Photosynthetic water oxidation: binding and activation of substrate waters for O–O bond formation

Abstract

Photosynthetic water oxidation occurs at the oxygen-evolving complex (OEC) of Photosystem II (PSII). The OEC, which contains a Mn₄CaO₅ inorganic cluster ligated by oxides, waters and amino-acid residues, cycles through five redox intermediates known as S_i states (i = 0–4). The electronic and structural properties of the transient S₄ intermediate that forms the O–O bond are not well understood. In order to gain insight into how water is activated for O–O bond formation in the S₄ intermediate, we have performed a detailed analysis of S-state dependent substrate water binding kinetics taking into consideration data from Mn coordination complexes. This analysis supports a model in which the substrate waters are both bound as terminal ligands and react via a water-nucleophile attack mechanism.

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1. Introduction

The climate, biology and geology of Earth were transformed by the evolution of oxygenic photosynthesis approximately three billion years ago.¹ All photosynthetic organisms, from the earliest cyanobacteria to modern vascular plants, use solar energy to oxidize water to molecular oxygen (O₂), protons and electrons at the oxygen-evolving complex (OEC) of Photosystem II (PSII).^2,3 PSII is a membrane-bound pigment–protein complex that generates a solar light-induced charge separation in order to oxidize water and reduce plastoquinone. The charge separation is initiated by the primary chlorophyll-a electron donor, P₆₈₀, forming a powerful oxidant, P₆₈₀⁺, that advances the oxidation state of the Mn₄CaO₅ cluster in the OEC (Fig. 1) via a redox-active tyrosine, Y_Z, that mediates electron transfer from the OEC to P₆₈₀⁺. The overall four-electron process leads to the formation of O₂ that is released as a byproduct and has accumulated in the biosphere and the release of protons that contribute to the trans-membrane proton motive force.⁴ The availability of O₂ as an electron sink has since powered oxygenic respiration to give rise to Earth's current biological diversity.

Fig. 1 Quantum mechanics/molecular mechanics (QM/MM) optimized S₁ structure of the OEC⁵ based on the 1.9 Å resolution crystal structure by Shen and coworkers.⁶

The catalytic cycle of the OEC involves five metastable redox intermediates known as S_i states (i = 0–4), as first described by Kok and coworkers.^7,8 The dark-stable intermediate, S₁, contains the Mn oxidation state pattern (III,IV,IV,III)⁹ (order of oxidation states is based on the numbering of the Mn ions in Fig. 1) and is converted to S₂ upon the loss of one electron. S₂ is present as two spin isomers with oxidation states of (III,IV,IV,IV) (S = 1/2) or (IV,IV,IV,III) (S = 5/2).¹⁰ The next oxidation event results in the loss of one proton and one electron from the OEC to form S₃, in which all four Mn ions are in the +4 oxidation state (S = 3).^11,12 Oxidation of Y_Z following the next charge separation causes release of a proton from S₃ to form a modified Y_Z˙ S₃′ state,¹³ which then forms the S₄ state. The structures of S₃′ and S₄ are poorly understood. S₄ spontaneously produces O₂, binds substrate water(s), and releases a proton to form S₀, thus resetting the catalytic cycle.

The general structure of the OEC has been revealed through a series of X-ray diffraction (XRD) studies.^6,14–17 However, the atomic structure of any single Kok cycle intermediate has not been determined through these experiments. Early XRD structures of the OEC suffered from X-ray induced reduction of the high-valent Mn centers.¹⁸ Nevertheless, the availability of metal–metal distances from extended X-ray absorption fine structure (EXAFS) spectroscopy^19,20 allowed computational studies (QM/MM and density functional theory (DFT)) to refine XRD models to structures corrected for radiation damage.^5,21 At present, the PSII research community is in general (but not universal³) agreement on the structures of the S₀,⁵ S₁,^5,10 and S₂¹⁰ intermediates and the Mn oxidation states of all S states.⁹

