In addition, dichroism studies on oriented PS II membranes have provided angular information about the Mn-Mn and Mn-Ca vectors. Based on these X-ray spectroscopy data, refined models for the structure of the OEC and a mechanism for oxygen evolution by the OEC are presented.
Photosystem II (PSII) contains two accessory chlorophylls (Chl(Z), ligated to D1-His118, and Chl(D), ligated to D2-His117), carotenoid (Car), and heme (cytochrome b(559)) cofactors that function as alternate electron donors under conditions in which the primary electron-donation pathway from the O(2)-evolving complex to P680(+) is inhibited. The photooxidation of the redox-active accessory chlorophylls and Car has been characterized by near-infrared (near-IR) absorbance, shifted-excitation Raman difference spectroscopy (SERDS), and electron paramagnetic resonance (EPR) spectroscopy over a range of cryogenic temperatures from 6 to 120 K in both Synechocystis PSII core complexes and spinach PSII membranes. The following key observations were made: (1) only one Chl(+) near-IR band is observed at 814 nm in Synechocystis PSII core complexes, which is assigned to Chl(Z)(+) based on previous spectroscopic studies of the D1-H118Q and D2-H117Q mutants [Stewart, D. H., Cua, A., Chisholm, D. A., Diner, B. A., Bocian, D. F., and Brudvig, G. W. (1998) Biochemistry 37, 10040-10046]; (2) two Chl(+) near-IR bands are observed at 817 and 850 nm in spinach PSII membranes which are formed with variable relative yields depending on the illumination temperature and are assigned to Chl(Z)(+), and Chl(D)(+), respectively; (3) the Chl and Car cation radicals have significantly different stabilities at reduced temperatures with Car(+) decaying much faster; (4) in Synechocystis PSII core complexes, Car(+) decays by recombination with Q(A)(-) and not by Chl(Z)/Chl(D) oxidation, with multiphasic kinetics that are attributed to an ensemble of protein conformers that are trapped as the protein is frozen; and (5) in spinach PSII membranes, Car(+) decays mainly by recombination with Q(A)(-), but also partly by formation of the 850 nm Chl cation radical. The greater stability of Chl(Z)(+) at low temperatures enabled us to confirm that resonance Raman bands previously assigned to Chl(Z)(+) are correctly assigned. In addition, the formation and decay of these cations provide insight into the alternate electron-donation pathways to P680(+).