Structure, ligands and substrate coordination of the oxygen-evolving complex of photosystem II in the S₂ state: a combined EPR and DFT study

Abstract

The S₂ state of the oxygen-evolving complex of photosystem II, which consists of a Mn₄O₅Ca cofactor, is EPR-active, typically displaying a multiline signal, which arises from a ground spin state of total spin S_T = 1/2. The precise appearance of the signal varies amongst different photosynthetic species, preparation and solvent conditions/compositions. Over the past five years, using the model species Thermosynechococcus elongatus, we have examined modifications that induce changes in the multiline signal, i.e. Ca²⁺/Sr²⁺-substitution and the binding of ammonia, to ascertain how structural perturbations of the cluster are reflected in its magnetic/electronic properties. This refined analysis, which now includes high-field (W-band) data, demonstrates that the electronic structure of the S₂ state is essentially invariant to these modifications. This assessment is based on spectroscopies that examine the metal centres themselves (EPR, ⁵⁵Mn-ENDOR) and their first coordination sphere ligands (¹⁴N/¹⁵N- and ¹⁷O-ESEEM, -HYSCORE and -EDNMR). In addition, extended quantum mechanical models from broken-symmetry DFT now reproduce all EPR, ⁵⁵Mn and ¹⁴N experimental magnetic observables, with the inclusion of second coordination sphere ligands being crucial for accurately describing the interaction of NH₃ with the Mn tetramer. These results support a mechanism of multiline heterogeneity reported for species differences and the effect of methanol [Biochim. Biophys. Acta, Bioenerg., 2011, 1807, 829], involving small changes in the magnetic connectivity of the solvent accessible outer Mn_A4 to the cuboidal unit Mn₃O₃Ca, resulting in predictable changes of the measured effective ⁵⁵Mn hyperfine tensors. Sr²⁺ and NH₃ replacement both affect the observed ¹⁷O-EDNMR signal envelope supporting the assignment of O5 as the exchangeable μ-oxo bridge and it acting as the first site of substrate inclusion.

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1 Introduction

In oxygenic photosynthesis, light-driven water oxidation is catalysed by the oxygen-evolving complex (OEC) of the transmembrane protein complex photosystem II (PSII). The OEC consists of a μ-oxo-bridged tetramanganese-calcium cofactor (Mn₄O₅Ca), embedded in its protein matrix. This matrix includes the redox-active tyrosine residue Y_Z (D1-Tyr161), which couples electron transfer between the Mn₄O₅Ca cluster and P680˙⁺, the photooxidant of the PSII reaction centre. The four-electron water oxidation reaction requires four consecutive light-induced charge separation events, driving the catalytic cycle of the OEC.^1,2 This cycle involves five redox intermediates, the S_n states, where n = 0–4 indicates the number of stored oxidizing equivalents. All S-state transitions represent oxidations of the Mn₄O₅Ca cluster by Y_Z˙ with the exception of the regeneration of S₀ from S₄, which proceeds spontaneously under the release of molecular triplet oxygen and the rebinding of at least one substrate water molecule. The rate-limiting step, oxidation of S₃ by Y_Z˙, has prevented the transient, fast-decaying S₄ state from being characterized yet. For a general introduction into water oxidation by the OEC, see ref. 3–8.

The structure of the Mn₄O₅Ca inorganic core⁹ resembles a ‘distorted chair’ where the base is formed by a μ-oxo-bridged cuboidal Mn₃O₄Ca unit (Fig. 1), while the fourth, ‘outer’ Mn, Mn_A4 is attached to this core structure via two μ-oxo bridges, O4 and O5. Spectroscopic evidence suggests that all five oxygen bridges are deprotonated in the S₂ state.^10–15 In addition to the μ-oxo-bridged network, the Mn₄O₅Ca scaffold is held together by six carboxylate ligands, four of which (D1-Asp342, D1-Asp170, D1-Glu333 and CP47-Glu354) form μ-carboxylato bridges between Mn sites. The cluster has only one immediate nitrogen ligand, the imidazole imino-N of D1-His332.

Fig. 1 (a) DFT cluster model of the native OEC and its immediate surrounding in the S₂ (S_T = 1/2) state. Mn ions are depicted in purple, O in red, Ca in yellow, C in light grey, N in blue and H in white; most H atoms are omitted for clarity. (b) Mn₄O₅Ca/Sr–H₂O/NH₃ cores showing the atom and ligand labels used in this work.

An understanding of the mechanism of water oxidation catalysis is intimately linked to an understanding of the electronic states of the paramagnetic tetranuclear Mn complex. Electron paramagnetic resonance (EPR) spectroscopy represents a powerful methodology in this endeavour.^16–19 The S₂ state, being readily generated from the dark-stable S₁ state, is the most extensively characterized S state by EPR. It is intrinsically heterogeneous,^20–22 existing in two ground state configurations. The lowest energy form exhibits a ground state of total spin S_T = 1/2. This spin configuration gives rise to the well-known S₂ state EPR multiline signal centred at g ≈ 2.0, with a hyperfine pattern of at least 24 peaks, with a line spacing around 87 G.²³ Depending on the conditions used, the S₂ state can also exhibit other broad EPR signals, centred at g ≥ 4.1, which have been assigned to (an) S_T ≥ 5/2 spin state(s).^20,24–28 The g ≥ 4.1 signal can also be induced by near-infrared illumination of the S₂ multiline state at temperatures ≤160 K.^24,25,29 Pulse electron nuclear double resonance (ENDOR) experiments,³⁰ especially at Q-band frequencies,^31–34 have enabled a more detailed examination of the electronic structure by uniquely determining four ⁵⁵Mn hyperfine interactions that give rise to the multiline pattern of the corresponding S_T = 1/2 EPR signal. This ENDOR analysis has strongly suggested that the OEC contains one Mn^III ion and three Mn^IV ions when poised in the S₂ state.^30–34

OEC models developed from broken-symmetry (BS) density functional theory (DFT) have been crucial for the interpretation of EPR and related magnetic resonance results.^{12,14,18,33,35–44} These calculations now allow different S-state models to be assessed based on the spin multiplicities and relative energies of their lowest magnetic levels, and, by means of the spin projection formalism (see ref. 18 and 36), the metal and ligand hyperfine couplings. This strategy enables the assignment of individual Mn oxidation states and coordination geometries and represents a method to develop unified models of the OEC that combine constraints from X-ray diffraction, EXAFS and magnetic resonance.

Site perturbation of the OEC provides a means to further characterize the global geometric and electronic structure of the Mn tetramer and obtain information about localized changes associated with the introduced modification. The two best described OEC perturbations in the literature are (i) the replacement of the Ca²⁺ with a Sr²⁺ ion^33,45–47 and (ii) the binding of NH₃ to the cluster.^48–55 Both of these perturbations do not inhibit functional turnover of the catalyst, but do modify the kinetics of O–O bond formation, substrate water uptake and its subsequent deprotonation. A brief description of these two modified OEC forms is given below:

(i) Sr²⁺ can be introduced into the OEC through chemical removal of the Ca²⁺ ion^45,46,56 or alternatively by biosynthetic incorporation in cyanobacterial cultures grown in the presence of SrCl₂ instead of CaCl₂.⁴⁷ Without Ca²⁺ (or Sr²⁺) the OEC is inhibited,^56–63 not progressing further than a modified form of the S₂ state, i.e. the S₂′Y_Z˙ state.^60–62 Sr²⁺ is unique as it is the only ion which can replace the Ca²⁺ ion while retaining catalytic activity, albeit at a lower enzymatic rate.^45,47,56 Presumably this is because Sr²⁺ has a similar size and Lewis acidity as Ca²⁺.⁶⁴ While slowing the turnover rate of the catalyst, Sr²⁺ substitution at the same time enhances the exchange rate with bulk water of at least one of the bound substrates,^65,66 as observed by time-resolved membrane inlet mass spectrometry (MIMS).^67,68 This behaviour suggests the Ca²⁺ ion may play an important role in substrate water binding and possibly proton release (for reviews, see ref. 3 and 69).

(ii) Ammonia binding to the Mn cluster (in the presence of high Cl⁻ concentrations)^48,50,51,54 only occurs upon formation of the S₂ state. It is subsequently released at some point during the S-state cycle (S₃ → S₀ → S₁), such that it is not bound upon return to S₁.⁵¹ As with Sr²⁺ replacement, NH₃ binding does not inhibit catalytic function. In the higher plant electron spin echo envelope modulation (ESEEM) study of Britt et al.,⁷⁰ NH₃ was shown to bind as a direct ligand of the Mn tetramer. The precise binding site and coordination mode of the NH₃ molecule was the subject of a recent study on cyanobacterial PSII from our laboratory.⁴⁴ By employing electron electron double resonance (ELDOR)-detected NMR (EDNMR), it was concluded that NH₃ replaces the water ligand of Mn_A4trans to the O5 bridge (W1, Fig. 1b). As the binding of NH₃ was also shown not to affect substrate exchange rates, these results suggest W1 does not represent a substrate water. One or more additional NH₃ binding sites, which are inhibitory, are known but are less well characterized.^49,50,71

Here, we present an extension of our earlier multifrequency EPR studies^33,34,44 of different ‘archetypal’ multiline forms, namely the native (Mn₄O₅Ca), Sr²⁺ substituted (Mn₄O₅Sr) and NH₃-treated (Mn₄O₅Ca–NH₃) S₂ states, providing a comprehensive analysis of all present X-, Q- and W-band data of ⁵⁵Mn, ¹⁴N/¹⁵N and ¹⁷O signals and for the first time including additional data on the doubly modified (Mn₄O₅Sr–NH₃) S₂ state. Improved ⁵⁵Mn ENDOR experimental conditions provide more reliable spectral line shapes than before, confirming our previous general S₂ state model. Extended BS-DFT models are shown to reproduce all EPR, ⁵⁵Mn and ¹⁴N magnetic spectroscopic observables for the native and the modified systems, a feature not achieved previously. The experimental results and calculations for ¹⁴N/¹⁵N ligands of the various S₂ state forms serve to prove that the basic electronic structure is not perturbed by these modifications, a result crucial for the interpretation of concomitant perturbations of the ¹⁷O EDNMR signal envelope. This combined experimental and theoretical approach supports our qualitative model for multiline heterogeneity, demonstrating that the magnetic connectivity between the two subunits and also within the trimeric moiety governs the structure of the multiline signal.³⁴ This basic structural template also explains the apparent orientations of the ⁵⁵Mn hyperfine tensors, as inferred from spectral simulations and single crystal measurements,³² and potentially provides a framework to further examine substrate binding. The different OEC forms represent a starting point to examine the energetics of higher S-state transitions, as they differ with regard to substrate binding and the kinetics of O–O bond formation and O₂ release.