The problem of X-ray induced Mn reduction can be completely avoided by using femtosecond pulses from X-ray free electron laser (XFEL) radiation sources.^22,23 XFEL studies using PSII nanocrystals have produced structures of dark-adapted PSII at 6.5–4.9 Å resolution.^24–27 Structures of S₂,^25,26 S₃,^26,27 and S₀²⁶ have also been reported at low resolution, which limits their interpretation. However, QM/MM methods combined with electron density map analysis have shown that the S₁ → S₂ transition involves significant displacement (and oxidation) of Mn4 (also referred to as the “dangler” Mn).²⁸ A 1.95 Å resolution XFEL structure of dark-adapted PSII was recently reported by Shen and coworkers.²⁹ However, the resulting XRD model of the OEC does not agree well with EXAFS data of S₁,¹⁹ which is likely the result of inaccuracy in positioning of the bridging oxo ligands and significant accumulation of S₀.³⁰

The S₄ state decays faster than it is formed and, therefore, cannot be observed as a kinetic intermediate. Consequently, no direct experimental evidence for the nature of O–O bond formation in S₄ has been produced. However, insights from inorganic and computational chemistry have produced two competing mechanisms: water-nucleophile attack and oxo–oxyl radical coupling.

In the oxo–oxyl radical coupling mechanism,^31–33 a Mn(IV)–oxyl radical reacts with a Mn-bridging oxo to generate O₂ (Fig. 2A). Extensive calculations by Siegbahn suggest that one of the substrate waters first binds during the S₂ → S₃ transition and then is oxidized to an oxyl radical in S₄ to carry out the reaction.³²

Fig. 2 Proposed mechanisms of O–O bond formation in the S₄ intermediate. (A) Oxo–oxyl radical coupling and (B) water-nucleophile attack. Substrate waters are shown in red.

A water-nucleophile attack mechanism for the OEC^34–37 is most consistent with synthetic water oxidation catalysts.³⁸ As shown in Fig. 2B, such a mechanism would involve a terminal water-nucleophile (preferably bound to calcium, see discussion below) and a formally Mn(V)–oxo electrophile.

These mechanisms can be distinguished by the positions of the substrate waters involved. Kinetics of substrate water exchange throughout the Kok cycle have been determined by membrane inlet mass spectrometry (MIMS) measurements following rapid mixing of PSII with H₂¹⁸O. This technique, which was pioneered in the Wydrzynski laboratory, allows ³⁴O₂ and ³⁶O₂ release by PSII in specific S states (advanced by single-turnover flashes) to be monitored as a function of incubation time with H₂¹⁸O.^39,40 The resulting rates of substrate water exchange are summarized in Table 1. In S₀, only one slowly exchanging water (W_s) is resolved with a rate constant of approximately 10 s⁻¹. The exchange rate of W_s is then dramatically slowed to 0.02 s⁻¹ in S₁. In S₂ and S₃, two phases of substrate water exchange kinetics are resolved; the slower and faster exchanging waters are referred to as W_s and W_f, respectively. W_s exchanges at a nearly identical rate in S₂ and S₃, but the rate is much faster than in S₁. W_f exchanges at decreasing rates in S₁, S₂, and S₃.

Table 1 Substrate water exchange rates in spinach thylakoids at 10 °C.^39,41

	k s, s^−1	k f, s^−1
S0	∼10	>120
S1	∼0.02	>120
S2	∼2	∼120
S3	∼2	∼40

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Determining the molecular mechanism of photosynthetic water oxidation is crucial for applying the principles of Nature's design to synthetic systems for solar fuel production. In the following analysis, we detail models for substrate binding and O–O bond formation in the OEC in light of experimental evidence and comparisons to well-studied inorganic systems.

2. Where are the substrate binding sites?

Kinetics and mechanism of terminal water vs. μ-oxo ligand exchange from model chemistry

Given our current understanding of the structure of the OEC in the S₀, S₁, and S₂ states, we can predict that the substrate waters that give rise to O₂ are bound either as terminal ligands to Mn4 and Ca²⁺, or as μ-oxo/hydroxo bridges. In order to understand the substrate water exchange kinetics of the OEC, we first look to inorganic model systems.

The exchange rate of a terminal water ligand on Mn depends greatly on the oxidation state, protonation, ancillary ligands, and geometry. As shown in Table 2, both the water exchange rate and pK_a of a terminal aqua ligand decrease dramatically with increasing oxidation state from Mn²⁺ to Mn³⁺ to Mn⁴⁺. The hexaaqua Mn(IV) complex has an especially slow water ligand exchange rate (<10⁻⁴ s⁻¹), as expected for a high-spin d³ ion with octahedral geometry for which the ligand field stabilization energy disfavors ligand dissociation leading to very slow ligand exchange. However, exchange of the terminal water ligands of the [Mn₄^IV,IV,IV,IV(μ-O)₅(terpy)₄(H₂O)₂]⁶⁺ (terpy = 2,2′:6′,2′′-terpyridine) complex occurs faster than the mixing time for the mass spectrometry measurement (∼10 s, k_ex > 10⁻¹ s⁻¹), despite the high-spin d³ configuration of the Mn(IV) ions. This can be explained by the reduced symmetry of the Mn(IV) ions in the tetrameric oxomanganese–terpy complex. Deviations from octahedral geometry of the Mn^IV centers resulting from the <90° bite angle of the tridentate terpy ligand and the asymmetry associated with oxo and pyridyl ligands result in loss of degeneracy of the t_2g and e_g orbitals, and can lower the barrier for exchange of the terminal water ligands. Indeed, computational studies have found low reaction barriers for the dissociative exchange of terminal waters in μ-O bridged Mn(IV) dimers.⁴²

Table 2 pK_a values and exchange rates of terminal waters. Calculated values appear in italics

	pKa	Reference	k ex, s^−1	Reference
a mes-terpy = 4′-mesityl-2,2′:6′,2′′-terpyridine. b terpy = 2,2′:6′,2′′-terpyridine. c L = 2-hydroxy-1,3-bis(3,5-X2-salicylideneamino)propane. d nd = not determined.
[Ca(H2O)6]^2+	12.8	43	∼10^8	44
[Sr(H2O)6]^2+	13.2	43	∼10^9	44
[Mn(H2O)6]^2+	10.6	45	2 × 10^7	46
			1 × 10^7	47
[Mn(H2O)6]^3+	0.7	45	2 × 10^3	47
			0.01–1	39 and 48
[Mn(H2O)6]^4+	<0		10^−6 to 10^−4	39
			10^−7 to 10^−8	48
[Mn2^III,IV(μ-O)2(mes-terpy)2(H2O)2]^3+^a	nd^d		>10^−1	49
[Mn4^IV,IV,IV,IV(μ-O)5(terpy)4(H2O)2]^6+^b	nd		>10^−1	49
[Mn2^IV,IV(μ-O)2(terpy)2(H2O)2]^4+	1.8	50	nd
[Mn2^III,III(L)2(H2O)]^+^c	19–20	51	nd
[Mn2^III,IV(L)2(H2O)]^2+^c	10–11	51	nd

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Generally, ligand exchange for high-valent first-row transition ions involves a dissociative mechanism, especially for Mn(IV) which is normally six-coordinate and has a small ionic radius that makes an associative mechanism unfavorable. For terminal water ligands, dissociation involves removing a polar but neutral ligand from the cationic metal center, which has a modest barrier.⁵² However, dissociative exchange of a μ-oxo ligand involves removing an anionic ligand from the cationic metal center, which has a much higher barrier. Therefore, the exchange of μ-oxo ligands with bulk water is thermodynamically more challenging than the exchange of terminal water ligands.

The mechanism of μ-oxo exchange has been investigated for di-μ-oxo di-Mn(III,IV) complexes.⁵³ These studies have shown that μ-oxo exchange requires protonation of the oxo before dissociation and exchange; therefore, μ-oxo pK_as are good predictors of their rates of exchange. The μ-oxo pK_a decreases dramatically with increasing Mn oxidation state in Mn model complexes (Table 3). The rate of μ-oxo exchange is very fast for Mn(III,III) dimers, owing to the high pK_a of the μ-oxo ligands. Indeed, di-μ-oxo di-Mn(III,III) complexes are generally not stable to hydrolysis. For di-μ-oxo di-Mn(III,IV) complexes, the rate of μ-oxo exchange is on a timescale of minutes (t_1/2 = 21 min for the [Mn₂^III,IV(μ-O)₂(bpy)₄]³⁺ complex, bpy = 2,2′-bipyridine).⁵³ However, no μ-oxo exchange could be detected on a one-day timescale for all-Mn(IV) complexes, such as [Mn₄^IV,IV,IV,IV(μ-O)₅(terpy)₄(H₂O)₂]⁶⁺, reflecting the very low pK_a of the μ-oxo ligands in all-Mn(IV) complexes (Table 3).