2 Materials and methods

2.1 PSII sample preparation

Ca²⁺- and Sr²⁺-containing PSII core complex preparations from T. elongatus⁷² were isolated as reported before^47,73,74 with the same modifications for the X-band samples as described in ref. 44. Universal ¹⁵N-labelling of the PSII proteins was achieved by growing the cyanobacteria in a modified BG11 or DTN medium that contained ¹⁵NH₄Cl and ¹⁵NO₃⁻ salts as the sole nitrogen source.⁷⁵ PSII preparations were stored at −80 °C until use. Dark-adapted samples were placed in X-, Q- or W-band quartz tubes with inner diameters of 3.0, 1.6 and 0.6 mm, respectively, and kept at 77 K (liquid N₂) until use. A sample concentration of 3.0–4.0 mg chlorophyll per ml was used throughout this study. All work was conducted in the dark or under dim green light. NH₃ modification was conducted as described in ref. 44, see also Section S1 of the ESI.† Isotopically enriched H₂¹⁷O buffer exchange was achieved as described in ref. 14.

2.2 X-, Q-, and W-band EPR and ENDOR measurements

X-band (≈9 GHz) continuous-wave (CW) EPR spectra were recorded at liquid He temperatures on a Bruker ELEXSYS E500 spectrometer, equipped with an ESR 900 liquid helium flow cryostat and an ITC503 helium flow temperature controller (Oxford Instruments Ltd). X-band pulse EPR measurements were carried out at 4.3 K using a Bruker ELEXSYS E580 spectrometer, equipped with a CF935 cryostat and an ITC503 temperature controller. Q-band (≈34 GHz) pulse EPR measurements were performed around 5 K using an ELEXSYS E580 spectrometer, equipped with a homebuilt TE₀₁₁ microwave cavity,⁷⁶ a CF935 liquid helium cryostat, an ITC-503 temperature controller and a radiofrequency (RF) amplifier ENI 5100L. W-band (≈94 GHz) EPR experiments were performed at 4.8 K using a Bruker ELEXSYS E680 EPR spectrometer. All W-band experiments were carried out using a homebuilt ENDOR microwave cavity, which contained a solenoid of Teflon coated silver wire integrated into a commercial ENDOR probe head (Bruker). In order to ensure broadband microwave excitation and minimize distortions, the loaded quality factor Q_L was lowered to 700 to obtain a microwave frequency bandwidth of 130 MHz.

Electron spin echo-detected (ESE) field-swept EPR spectra were measured using the pulse sequence t_p–τ–2t_p–τ–echo,⁷⁷ three-pulse ESEEM spectra by use of t_p–τ–t_p–T–t_p–τ–echo⁷⁸ and hyperfine sublevel correlation (HYSCORE) spectra by employing t_p–τ–t_p–T₁–2t_p–T₂–t_p–τ–echo.⁷⁹ The lengths of the π/2 microwave pulses were generally set to t_p = 16 ns (X-band), 12 ns (Q-band) and 24 ns (W-band), respectively. For ESE-detected EPR experiments, inter-pulse distances were τ = 260 ns (Q-band) and 300 ns (W-band). For the three-pulse ESEEM measurements, multiple τ values in the ranges τ = 136–248 ns (X-band) and 200–356 ns (Q-band) and an optimum τ = 260 ns for the HYSCORE experiments were chosen to account for blind-spotting artefacts. Q-band ⁵⁵Mn-ENDOR spectra were acquired employing the Davies-type pulse sequence t_inv–t_RF–T–t_p–τ–2t_p–τ–echo⁸⁰ using a length t_inv = 24 ns for the π inversion microwave pulse and a radio frequency π pulse length t_RF = 3.5 μs. The length of the π/2 microwave pulse in the detection sequence was generally set to t_p = 12 ns and the inter-pulse delays to T = 2 μs and τ = 268 ns. A shot repetition time of 1 ms was used for all measurements. EDNMR measurements were collected using the pulse sequence t_HTA–T–t_p–τ–2t_p–τ–echo.⁸¹ The high turning angle (HTA) microwave pulse was applied at microwave frequencies ν_mw. The Hahn echo detection pulse sequence t_p–τ–2t_p–τ–echo, at a microwave frequency ν⁽⁰⁾_mw matched to the cavity resonance, was set at a sufficient time T after the HTA pulse to ensure near-complete decay of the electron spin coherencies. The π/2 pulse length used for detection was t_p = 200 ns (¹⁴N, ¹⁷O) or 80 ns (¹⁵N) and an inter-pulse separation of τ = 500 ns was used. The echo was integrated ≈600 ns around its maximum. The spectra were acquired via continuously sweeping the HTA frequency ν_mw at a fixed magnetic field in steps of 78.1 kHz (¹⁴N), 128.9 kHz (¹⁵N) or 162.1 kHz (¹⁷O). A HTA microwave pulse of length t_HTA = 14 μs (¹⁴N, ¹⁷O) and 8 μs (¹⁵N) and an amplitude ω₁ = 12–16 × 10⁶ rad s⁻¹ was used.

2.3 Spectral simulations

Spectra were fit assuming an effective spin S_T = 1/2 ground state (Section S4.2, ESI†). The basis set that describes the ⁵⁵Mn tetramer-single electron spin manifold (eqn (1)) and the ¹⁴N, ¹⁵N and ¹⁷O single nucleus-single electron spin manifolds (eqn (2)) can be built from the product of the eigenstates of the interacting spins:

|½MI₁m₁I₂m₂I₃m₃I₄m₄〉 (1)

|½MIm〉 (2)

Here, M refers to the electronic magnetic sublevel, ±½; I takes the values 5/2 for ⁵⁵Mn, 1 for ¹⁴N, ½ for ¹⁵N and 5/2 for ¹⁷O; m_i takes the values −I_i, 1 − I_i,…, I_i − 1, I_i. The spin manifolds can be described by the following spin Hamiltonian:It contains (i) the Zeeman term for the total electronic spin, (ii) the hyperfine and (iii), except for the EPR spectra, nuclear Zeeman terms for either the metal ⁵⁵Mn nuclei, or the ligand ¹⁴N, ¹⁵N or ¹⁷O nuclei and (iv), for the ¹⁴N nuclei (I = 1), the nuclear quadrupole interaction (NQI) term; the NQI splitting is not resolved in the ⁵⁵Mn and ¹⁷O spectra. Spectral simulations were performed numerically using MATLAB® (R2010a, The MathWorks, Natick, MA, USA), a vector-based linear algebra package, and the EasySpin toolbox.⁸² For further information on data processing, details of the simulations and theory, see Sections S2, S3 and S4 (ESI†), respectively.

2.4 DFT calculations

All calculations were performed with ORCA.⁸³ The DFT models of the OEC systems consist of 238 or 239 atoms (with H₂O or NH₃ at the W1 position, respectively) and were constructed as described in Pantazis et al.⁴⁰ Alternative ammonia binding modes, including terminal or bridging amido and imido substitution, can be rejected on energetic grounds alone (see Fig. S2, ESI†). Geometry optimizations of the cluster models used the BP86 density functional^84,85 with the zeroth-order regular approximation (ZORA)^86–88 and specially adapted segmented all-electron relativistically recontracted basis sets⁸⁹ (ZORA-TZVP for Mn, O and N; ZORA-SVP for C and H). The conductor-like screening model (COSMO)⁹⁰ was used with a dielectric constant ε = 8.0. D3 dispersion corrections⁹¹ were applied in all calculations. Exchange coupling constants, assuming an isotropic Heisenberg Hamiltonian of the formand all EPR properties were computed with the TPSSh hybrid meta-GGA functional^92,93 from BS-DFT calculations.^35,94–98 The resolution of identity (RI)⁹⁹ approximation was used in the calculation of Coulomb integrals and the chain-of-spheres approximation (COSX)¹⁰⁰ was used for Hartree–Fock exchange, employing completely decontracted def2-TZVP/J auxiliary basis sets.¹⁰¹ Tight SCF convergence criteria and increased integration grids (Grid6 and GridX6) were applied throughout.