Table 3 μ-oxo pK_a values of [Mn₂(μ-O)₂(bpy)₄]ⁿ⁺. Calculated values appear in italics

	pKa	Reference
[Mn2^III,III(μ-O)2(bpy)4]^2+	12.4	55
[Mn2^III,IV(μ-O)2(bpy)4]^3+	2.3	49
[Mn2^IV,IV(μ-O)2(bpy)4]^4+	−6.5	55

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The [Mn₂^III,IV(μ-O)₂(terpy)₂(H₂O)₂]³⁺ complex contains terminal water ligands adjacent to the μ-oxo ligands. It has been found that the terminal water ligands accelerate the rate of μ-oxo exchange.⁵³ Because the pK_a of the water ligand bound to a high-valent Mn center is lower than solvent water (Table 2), a μ-oxo can be more readily protonated by ligated water, which opens up the μ-oxo bridge and lowers the energetic barrier for dissociation.⁵³ In this mechanism, the terminal water and μ-oxo are in dynamic equilibrium with bulk water. For Mn dimers without terminal water ligands, the exchange is much slower, such as in [Mn₂^III,IV(μ-O)₂(bpy)₄]³⁺, because intramolecular proton transfer between terminal-bound water and μ-oxo bridges is eliminated.⁵³

There have also been measurements of terminal water and μ-oxo ligand exchange of the di-Mn center in Mn-catalase.⁵⁴ The active site of Mn-catalase contains a redox-active di-Mn center for which ligand exchange has been studied in the di-μ-oxo di-Mn(III,IV) state. It was found that the terminal water ligand exchanges within the mixing time of one minute, but the μ-oxo ligands require over one hour for exchange. These results parallel those obtained for Mn model complexes in solution, although the exchange rate of the μ-oxo ligands is much lower in the protein active site.

In summary, electrostatics play a dominant role in the dissociative ligand exchange reactions of high-valent oxo–manganese complexes. There is a large barrier for μ-oxo exchange due to the high negative charge of the oxo ligand. Thus, protonation of the μ-oxo ligand is required to promote ligand exchange. Because of the large pK_a shift of the μ-oxo ligands between di-μ-oxo di-Mn(III,IV) and di-μ-oxo di-Mn(IV,IV) complexes, μ-oxo ligands exchange at a modest rate for di-μ-oxo di-Mn(III,IV) complexes but do not exchange for di-μ-oxo di-Mn(IV,IV) centers. On the other hand, terminal water ligands exhibit fast rates of ligand exchange for both Mn(III) and Mn(IV) centers. Moreover, the rate of terminal water ligand exchange is similar for Mn(III) and Mn(IV) because both have similar electrostatic potential (ESP) charges.⁵²

Ammonia does not compete with substrate water

The water analog ammonia inhibits the OEC by binding to two sites in PSII. The first is in the outer coordination sphere of the OEC and upon binding of ammonia, alters the hydrogen-bonding networks in such a way that the S₂ state S = 5/2 isomer is favored.⁵⁶ The binding of ammonia to this site is competitive with chloride^57,58 and can be accessed by larger amines,^56,58 but its specific location has not been resolved.

Ammonia also binds directly to Mn in the OEC in the S₂ state resulting in the formation of an altered multiline g = 2 EPR signal.⁵⁹ However, the substrate water exchange kinetics of S₂ do not change when ammonia is bound.⁶⁰ Therefore, the binding site of ammonia cannot be the binding site of a substrate water.

Lubitz and coworkers have proposed that ammonia binds to Mn4 trans to O5 based on ¹⁷O-ELDOR-detected NMR (EDNMR) measurements.⁶⁰ In these experiments, dark-adapted PSII (poised in S₁) is incubated with H₂¹⁷O for tens of minutes and perturbations in the ¹⁷O-EDNMR signal are detected when ammonia (as NH₄Cl at pH 7.6) is present following advancement to the S₂ state by 200 K illumination and subsequent annealing at 260 K. In accompanying ¹H-ENDOR measurements, no change in the proton environment around the OEC was observed.⁶⁰

Other lines of evidence have suggested that ammonia binds as a bridging ligand between two Mn ions. This motif was first proposed by Britt and coworkers based on ESEEM measurements of the ammonia-bound S₂ state in higher plant PSII.⁶¹ The quadrupole coupling between the S = 1/2 S₂ state of the OEC and bound ¹⁴NH₃ was resolved as 1.61 MHz with η = 0.59. A very similar coupling was observed in cyanobacterial PSII (1.52 MHz, η = 0.47).⁶⁰ For comparison, a nitrogen nucleus with purely axial symmetry is characterized by η = 0, while ammonia (amino) ligands display η ≤ 0.3. Therefore, the measured quadrupole coupling of ammonia to S₂ is highly deviant from axial symmetry and could represent a deprotonated bridging ligand such as an imido (NH²⁻) or nitrido (N³⁻). However, Britt and coworkers have very recently revealed the origin of this anisotropy. In wild type cyanobacterial PSII, they measured the ¹⁴NH₃ coupling as 1.62 MHz with η = 0.40, but in a D1-D61A mutant, the interaction was nearly completely axial (1.54 MHz, η = 0.04).⁶² This work provides convincing evidence that bound ammonia has a strong hydrogen bond to Asp61 and is, therefore, a terminal ligand to Mn4.

In a recent study using QM/MM methods and EXAFS simulations, we have proposed that instead of replacing a terminal water or μ-oxo group, ammonia binds as a sixth ligand to Mn4 resulting in a complete octahedral coordination sphere.⁶³ In this model (Fig. 3), ammonia binds to the S = 5/2 S₂ state but then induces a redox switch stabilizing the S = 1/2 S₂ state by protonating O5 and deprotonating W2. This additional ligand would lower the reduction potential of ammonia-bound S₂ as has been observed by both thermoluminescence⁶⁴ and flash O₂ measurements.⁶⁵

Fig. 3 Proposed mechanism of ammonia binding in the S₂ intermediate. Ammonia binds as an additional ligand to the dangler Mn in the S = 5/2 S₂ state, which induces a redox switch to the S = 1/2 state. Adapted from ref. 63.

While both substrate waters are already bound in S₂,⁴¹ an additional water may bind during the S₂→S₃ transition as computationally predicted.³² We have proposed that ammonia, a “harder” Lewis base than water, binds in S₂ to the dangler Mn4 in an analogous site as water binds in S₃ to the dangler Mn4.⁶³ The addition of a ligand to Mn4 in either S₂ or S₃trans to O5 would cause the other terminal water ligands (W1 and W2) to shift their positions towards the cuboidal core of the OEC. In this model of ammonia binding, both water–nucleophile attack and oxo–oxyl radical coupling O–O bond formation mechanisms are feasible.

A model for substrate water exchange in the OEC

W2 exchanges rapidly. In agreement with previous analyses,^66,67 we assign the kinetic features of W_f primarily to the terminal water W2. This water is already bound in S₂ as resolved in measurements at reduced temperatures⁴¹ and in PSII cores from the red alga Cyanidioschyzon merolae.⁶⁸ In S₀ and S₁, W2 is present as a Mn³⁺-aqua species and would be rapidly exchanged (>120 s⁻¹), consistent with the predicted exchange kinetics of [Mn(H₂O)₆]³⁺ (Fig. 4, Table 2).

Fig. 4 Proposed mechanism of OEC turnover and substrate water exchange. W_s is shown in blue and W_f in red (in S₀ and S₁, W2 is shown in purple as it contributes to both the W_s and W_f kinetics).

For the water-nucleophile attack mechanism, the substrate waters are W2 and W3. W3, which is bound to Ca²⁺, is also expected to exchange rapidly (specifically in S₀ and S₁, see discussion below). Therefore, the W_f kinetic (measured by monitoring ³⁴O₂) may reflect contributions from both W2 and W3.