For the calculation of the hyperfine tensors, triple-zeta ZORA-recontracted basis sets were used on all atoms, while locally dense radial grids were used for Mn, N and O atoms (integration accuracy of 11 for Mn and 9 for N and O as per ORCA nomenclature). Picture change effects were applied for the calculation of EPR parameters and the complete mean-field approach was used for the spin–orbit coupling operator. The results were transformed into on-site or spin-projected values as detailed previously.³⁶ To compare computed ⁵⁵Mn hyperfine coupling constants using the methods described above with experimental results, a scaling factor of 1.78 was calculated from a set of twelve Mn^IIIMn^IV mixed-valence dimers.¹⁰²

3 Results and discussion

3.1 DFT models of different OEC forms in the S₂ state

Geometric parameters of optimized DFT cluster models of the S₂ state of the OEC in the S_T = 1/2 configuration⁴⁰ are shown in Fig. 2 (for coordinates, see Section S5, ESI†). Four variants were considered in this study: (i) the native cofactor system (Mn₄O₅Ca, also see Fig. 1), (ii) the Sr²⁺-substituted system obtained by replacing Ca²⁺ with Sr²⁺ (Mn₄O₅Sr), (iii) the NH₃-modified system obtained by replacing the H₂O in the W1 position with NH₃ (Mn₄O₅Ca–NH₃), and (iv) the combined Sr²⁺-substituted and NH₃-modified system (Mn₄O₅Sr–NH₃). In all models, W2 was considered to be an OH⁻ ligand, as determined previously.¹² Mulliken spin population analysis of all four variants confirms that the only Mn^III ion of the tetramanganese complex is Mn_D1. The three Mn^IV ions (Mn_A4, Mn_B3 and Mn_C2) represent coordinatively saturated, 6-coordinate octahedral sites, whereas the Mn_D1^III is 5-coordinate square-pyramidal, with a Jahn–Teller elongation along the axis of the Mn_D1-Asp342 carboxylate ligand, opposite to its open coordination site.

Fig. 2 Bond lengths and metal–metal distances in Å of the four geometry-optimized DFT models.

In accordance with previous DFT and QM/MM structures,^{12,37,38,40–42,103–106} the optimized Mn–Mn and Mn–Ca distances of the Mn₄O₅Ca model are consistent with those determined from EXAFS spectroscopy.^107–110 Only minor changes are observed between the Mn₄O₅Ca and the Mn₄O₅Sr models (Fig. 2). As a result of the larger radius of Sr²⁺, the O–Sr bond lengths increase by 0.04 Å, while the Mn–Sr distances also increase by 0.04 Å except for Mn_D1–Sr, which is 0.03 Å shorter than the Mn_D1–Ca distance. On average, this is in line with observations from EXAFS spectroscopy¹⁰⁸ and with the recent 2.1 Å resolution crystallographic model of Sr²⁺-substituted PSII.¹¹¹ The Mn–Mn distances are almost entirely unaffected, with the exception of Mn_D1–Mn_C2, which is shortened by 0.02 Å in the Mn₄O₅Sr model.

Upon NH₃ substitution of W1 (Mn₄O₅Ca–NH₃), only the Mn_D1–Mn_B3 distance and the Ca²⁺ distance from the terminal Mn ions change notably, albeit by less than 0.05 Å (Fig. 2). Only one structural element is more significantly perturbed, i.e. the position of O5, the μ-oxo bridge trans to the binding position of NH₃. The Mn_A4–O5 distance increases by 0.05 Å with concomitant decrease of the Mn_D1–O5 distance by 0.14 Å. Other ligands of Mn_A4, such as the second water-derived ligand, W2, remain unaffected. A similar modification to the connectivity of the Mn₄O₅ core was seen for the smaller OEC models reported in our previous study.⁴⁴ We note that EXAFS data for the NH₃-modified OEC have only been reported for samples purified from spinach, not from the cyanobacterial model systems T. elongatus/vulcanus, and suggested an elongation of one of the short Mn–Mn distances of 0.02 Å.¹¹³ This type of perturbation is not observed in our optimized Mn₄O₅Ca–NH₃ model. The structural model including both Sr²⁺ and NH₃ (Mn₄O₅Sr–NH₃) is found to replicate both effects seen in the singly modified structures.

3.2 Multifrequency EPR and ⁵⁵Mn-ENDOR of the S₂ states of the OEC variants

Multifrequency EPR/⁵⁵Mn-ENDOR experiments spanning the microwave range from ≈9 to ≈90 GHz were employed to experimentally characterize the electronic structures of the different S₂ state forms described above. Fig. 3A (black solid traces) depicts X-band CW EPR spectra of the S₂ state of native PSII (Mn₄O₅Ca),³³ Sr²⁺-substituted PSII (Mn₄O₅Sr),³³ NH₃-modified (annealed) native PSII (Mn₄O₅Ca–NH₃)⁴⁴ and NH₃-modified Sr²⁺-substituted PSII (Mn₄O₅Sr–NH₃). Shown are light-minus-dark spectra, generated by taking the difference between the illuminated spectrum (S₂) and the dark-state spectrum (S₁) in order to remove background signals, such as from the cytochromes b₅₅₉ and c₅₅₀. The modified multiline (Mn₄O₅Sr, Mn₄O₅Ca–NH₃) displays 26 lines of altered intensity as compared to the native multiline signal with 24 lines. The Mn₄O₅Sr–NH₃ S₂ state yields essentially the same modified multiline signal; the simulation superimposing this data trace uses the parameters that fit the Mn₄O₅Sr dataset.

Fig. 3 EPR and ⁵⁵Mn-ENDOR spectra of the S₂ states of the native (Ca), Sr²⁺-substituted (Sr), NH₃-modified (CaNH₃) and NH₃-modified Sr²⁺-substituted (SrNH₃) OEC in PSII isolated from T. elongatus. In panels A, B and D, the black solid traces depict the light-minus-dark subtractions of the experimental spectra. If present, the g ≈ 2 radical signal of Y_D˙ (D2-Tyr160) was removed from the EPR spectra for clarity of presentation. Least-squares fittings to the EPR and ⁵⁵Mn-ENDOR datasets using a model based on the spin Hamiltonian formalism (see Section 2.3 and Sections S3 and S4, ESI†) are represented by superimposing red dashed lines. In panel D, coloured dashed lines represent a decomposition of the simulation showing contributions from the individual ⁵⁵Mn nuclei. Simulations superimposed on the SrNH₃ spectra are those fitted to the Sr dataset. The optimized parameter sets are listed in Table 1. (A) X-band CW EPR. In the Ca and Sr samples, Y_D had been replaced by a phenylalanine, removing the Y_D˙ signal from the spectra,¹¹² which were taken from Cox et al.³³ The CaNH₃ spectrum was originally published in ref. 44. Experimental parameters: microwave frequencies: 9.4097 GHz (Ca), 9.4213 GHz (Sr), 9.4075 GHz (CaNH₃), 9.4970 GHz (SrNH₃); microwave power: 20 mW; modulation amplitude: 25 G; time constant: 82 ms; temperature: 8.6 K. (B) Q-band ESE-detected EPR. The experimental data are presented as pseudo-modulated, derivative-shaped spectra. Experimental parameters: microwave frequencies: 34.0368 GHz (Ca), 34.0430 GHz (Sr), 34.0162 GHz (CaNH₃); shot repetition time: 1 ms; microwave pulse length (π): 24 ns; τ: 260 ns; temperature: 5.2 K. (C) W-band ESE-detected EPR. Contaminating Mn²⁺, present in the samples in varying concentrations, is evident as over-rotated hyperfine features of negative signal intensity. Experimental parameters: microwave frequencies: 93.9894 GHz (Ca), 93.9781 GHz (Sr), 94.0669 GHz (CaNH₃), 94.0615 GHz (SrNH₃); shot repetition time: 1 ms (Ca, Sr), 5 ms (CaNH₃, SrNH₃); microwave pulse length (π): 48 ns; τ: 300 ns; temperature: 4.8 K. (D) Q-band Davies ENDOR. Experimental parameters: microwave frequencies: 33.9678 GHz (Ca), 33.9950 GHz (Sr), 34.0053 GHz (CaNH₃); magnetic field: 1220 mT; shot repetition time: 1 ms; microwave pulse length (π): 32 ns; τ: 268 ns; RF pulse length (π_RF): 3.5 μs; temperature: 4.8 K.

Fig. 3B shows the corresponding Q-band ESE-detected S₂ state multiline EPR signals of the Mn₄O₅Ca, Mn₄O₅Sr and Mn₄O₅Ca–NH₃ OEC forms. Pseudo-modulated (CW-like) spectra are shown in order to more clearly visualize differences in the hyperfine structures between the three forms. Compared to earlier published data by Cox et al.,³³ the spectra are essentially free of contaminating hexaquo-Mn²⁺ signals. Furthermore, there is a small difference of the centre positions of the multiline spectral envelopes, presumably due to inaccuracy in the microwave frequency calibration of this earlier study.

Fig. 3C shows the W-band ESE-detected multiline signals of the Mn₄O₅Ca, Mn₄O₅Sr, Mn₄O₅Ca–NH₃ and Mn₄O₅Ca–NH₃ S₂ state forms. These spectra do contain small hexaquo-Mn²⁺ contaminations. This contamination manifests itself as six inverted hyperfine lines centred at g ≈ 2, as the high-spin Mn²⁺ signal (S = 5/2) is over-rotated when using optimal instrumental settings to visualize the S = 1/2 multiline signal. No ⁵⁵Mn hyperfine structure is observable in the W-band multiline EPR spectra. Thus, these signals provide no additional information on the hyperfine matrices. The utility of these high-frequency data instead is to constrain the G tensor. While the spectra of the Mn₄O₅Ca and the Mn₄O₅Ca–NH₃ cluster show similar signals, centred at g = 1.975, the Mn₄O₅Sr spectrum is shifted to higher field and centred at g = 1.980, similar to the shift observed in W-band spectra from the Bittl laboratory.¹¹⁴ The NH₃-modified Sr²⁺-substituted S₂ state signal is centred at g = 1.979. The almost identical high-field shift, illustrated by the superimposed Mn₄O₅Sr simulation, indicates that the G-tensor shift is dependent only on the presence of Sr²⁺ but not NH₃. This is in contrast to the result at X-band, which showed that the hyperfine structure is approximately the same for both modifications.

Fig. 3D shows the Q-band Davies ⁵⁵Mn-ENDOR light-minus-dark spectra, measured at the central magnetic field position of the corresponding EPR spectra (Fig. 3B). The line shape of the ⁵⁵Mn-ENDOR signal shows only a small field dependence over the 1190 to 1260 mT range (not shown), consistent with its assignment to the tetranuclear Mn cluster.³¹ In contrast to the X-band CW EPR spectra described above (Fig. 3A), the S₂ states of the native, Sr²⁺-substituted, NH₃-annealed and doubly modified (not shown) OEC give rise to highly similar ⁵⁵Mn-ENDOR spectra. Five peaks are observed for all three sample types appearing at approximately the same frequency positions and of similar intensities. Small differences in the region of the largest peak (≈115 MHz) may represent residual Mn²⁺ contaminations. Importantly, no large difference is seen with regard to the total spectral breadth of the signal (≈55 to ≈195 MHz). For the NH₃-modified S₂ state, the results are nominally consistent with the earlier X-band ENDOR data of Peloquin et al.³⁰ The Q-band ENDOR spectra presented here do slightly differ from those presented in ref. 33, 34, 44 and 115 with regard to line intensities, discussed in detail in the Section S6 (ESI†).

Spectral simulations of the complete EPR and ⁵⁵Mn-ENDOR datasets using the spin Hamiltonian formalism are also shown in Fig. 3 (red dashed lines); the fitted effective G and hyperfine tensors A of the Mn clusters in the low-spin S₂ state are listed in Table 1. The effective G tensors of all three spin systems are in the same range as inferred from EPR measurements on PSII single crystals at W-band, i.e. G = [1.997, 1.970, 1.965]¹¹⁶ and G = [1.988, 1.981, 1.965].¹¹⁷ As found previously,^{30,31,33,34,44} the inclusion of four hyperfine tensors of approximately the same magnitude and near-axial symmetry is required to simultaneously fit the X-, Q- and W-band EPR and Q-band ⁵⁵Mn-ENDOR line widths and shapes. The z component represents the principal component for the fitted G and all four hyperfine tensors. Comparison of the fitted parameters demonstrates that the three samples basically exhibit the same electronic structure. The sets of the four isotropic values A_i,iso deviate only by ≤4% between the three different systems and the signs and magnitudes of the anisotropies A_i,aniso are broadly similar, suggesting that there are no significant differences in the electronic exchange coupling schemes of the Mn₄O₅Ca/Sr(–NH₃) clusters.

Table 1 Principal values of the effective G and ⁵⁵Mn hyperfine tensors A_i for the simulations of the S₂ state spectra of the Mn₄O₅Ca, Mn₄O₅Sr and Mn₄O₅Ca–NH₃ clusters in PSII from T. elongatus^a

		G	A i /MHz
			A 1	A 2	A 3	A 4
a All G and A tensors are collinear. b The equatorial and axial G and Ai values are defined as G⊥ = (Gx + Gy)/2, G∥ = Gz, and Ai,⊥ = (Ai,x + Ai,y)/2, Ai,∥ = Ai,z. c The isotropic G and Ai (i = 1–4) values are the averages of the individual values: Giso = (Gx + Gy + Gz)/3 and Ai,iso = (Ai,x + Ai,y + Ai,z)/3. d The anisotropy in the G and Ai values is expressed as the difference Ai,aniso = Ai,⊥ − Ai,∥.
Native	x	1.989	350	214	214	173
	y	1.978	329	195	184	157
	⊥^b	1.983	339	204	199	165
	z^b	1.956	321	282	282	251
	iso^c	1.974	333	230	227	194
	aniso^d	0.028	19	−78	−83	−87
Sr^2+-substituted	x	1.992	328	213	215	161
	y	1.981	347	201	180	175
	⊥^b	1.986	338	207	197	168
	z^b	1.963	320	283	270	224
	iso^c	1.978	332	232	221	187
	aniso^d	0.024	17	−76	−73	−56
NH3-modified	x	1.989	326	214	215	154
	y	1.978	345	195	187	175
	⊥^b	1.984	336	204	201	164
	z^b	1.956	322	283	275	229
	iso^c	1.975	331	231	225	186
	aniso^d	0.027	13	−79	−74	−65

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3.3 Calculated magnetic properties for the native and modified S₂ states of the OEC

The electronic structure of the coupled OEC spin system is defined by the set of six pairwise Mn–Mn exchange interaction terms J_ij, which can be calculated using BS-DFT. For all four computational models describing the set of native and chemically perturbed S₂ state clusters, the calculations reveal that the αββα spin configuration (Fig. 4A and B) is the lowest in energy. Sets of J_ij coupling constants are given in Table S1 of the ESI.† Diagonalization of the Heisenberg Hamiltonian to obtain the complete spin ladder confirms that all four models exhibit an effective total spin S_T = 1/2 ground state, as observed experimentally, and an S_T = 3/2 first excited state. The estimated energy differences between the two lowest states of the spin ladder are on the order of 24–26 cm⁻¹ for the Mn₄O₅Ca/Sr S₂ state structures, lowering by 7 cm⁻¹ upon exchange of W1 for NH₃ (Table S1, ESI†). These values are in the range inferred from experiments.‡ ^{33,34,118,119}

Fig. 4 The magnetism of the low-spin S₂ state. (A) Connectivity of the inorganic centres showing the three μ-oxo-bridged dimanganese units, defining the main electronic exchange pathways of the cluster. (B) Spin coupling scheme showing the main (black solid lines) and weak (grey dashed lines) exchange couplings J_ij within the OEC. The numbers next to the Mn ions represent their spin projection factors ρ_i,iso for the native S₂ state BS-DFT model. (C) Simplified spin coupling model in which the trimer unit Mn_B3Mn_C2Mn_D1 interacts with the outer Mn_A4via an effective coupling J_eff that may be approximated as J_AB.

For all four S₂ state OEC forms, the J-coupling topology consists of three main coupling pathways (Table S1, ESI† and Fig. 4B): (i) an antiferromagnetic coupling pathway between Mn_D1 and Mn_C2 (J_CD); (ii) a ferromagnetic coupling pathway between Mn_C2 and Mn_B3 (J_BC); (iii) and an antiferromagnetic coupling pathway between Mn_B3 and Mn_A4 (J_AB). The ferromagnetic exchange pathway J_BC = 19–28 cm⁻¹ is the largest in absolute magnitude, while the antiferromagnetic pathways J_CD = −16 to −18 cm⁻¹ and J_AB = −12 to −16 cm⁻¹ are slightly weaker. The remaining exchange coupling constants J_AC, J_AD and J_BD are small, as can be expected from geometric considerations (see Table S1, ESI†). J_CD and J_BC represent the two largest exchange interactions within the cuboidal trimer unit (Mn_B3Mn_C2Mn_D1) of the cluster, whereas J_AB can be considered to a good approximation as being representative of an effective exchange interaction between this cuboidal unit and the outer Mn_A4, as shown in Fig. 4C.

Systematic differences are observed for the exchange pathways upon the two chemical perturbations, replacement of Ca²⁺ by Sr²⁺ and NH₃ exchange at W1 (Table S1, ESI†). When comparing the structure pairs that differ in terms of the presence of Ca²⁺ or Sr²⁺, i.e. Mn₄O₅Ca vs. Mn₄O₅Sr and Mn₄O₅Ca–NH₃vs. Mn₄O₅Sr–NH₃, it is seen that only the major coupling pathways J_CD and J_BC are modified, decreasing by 2 cm⁻¹ and 5 cm⁻¹, respectively. J_AB remains unchanged. By contrast, for the corresponding structure pairs where NH₃ is exchanged for W1, the J_CD pathway is unchanged, while J_BC and J_AB increase by 4 cm⁻¹. It is noted that the perturbation of the O5 position upon NH₃ substitution, as shown in Fig. 2, results also in an enhancement of J_BD by 3 cm⁻¹. In both cases, the changes in the magnetic interactions can be understood within the geometric changes discussed in Section 3.1 (see Fig. 2): Sr²⁺ substitution mostly affects the structure of the cuboidal unit, thus perturbing principally the exchange pathways within the Mn-trimer unit, whereas NH₃ binding perturbs mostly the connectivity between the trimeric moiety and the outer Mn_A4 (Fig. 4C).

Calculated ⁵⁵Mn on-site (intrinsic/not spin-projected) hyperfine tensors as in the full (non-effective) spin Hamiltonian based on the BS-DFT models are listed in Table 2 for the four S₂ state variants. The calculated isotropic hyperfine values a_i,iso for the three Mn^IV ions fall within the range seen in Mn^IV model compounds experimentally, i.e. |a_iso| = 187–253 MHz (see ref. 30 and 33). The anisotropy of the calculated hyperfine tensors for these three sites is also small, of the order seen in octahedral Mn^IV model complexes, i.e. |a_aniso| < 30 MHz.³³ For the Mn_D1^III ion, the calculated isotropic hyperfine value (≈130 MHz) is smaller than that for Mn^IV, as expected, and lies just outside the range seen in Mn^III compounds, i.e. |a_iso| = 165–225 MHz.^30,33 As typical for Mn^III, it exhibits a significant hyperfine anisotropy, more pronounced than for the Mn^IV ions. However, it is noted that the calculated values for the Mn_D1^III site are unexpectedly large. Nevertheless, the computed parameters correlate with the inferred site geometry of Mn_D1, namely that of a square-pyramidal 5-coordinate Mn^III ion. Such a coordination environment generally yields a small isotropic ⁵⁵Mn on-site hyperfine coupling and a negative anisotropy (see Table 2), consistent with an effective local ⁵B₁ electronic ground state for the Mn_D1^III ion.^30,33,120

Table 2 Isotropic and anisotropic spin projection factors ρ_i,iso^a and ρ_i,aniso^b, and calculated and experimental isotropic and anisotropic on-site hyperfine values a_i,iso^a and a_i,aniso^b in MHz for the Mn ions of the BS-DFT models of the Mn₄O₅Ca, Mn₄O₅Sr and Mn₄O₅Ca–NH₃ clusters in the low-spin S₂ state configuration^c

Mn ion	Structure	Spin projections		BS-DFT		Experiment
		ρ i,iso	ρ i,aniso	a i,iso	a i,aniso	a i,iso	a i,aniso
a The isotropic ρi,iso and ai,iso values are the averages of the individual tensor components ρi,iso = (2ρi,⊥ + ρi,∥)/3 and ai,iso = (ai,x + ai,y + ai,z)/3. b The anisotropies of the ρi and ai tensors are expressed as the differences ρi,aniso = ρi,⊥ − ρi,∥ and ai,aniso = ai,⊥ − ai,∥, i.e. between the perpendicular and parallel tensor components. c The intrinsic fine structure values of Mn^IV ions were assumed to be dA4 = dB3 = dC2 = 0 cm^−1. For the MnD1^III ion, a value of dD1 = –1.43 cm^−1 was fitted, with eD1/dD1 = 0.
MnA4 (Mn^IV)	Mn4O5Ca	1.11	0.23	−247	−33	−206	25
	Mn4O5Sr	1.13	0.19	−247	−32	−204	31
	Mn4O5Ca–NH3	0.94	0.36	−242	−30	−245	−9
MnB3 (Mn^IV)	Mn4O5Ca	−0.93	0.29	194	−22	207	26
	Mn4O5Sr	−0.92	0.24	194	−21	202	8
	Mn4O5Ca–NH3	−0.86	0.44	193	−19	221	−33
MnC2 (Mn^IV)	Mn4O5Ca	−1.00	0.32	212	−17	226	9
	Mn4O5Sr	−0.99	0.27	212	−18	224	11
	Mn4O5Ca–NH3	−1.01	0.46	213	−16	226	−26
MnD1 (Mn^III)	Mn4O5Ca	1.81	0.38	−128	−142	−186	−46
	Mn4O5Sr	1.78	0.32	−138	−144	−188	−41
	Mn4O5Ca–NH3	1.92	0.54	−127	−142	−176	−52

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The effective hyperfine couplings measured by EPR spectroscopy for oligonuclear metal complexes reflect the on-site hyperfine couplings of the individual metal ion nuclei scaled by the contribution of the electronic spin of each metal ion to the effective spin state: A_i = ρ_ia_i. The set of scaling factors ρ_i, termed spin projection coefficients, are primarily determined by the set of pairwise exchange couplings as detailed in ref. 18, 30, 31, 33, 34, 36, 37 and 63. However, additional terms must be included to correctly estimate such spin projections for the OEC, specifically the relevant on-site fine structure parameters d_i for the individual Mn_i ions,^{18,30,33,34,63} yielding what are more accurately described as spin projection tensors. As the coordination geometries of the three Mn^IV ions of the S₂ state are all octahedral, their local electronic structure should be of approximate spherical symmetry, their orbitals of t_2g origin (d_xy, d_xz and d_yz) being half-filled (local high-spin d³ configuration). As such, the Mn^IV ions are expected to only display small fine structure parameters d_i (<0.3 cm⁻¹)¹²¹ and hence do not need to be explicitly considered. Thus, the set of parameters which define the spin projection tensors in the S₂ state are the six pairwise exchange interaction terms and the fine structure parameter of the Mn^III ion, d_D1.

Using these spin projection tensors, the fitted projected ⁵⁵Mn hyperfine tensors were scaled back to on-site hyperfine tensors to allow comparison to the BS-DFT values discussed above (Table 2). The only plausible assignment for all three forms of the OEC is that A₁, A₂, A₃, and A₄ correspond to a_D1, a_A4, a_C2 and a_B3, respectively. In our previous work,³³ using BS-DFT structural models predating the latest crystal structure,⁹ values of −1.2 to −1.3 cm⁻¹ were estimated for a supposedly axially symmetric d_D1 in the native and Sr²⁺-substituted S₂ states. Using the same approach, d_D1 was re-estimated. It was possible to obtain on-site hyperfine anisotropies in the ranges characteristic for Mn^III and Mn^IV ions employing a single value of −1.43 cm⁻¹ for the three OEC systems, well within the range typically seen for Mn^III model complexes. As discussed above with regard to the hyperfine tensor anisotropy of Mn_D1^III, a negative d value requires an effective local ⁵B₁ state for the Mn_D1^III ion and is thus consistent with the square-pyramidal 5-coordinate ligand geometry of Mn_D1^III, as present in all computational models. The experimental on-site hyperfine tensor values (Table 2) generally agree well with the BS-DFT estimates and Mn^III and Mn^IV model compounds, with smaller isotropic values a_i,iso for the Mn^III ion than for the Mn^IV ions. In the native and Sr²⁺-substituted models, Mn_A4 displays a smaller a_A4,iso than computed and in the NH₃-modified system, where it exhibits the largest coupling of the Mn^IV ions. Compared to the calculations, the anisotropic components for the Mn^IV ions show a larger variance within |a_i,aniso| ≲ 30 MHz. For Mn_D1^III, a_D1,aniso < −40 MHz is less negative than calculated. Overall, the experimental results confirm that the computed spin coupling schemes serve as a valid description of the native and modified S₂ states.

3.4 The Mn_D1–His332-imino-N interaction

Three-pulse ESEEM measurements were performed to characterize the imino-N signal of His332 associated with the OEC variants in the S₂ state. Fig. S4 and S5 (ESI†), respectively, depict τ- and magnetic-field-dependent (g ≈ 2.10–1.90) light-minus-dark- subtracted spectra and simulations of the native Mn₄O₅Ca (A, B), Sr²⁺-substituted Mn₄O₅Sr (C, D) and NH₃-modified Mn₄O₅Ca–NH₃ (E, F) S₂ state samples. As noted in Pérez Navarro et al.,⁴⁴ the ¹⁴N nitrogen signal observed for the native S₂ state from T. elongatus is very similar to that measured in PSII from both higher plants (spinach)¹²² and the mesophilic cyanobacteria Synechocystis sp. PCC 6803,¹²³ assigned to the imino-N of His332 via mutagenesis.^123,124 The signals are essentially the same in the native, Sr²⁺-substituted and NH₃-modified OEC clusters with regard to both their τ and magnetic-field dependence. The His332 imino-¹⁴N signal at Q-band nearly fulfils the cancellation condition, where A_iso is twice the ¹⁴N nuclear Larmor frequency (ν_n = 3.75 MHz at 1.22 T). The spectra are characterized by three features: the lines centred at frequencies below 2.5 MHz (ν_α = ν_n − |A_iso|/2), single-quantum transitions around 7.5 MHz (ν_β = ν_n + |A_iso|/2) and smaller double-quantum resonances around 15 MHz (ν_2β = 2ν_n + |A_iso|). The line structuring is defined both by the ¹⁴N hyperfine anisotropy and the NQI.

HYSCORE spectroscopy (a two-dimensional ESEEM technique) was performed on the three S₂ state OECs at different magnetic-field positions (g ≈ 2.07–1.93) of the corresponding Q-band EPR envelopes to further constrain the ¹⁴N hyperfine and quadrupolar interaction matrices. Panels A, C and E in Fig. 5 show the Fourier-transformed spectra and simulations at the centre field position; low- and high-field spectra and simulations are presented in Fig. S6 and S7 in the ESI.† As seen for the three-pulse ESEEM spectra, their appearance is highly similar for all three variants of the OEC in the S₂ state. In two dimensions, the three features that comprise the Q-band ESEEM spectra appear as cross peaks at corresponding frequencies both in the (−,+) and the (+,+) quadrants. As the ¹⁴N hyperfine coupling matches the cancellation condition, the cross peaks are shifted away from the diagonal, instead appearing near the frequency axes. Overall, virtually no orientation dependence is seen comparing the spectra at the three different magnetic fields (Section S8.2, ESI†), consistent with the electron–nuclear interaction being dominated by the isotropic component of the hyperfine coupling as compared to the anisotropic part and the traceless NQI, as in ref. 122, 123 and 125. Thus, the orientation of the His332 imino-¹⁴N hyperfine tensor relative to the G tensor cannot be determined from this dataset.

Fig. 5 (−,+) and (+,+) quadrants of the Fourier-transformed Q-band HYSCORE experimental spectra (A, C, E) and spin Hamiltonian-based simulations (B, D, F) of the S₂ state Mn₄O₅Ca (A, B), S₂ state Mn₄O₅Sr (C, D) and annealed S₂ state Mn₄O₅Ca–NH₃ (E, F) clusters in PSII samples isolated from T. elongatus at central magnetic field. SQ and DQ point out the regions of single- and double-quantum transitions, respectively. The optimized parameter sets for the simulations, as described in Section 2.3 and Sections S3, S4 and S8.4 (ESI†), are listed in Table 3 and, in detail, in Table S2 (ESI†). Experimental parameters: microwave frequencies: 34.0370 GHz (Ca), 34.0433 GHz (Sr), 34.0151 GHz (NH); magnetic fields: 1220 mT (Ca), 1222 mT (NH₃, Sr); shot repetition time: 1 ms; microwave pulse length (π/2): 12 ns; τ: 260 ns; ΔT: 100 ns; temperature: 5.2 K.

Fitted spin Hamiltonian parameters derived from the simultaneous simulation of both the ESEEM and HYSCORE datasets are given in Table 3 together with BS-DFT estimates. To directly compare DFT values with experiment, the calculated site hyperfine tensor for the His332 was multiplied by the axial Mn_D1 spin projection tensor described in Section 3.3. All DFT models yield virtually the same hyperfine and quadrupole values. The calculated A_iso underestimates experimental results by <20%, but the dipolar component A_dip and the rhombicity A_η nominally agree with experiment. It is noted that the on-site ¹⁴N hyperfine tensor a is expected to be axial with its unique component a₁ aligned along the Mn_D1–N bond, as seen in our calculations (Table S2, ESI†). As such, the axial ¹⁴N hyperfine component a₁ is essentially rotated 90° relative to that of the Mn_D1 spin projection tensor, which is expected to coincide with the Jahn–Teller axis of Mn_D1^III. This 90° rotation explains why the ¹⁴N hyperfine tensor A is rhombic in the projected (experimental) reference frame. For a more detailed description of the simulations, see Section S8.4 (ESI†). Importantly, the near-invariance of the imino-N spin Hamiltonian parameters for the three S₂ state forms requires the His332 ligation, the electronic structure of the Mn_D1^III ion and by extension the whole tetranuclear Mn cluster, to not be significantly perturbed by Ca²⁺/Sr²⁺ and NH₃/W1 exchange, in line with the EPR/⁵⁵Mn ENDOR results described in Section 3.2.

Table 3 Fitted and calculated effective/projected ¹⁴N hyperfine and NQI tensors in MHz for the electron–nuclear couplings of the His332 imino-N and of NH₃ with the various cluster forms studied in the S₂ state in PSII from T. elongatus

S2 state	^14N	Method	|Aiso^a|	A dip	A η	|e^2Qq/h|	η
a A iso is defined as the average of the principal components of the hyperfine tensor: Aiso = (A1 + A2 + A3)/3. b A dip is defined in terms of T1, T2, and T3 as Adip = (T1 + T2)/2 = –T3/2. c The rhombicity is defined by Aη or η = (T1 − T2)/T3, respectively. T1, T2, and T3 represent the three principal components of the hyperfine tensors minus Aiso and of the NQI tensors and are labelled such that |T1| ≤ |T2| ≤ |T3|. d The Euler rotation angles [α, β, γ] of the NQI relative to the A tensors are [20, 12, 0]°, [18, 9, 0]° and [16, 16, 0]° for the Mn4O5Ca, Mn4O5Sr and Mn4O5Ca–NH3 clusters, respectively.
Native	His332^d	Exp.	7.1	0.75	0.81	1.97	0.75
		DFT	5.8	0.59	0.74	1.65	0.91
Sr^2+-substituted	His332^d	Exp.	7.3	0.69	0.83	1.98	0.79
		DFT	5.8	0.57	0.61	1.65	0.91
NH3-modified	His332^d	Exp.	7.2	0.75	0.89	1.96	0.80
		DFT	6.1	0.71	0.99	1.68	0.88
	NH3	Exp.	2.36	0.33	0.22	1.52	0.47
		DFT	2.68	−0.65	0.02	0.94	0.87
NH3-modified, Sr^2+-substituted	NH3	Exp.	2.37	0.28	0.23	1.58	0.45
		DFT	2.68	−0.59	0.03	0.93	0.87

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While the orientation of the hyperfine tensor relative to the G tensor cannot be determined using ESEEM/HYSCORE at Q-band frequencies, it can be measured at W-band, e.g. using EDNMR. In our earlier study,¹⁴ it was found that the hyperfine tensor is orientated such that its principal, i.e. the smallest component A₁ is aligned such that it is mid-way between G_x, and G_z. Importantly though, it is noted that the set of spin Hamiltonian parameters deduced from Q-band ESEEM/HYSCORE (Table 3) does not reproduce the W-band data sets (Section S8.3, ESI†). This is not due to the inclusion/exclusion of the NQI term, which, for the W-band EDNMR data, mainly contributes to the spectral line width. To reproduce the field dependence of the ¹⁴N- and ¹⁵N-EDNMR signals (Fig. S8 and S9, ESI†), the values determined from Q-band ESEEM/HYSCORE needed to be scaled: A_iso was decreased by 10%, whereas A_dip was increased by a factor of two (Table S2, ESI†). The same results were observed for all three S₂ state forms, which basically exhibit the same ¹⁴N-EDNMR spectra. A possible reason for this difference comes from the observation that the ground spin state, an effective spin S_T = 1/2 state, is not very well separated energetically from higher spin states in the regime of the W-band excitation energy (≈3 cm⁻¹), consistent with DFT estimates for the ground-to-first excited state energy splitting ΔE (Section S7, ESI†). Excited-state mixing due to a small ΔE has the consequence of altering spin Hamiltonian observables such as effective ⁵⁵Mn and ¹⁴N hyperfine tensors. Alternatively, the rhombicity of the effective G tensor as inferred from the EPR/⁵⁵Mn-ENDOR simulations may be artificial, a consequence of using collinear G and ⁵⁵Mn hyperfine tensors. This latter suggestion would also explain why the G tensors inferred from W-band measurements on PSII single crystals^116,117 differ from those inferred from our multifrequency measurements on frozen solution PSII samples.

The lack of agreement between the two ¹⁴N datasets brings into question whether the W-band ¹⁴N/¹⁵N-EDNMR signals can be used to assign the exchangeable μ-oxo bridge ¹⁷O signal based on the relative orientations of the ¹⁴N and ¹⁷O hyperfine tensors, as suggested by Rapatskiy et al.¹⁴ Thus, further experimental results, particularly from single crystals of PSII, are needed to test this proposal (see Section 3.6).

3.5 NH₃ binding to the Ca²⁺- and the Sr²⁺-containing OEC

In the NH₃-modified S₂ state, a second nitrogen nucleus is bound to the Mn cluster as a terminal ligand, as described in Pérez Navarro et al.⁴⁴ Its binding can be observed using X-band (three-pulse) ESEEM, as shown in Fig. S11 (ESI†) for ¹⁴NH₃/¹⁵NH₃-bound, Ca²⁺- and Sr²⁺-containing PSII. The ¹⁴NH₃ resonances comprise three characteristic single-quantum lines at 0.5, 0.95 and 1.45 MHz split by the NQI and smaller double-quantum transitions centred at 4.9 MHz, highly similar to the higher plant data.⁷⁰ Due to the lack of the NQI, the ¹⁵NH₃ signal is clearly less complicated, consisting only of one single-quantum hyperfine peak centred at 0.3 MHz. As seen for the His332 imino-¹⁴N signal at Q-band, the NH₃ interaction at X-band fulfils the cancellation condition, leading to a narrow ν_α line while the ν_β line is broadened beyond detection.⁷⁰ Most importantly, the spectra of the ¹⁴NH₃-modified Ca²⁺- and Sr²⁺-containing ¹⁴N-PSII samples are essentially identical. Thus, NH₃ binding to the Sr²⁺-substituted S₂ state cluster is the same as in the native S₂ state.

In our first report on NH₃ binding to the OEC, only the ¹⁴NH₃ interaction was considered.⁴⁴ Here, we simultaneously fit the spectra of both the ¹⁴NH₃-modified ¹⁵N-PSII and the ¹⁵NH₃-modified ¹⁴N-PSII in the S₂ state (Table 3, Fig. S11 and Table S3, ESI†). This resulted in an optimized hyperfine tensor A = [2.76 2.62 1.69] MHz for ¹⁴NH₃ (and for ¹⁵N scaled by the ratio of the nuclear g values of ¹⁵N and ¹⁴N) and ¹⁴N NQI parameters e²Qq/h = 1.52 MHz and η = 0.47. Highly similar hyperfine and NQI values reproduce the NH₃ signal observed in the annealed S₂ state of Sr²⁺-containing ¹⁴N-PSII. The isotropic ¹⁴N hyperfine coupling A_iso = 2.36 MHz and A_dip = 0.33 MHz, e²Qq/h and the asymmetry parameter η are the same as reported before for PSII from T. elongatus⁴⁴ and similar to the values from the analysis of higher plant X-band ESEEM spectra⁷⁰ (Table 3).

An axial projected ¹⁴NH₃ hyperfine tensor is obtained from BS-DFT calculations, as seen in the experiment. This is because (i) the on-site ¹⁴NH₃ hyperfine tensor is axial, and (ii) its axial and equatorial components are essentially coincident with those of the Mn_A4 spin projection tensor (Table S3, ESI†), unlike the case for the His332 imino-¹⁴N a tensor (see Section 3.4). The BS-DFT calculations also reproduce the comparably large and rhombic NQI parameters (Table 3), although the sign of the hyperfine anisotropy is inverted compared to experiment. For more details, see Section S9 in the ESI.†

3.6 Interactions with exchangeable ¹⁷O species

As we have recently shown,^14,19 EDNMR spectroscopy at W-band, due to its comparatively high sensitivity, is the preferred method to measure the interactions of exchangeable ¹⁷O nuclei with fast-relaxing electronic species such as the S₂ state of the OEC. Fig. 6 shows these spectra and simulations (see Section 2.3 and Sections S3 and S4, ESI† for details) of the single-quantum region for the native, the Sr²⁺-substituted, the NH₃-annealed and the Sr²⁺- and NH₃-modified S₂ state variants after H₂¹⁷O buffer exchange in the S₁ state (see Fig. S12 (ESI†) for the double-quantum region). The spectrum of the native system exhibits the single- and double-quantum resonances of the imino-¹⁴N of His332 (blue) and of three different classes of ¹⁷O species,¹⁴i.e. (i) a strongly coupled, bridging species (green), (ii) an intermediately coupled terminal O-ligand (orange), and (iii) a weakly coupled terminal class (cyan). These were assigned to the μ-oxo bridge O5, the hydroxide ion W2¹² and the H₂O matrix (comprising ligand W1 of Mn_A4 and two H₂O ligands at the Ca²⁺ ion), respectively. NH₃ binding causes a narrowing of the ¹⁷O single- and double-quantum envelopes, reproduced by a decrease of the hyperfine couplings of O5 and W2 and concomitant reduction of the matrix line intensity, which was interpreted by NH₃ binding to Mn_A4 in exchange for W1.⁴⁴ Comparing these two spectral forms to those of the corresponding Sr²⁺-substituted W1- and NH₃-containing clusters (Fig. S13, ESI†), we see a systematic narrowing of the single-quantum envelope by ≈0.5 MHz and a corresponding narrowing of the double-quantum envelope. This can be reproduced by spectral simulations in which the hyperfine couplings of the μ-oxo bridge are reduced accordingly (W1: 9.2 MHz vs. 9.7 MHz, NH₃: 6.5 vs. 7.0 MHz), while the other ¹⁷O interactions remain unaltered (for a complete set of hyperfine parameters, see Table S4, ESI†). Although weaker than the NH₃ effect, the narrowing was found to be reproducible in all Sr²⁺-substituted PSII samples. It clearly shows that Ca²⁺/Sr²⁺ exchange perturbs the μ-oxo bridge, in addition to a simultaneous modification by NH₃ binding.

Fig. 6 ¹⁷O-EDNMR spectra of the native (Ca) the Sr²⁺-substituted (Sr), the NH₃-annealed (CaNH₃) and the Sr²⁺-substituted NH₃-annealed (SrNH₃) S₂ states in PSII samples isolated from T. elongatus. Black solid traces depict the single-quantum region of background-corrected experimental spectra; superimposing red dashed traces represent simulations based on the spin Hamiltonian formalism as outlined in Section 2.3 and Sections S3 and S4 (ESI†). Coloured dashed lines represent a decomposition of the simulation showing contributions from the individual ¹⁴N and ¹⁷O nuclei. Black dashed lines highlight the decrease of the strong ¹⁷O interaction upon Ca²⁺/Sr²⁺ exchange. The optimized parameter sets are listed in Table S4 (ESI†). Double-quantum regions are shown in Fig. S12 (ESI†). Inset: a section of the X-band CW EPR spectra of the Sr²⁺-substituted S₂ state in PSII samples in the absence (black) and presence (red) of H₂¹⁷O showing no line broadening upon ¹⁷O exchange. Experimental parameters: see Fig. S12 and for the inset Fig. S14 (ESI†).

The inset in Fig. 6 depicts a section of the X-Band CW EPR spectra of the Sr²⁺-substituted S₂ state, which exhibits an intrinsically smaller average line width (≈3.6 mT peak-to-peak) than the native form (≈4 mT), in the presence and absence of ¹⁷O (see Fig. S14 (ESI†) for the entire spectra). No EPR line broadening is observed upon ¹⁷O exchange. This demonstrates that the largest ¹⁷O coupling represents only one exchangeable oxo bridge. In the case of two hyperfine interactions of ≈10 MHz, the effective line broadening due to the combined ¹⁷O couplings would be larger than 120 MHz or 4.3 mT, exceeding the actual line width.

4 Discussion

4.1 A common electronic structure of the S₂ state variants

Our DFT results show that the Sr²⁺-substituted, the NH₃-annealed and the Sr²⁺- and NH₃-modified low-spin S₂ states basically represent the same structure on both a geometric and electronic level. This result is not immediately obvious from their X-band EPR signals. Indeed historically, the Sr²⁺-substituted and NH₃-modified forms were explained in terms of a change of the valence state distribution within the Mn tetramer and thus of the coordination environment of the Mn^III ion.¹²⁶ The comprehensive approach pursued in this study conclusively rules out such a mechanism for electronic structure perturbation. Instead, as proposed by our group,^33,34,63 multiline heterogeneity reflects rather subtle changes of the Mn-tetramer structure. The similarity of the perturbed multiline forms suggest a common mechanism for electronic perturbation, which probably also explains S₁ state heterogeneity. This is discussed below, with reference to solvent access, substrate binding and exchange.

4.1.1 The mechanism of structural perturbation. In Su et al.,³⁴ a qualitative model for multiline heterogeneity was proposed. In this model, the electronic structure of the S₂ state was considered in terms of a simplified model of two spin fragments: (i) a cuboidal trimer unit, made up of Mn_D1, Mn_C2 and Mn_B3, and (ii) a ‘monomeric’ Mn unit, consisting of the outer Mn_A4. Therein, variation of the electronic structure in S₂ was attributed to changes in the connectivity of the outer Mn_A4 to the cuboid, changing the properties of the electronic ground state by altering the mixing-in of excited spin state character. The physical rationale for this observation was that the outer Mn_A4 represents the solvent accessible end of the cluster. It has two water-derived ligands and solvent channels that begin or terminate at this site.^9,127 As such, it is this site and its connection to the rest of the cluster that is most likely to vary amongst different sample conditions and possibly different PSII species. In terms of this ‘monomer–trimer’ model with regard to the two modifications discussed here, Ca²⁺/Sr²⁺ forms part of the linkage between the cuboidal and outer fragments, as mediated by the μ-oxo bridge O5, whereas NH₃ binds to the outer Mn_A4, also perturbing O5, the bridge trans to its binding position W1.⁴⁴

The magnetic observable that is altered upon Ca²⁺/Sr²⁺ replacement and/or W1/NH₃ exchange, leading to the perturbed multiline forms is the ⁵⁵Mn hyperfine anisotropy. Small perturbations of the four hyperfine tensors result in a change in the hyperfine peak superposition, altering the apparent structure of the X-band EPR signal (Fig. 3A). Importantly these changes are subtle, as demonstrated by the invariance of the ⁵⁵Mn-ENDOR spectra (Fig. 3D). The ⁵⁵Mn hyperfine anisotropy is not a site property, but instead an indirect measure of the fine structure splitting of the Mn^III ion^30,33,34,63 or, in the ‘monomer–trimer’ model, the zero-field splitting of the whole trimer unit. Within this model, its contribution is modulated by the electronic connectivity between the two fragments, predominantly the exchange pathway J_AB, the coupling that mostly defines the energy splitting ΔE between the ground state and the first excited state (Fig. 4C). Our BS-DFT results support this basic mechanism for electronic structure perturbation and, for the first time, describe the changes on the molecular level that impart this variation, and which differ for the two modifications. Upon replacement of Ca²⁺ by Sr²⁺, the slight distortion of the cuboidal moiety leads to a perturbation of the intra-cuboidal exchange network and possibly the Mn_D1^III site fine structure splitting. It is noted that this, besides changing the ⁵⁵Mn hyperfine anisotropy, also manifests itself in terms of the G tensor, also contributing to the altered multiline appearance and the g shift of the W-band EPR signal. Exchange of W1 by NH₃ affects the connectivity of the outer Mn_A4 to the cuboidal unit, as modulated by the μ-oxo bridge O5, perturbing the J_AB exchange pathway, thus changing the ⁵⁵Mn hyperfine anisotropy. In the case of the S₂ state variant that contains both these modifications, their effects on the electronic structure are additive. It is noted that it is the properties of the cuboidal unit that define the G tensor as opposed to the outer Mn_A4, which presumably has an isotropic on-site g value. This is expected, as it is the Mn_D1^III ion, which is part of the trimer fragment in the S = 1/2 configuration, that should form the dominant contribution to the anisotropy of the G tensor in all four systems.

4.1.2 The Mn_D1–His332-imino-N and Mn_A4–NH₃ interactions as local probes for the electronic structure.
4.1.2a The Mn_D1–His332-imino-N interaction. As described by Stich et al.,¹²³ the magnitude of the Mn_D1–His332 imino-N hyperfine interaction, as compared against mixed-valence Mn^IIIMn^IV model compounds and protein cofactors with imidazole ligands to Mn^III (A_iso ≤ 13 MHz) and Mn^IV ions (A_iso = 1.5–3.3 MHz),^128–134 favours assigning Mn_D1 as the only Mn^III ion of the S₂ state, consistent with the EPR/⁵⁵Mn-ENDOR/DFT results already reported in the literature and detailed above (Sections 3.1–3.3). The large hyperfine couplings seen for ligands coordinating to Mn^III in S₂ (and model systems) comes from the fact that the Mn^III ion carries the largest spin projection coefficient, i.e. in Mn dimers ρ_iso(Mn^III) = 2 and ρ_iso(Mn^IV) = –1. Interestingly, the hyperfine and quadrupole couplings of imidazole ligands of Mn^III ions differ depending on whether they represent axial (A_iso = 9–13 MHz, e²Qq/h = 2.1–3.0 MHz)^128–132 or equatorial ligands (A_iso = 1.5–6.6 MHz, e²Qq/h = 1.5–2.5 MHz).^131–134 The values seen for the His332 imino-N (A_iso = 7.1 MHz, e²Qq/h = 1.97 MHz) fall closer to the equatorial range supporting its assignment as an equatorial ligand consistent with DFT structural models. In such models,^{12,37,40,103,106} the local Jahn–Teller axis of the Mn_D1^III ion is aligned along the open coordination site, thus considered a pseudo-Jahn–Teller axis, perpendicular to the Mn_D1–N bond. It is supposed that the reason why the ¹⁴N couplings measured for the His332 do not exactly fall within the range seen in model complexes is that all model complexes measured thus far represent 6-coordinate Mn^III ions whereas the Mn_D1^III ion in the S₂ state is 5-coordinate.
4.1.2b The Mn_A4–NH₃ interaction. As recently shown in Pérez Navarro et al.,⁴⁴ the binding site of NH₃ is likely the W1 site. The small effective isotropic ¹⁴N hyperfine coupling (A_iso = 2.36 MHz) and the axiality of the hyperfine tensor are both consistent with a terminal ligand to a Mn^IV (d³) ion.^44,70 The similar A_iso in the Mn₄O₅Sr–NH₃ cluster confirms that the oxidation state of the Mn_A4 ion is not altered by Sr²⁺ substitution. Moreover, the binding mode and perturbation mechanism of NH₃ is the same in the Ca²⁺- and Sr²⁺-containing Mn clusters. The non-axiality of the electric field gradient (η = 0.47) is characteristic for this ligand. A large asymmetry parameter is uncommon for a terminal ligand of Mn^IV (although our value is already ≈20% smaller than that reported earlier⁷⁰). The latest crystal structure⁹ suggests that such an asymmetric distortion could be present for the W1 site due to the charged residue D1-Asp61, in H-bonding distance to W1/NH₃, as seen for other protein systems.¹³⁵ Indeed, upon inclusion of the Asp61 residue, which was not included in our previous, smaller BS-DFT model,⁴⁴ the asymmetric quadrupole tensor is now reproduced, and the hyperfine coupling constant shows better agreement with experiment (Fig. 7 and Table 3). In contrast, such an asymmetric distortion is not seen for the W2 ligand as a similar charged amino-acid residue partner is not present to provide a H-bond.

Fig. 7 Hydrogen bonding network in the vicinity of the Mn_A4-bound NH₃, as observed in the optimized DFT structure of the Mn₄O₅Ca–NH₃ model. Mn ions are depicted in purple, O in red, Ca in yellow, C in light grey, N in blue and H in white. Only relevant residues, water molecules and protons are shown.

4.2 The exchangeable μ-oxo bridge

Both modifications investigated, Ca²⁺/Sr²⁺ substitution and NH₃/W1 replacement, perturb the ¹⁷O-EDNMR signals of exchangeable oxygen species of the OEC, specifically the exchangeable μ-oxo bridge. It is this bridge that likely represents one of the substrate water sites of the Mn tetramer. As the electronic structure of the OEC is essentially invariant for all four OEC forms, the change in hyperfine coupling for this μ-oxo bridge must represent a site modification, near or at the oxygen nucleus. NH₃ binding primarily affects the connectivity of the outer Mn_A4 to the cuboidal trimer, whereas Sr²⁺ substitution instead perturbs the exchange network within the cluster. Thus, it can be surmised that the exchangeable μ-oxo bridge must both coordinate to the outer Mn_A4 and be associated with the Ca²⁺/Sr²⁺ ion itself as a structural element of the cuboidal trimer. Only the bridge O5 fulfils both these criteria. As a ligand to the Ca²⁺/Sr²⁺ ion, O5 is affected by the exchange of these ions of the same charge but different sizes. Similarly, as argued in Pérez Navarro et al.,⁴⁴ NH₃/W1 exchange perturbs O5 by binding trans to this bridge position, distorting the Mn_A4–O5 bond length.

It is noted that these results exclude the possibility that NH₃ displaces the exchangeable μ-oxo bridge as a bridging –NH₂– species, an alternative rationale for the narrowing of the ¹⁷O signal envelope in line with earlier suggestions.⁷⁰ Ca²⁺/Sr²⁺ and W1/NH₃ exchange are additive in terms of their effect on the width of the ¹⁷O-EDNMR envelope, modelled here as defined by the μ-oxo bridge hyperfine coupling A_iso. This result mirrors the structural modifications observed for the doubly modified Mn₄O₅Sr–NH₃ OEC model; i.e., the model contains additive structural modifications reflecting both singularly modified Mn₄O₅Sr and Mn₄O₅Ca–NH₃ structures. If instead the NH₃ did indeed replace the bridge, the width of the ¹⁷O-EDNMR envelope would be now defined by the W2 hyperfine coupling, and as such should be invariant to Ca²⁺/Sr²⁺ substitution. It is also noted that NH₃ replacement of the exchangeable bridge O5 cannot quantitatively explain the virtually unaltered ¹⁴NH₃ signal upon exchange of the O5-binding Ca²⁺, and the ¹⁷O hyperfine changes. Assuming an unaltered spin density on the bridge position, as follows from the similar spin projection factors for the four Mn ions, the measured ¹⁴N hyperfine coupling for the bound ammonia at this position is far too small. On the other hand, the ¹⁷O coupling of 6.5–7 MHz seen for the NH₃/W1-exchanged system is in the range of those observed in Mn model complexes with a N-ligand trans to the oxo bridge.¹⁴

5 Conclusions

Time-dependent mass spectrometry experiments indicate that the early binding substrate (W_S) is associated with all intermediate states of the OEC.^66,136 Furthermore, the relatively slow exchange and the S-state dependence of this bound substrate with bulk water suggests that it represents a ligand of (a) Mn ion(s). As Ca²⁺/Sr²⁺ substitution also perturbs its exchange rate, W_S is also supposed to coordinate to the Ca²⁺ ion.^65,66 Of the exchangeable oxygen species identified here by ¹⁷O-EDNMR, only O5 is a ligand to both Mn and Ca²⁺. Similarly, only the O5 spectral signature is perturbed by Ca²⁺/Sr²⁺ exchange. Thus, O5 is the most likely candidate for W_S. This assignment limits the possible reaction pathways for photosynthetic water splitting, and lays a foundation for studies of higher oxidized S states, which will serve to identify the second, fast exchanging substrate and eventually elucidate the mechanism of O–O bond formation. Currently two pathways are envisaged: O–O bond formation could proceed as a coupling between O5 and either (i) Mn_A4-bound W2 or Ca²⁺-bound W3, or (ii) a further oxygen not present yet in the S₂ state.

Acknowledgements

Financial support was provided by the Max-Planck-Gesellschaft, the “Bioénergie” program of the Commissariat à l'Énergie Atomique et aux Énergies Alternatives, the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-01 program, and the EU/Energy Network project SOLAR-H2 (FP7 contract 212508). T.L. was supported by the Federal Ministry of Education and Research of Germany (BMBF) in the framework of the Bio-H2 project (03SF0355C). M.P.N. acknowledges support from the Spanish Ministerio de Economia y Competitividad (MAT 2008-03461).

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Footnotes

† Electronic supplementary information (ESI) available: PSII sample preparation; data processing: baseline correction and light-minus-dark subtraction; spectral simulations; theoretical background; DFT calculations: NH₃ binding modes and spatial coordinates of the S₂ state models; Q-band ⁵⁵Mn-ENDOR: comparison of instrumental settings; electronic structures: exchange couplings and spin states of the BS-DFT models; the Mn_D1–His332-imino-N interaction: Q-band three-pulse ESEEM, Q-band HYSCORE, W-band EDNMR and simulations; X-band ¹⁴N- and ¹⁵N-three pulse ESEEM experiments on the NH₃-modified S₂ state; exchangeable ¹⁷O species: W-band EDNMR and X-band EPR. See DOI: 10.1039/c3cp55017f

‡ Differences in the experimental numbers between the Mn₄O₅Ca–NH₃ (≥30 cm⁻¹) and the Mn₄O₅Ca/Sr (≈25 cm⁻¹) S₂ states are likely due to different sample types, i.e. higher plant vs. cyanobacterial PSII with and without EtOH, respectively.

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