The exchange rate of W2 is first resolved after the S₁ → S₂ transition with a value in the S₂ state of k_f ∼120 s⁻¹. As discussed above, S₂ is present as two spin isomers. Given the changes seen in XFEL experiments at the Mn4 position measured at physiological temperature, we have asserted that the S = 1/2 isomer is dominant.²⁸ Therefore, W_f would be bound as a terminal Mn⁴⁺-aqua species in the S₂ state and exchanges at a rate comparable to those measured in model complexes (Table 2).

As modeled in Fig. 4, the formation of S₃ involves the release of a proton from W2 and a 3-fold slowing of its exchange rate (Table 1). This change is qualitatively consistent with the differences expected between Mn⁴⁺-aqua and Mn⁴⁺-hydroxo species.

W_s in S₀ and S₁ involves O5/W2 exchange. Identifying the origin of the substrate water exchange rates for the W_s kinetic phase is more complex. The water exchanges fastest in S₀, dramatically slows down in S₁, and then dramatically speeds up again in S₂ and S₃. These observations are difficult to explain based on exchange at a single site. Herein, we propose a new model for W_s exchange that involves contributions from both terminal and bridging ligands (W2 and O5, respectively) whose interconversion is possible only in the S₀ and S₁ states owing to the decreasing pK_a of the μ-oxo species, O5, with increasing Mn oxidation states.

It has been determined that only one of the μ-oxo bridges denoted O5 slowly exchanges with H₂¹⁷O in the dark-stable S₁ state over tens of seconds.^54,69 We assume that O5 also exchanges with bulk water in S₀ (oxidation states III,IV,III,III). QM/MM models of the S₀ state⁵ predict that O5 is protonated, as expected if its pK_a is compared to Mn(III,III) dimers (Table 3). Consequently, O5 exchange would be expected to occur relatively rapidly in S₀ (10 s⁻¹) because it is already protonated at physiological pH. During exchange, protonated O5 may dissociate from Mn3 becoming a terminal water ligand to Mn4. Thus, in the S₀ state, W2 (purple, Fig. 4) and O5 (blue, Fig. 4) are expected to be in rapid equilibrium with each other.

In S₁ (oxidation states III,IV,IV,III), O5 is present in a chemical environment analogous to Mn(III,IV) dimer complexes and the pK_a of O5 in S₁ is predicted to be much lower than in S₀ (Table 3). Based on QM/MM models, O5 is not protonated in the S₁ state^5,21 and, thus, would have a higher energetic barrier for exchange. This change is proposed to be the reason for the 500-fold decrease in the exchange rate of W_s observed in S₁ as compared to S₀.

W_f is not resolved in S₀ and S₁ and reflects the rapid exchange of W2 (and W3, discussed below). Because μ-oxo exchange involves subsequent dissociation, binding, and association steps,⁵³ isotopically labeled substrate will appear in both the O5 and W2 positions. Therefore, W2 is labeled with two rates. The first is rapid (>120 s⁻¹) and reflects direct exchange with bulk water. The second kinetic is slow (0.02–10 s⁻¹) and reflects the interconversion of labeled O5 with W2. Crucially, this mechanism implies that the rate of W_sexchange is controlled by the rate of O5 exchange in S₀and S₁, but that O5 is not necessarily a substrate water.

For the dominant S₂ state S = 1/2 spin isomer (oxidation states IV,IV,IV,III), the O5 chemical environment is analogous to Mn(IV,IV) dimer complexes in which the μ-oxo pK_a is very low (Table 3). Given the large energetic barrier for μ-oxo exchange, the measured rate of W_s in S₂ (2 s⁻¹) is too fast to be assigned to O5.

W_s in S₂ and S₃ involves a terminal water bound to Ca²⁺. In S₀ and S₁, W3, which is a substrate water in the proposed water-nucleophile attack mechanism (Fig. 2B and 4), exchanges at a rate >120 s⁻¹ and its kinetic features are merged with those of W2. However, the S₁ → S₂ transition does not involve the release of a proton to the bulk, but does involve an oxidation. The result is the build-up of a positive charge and a significant contraction of the OEC as recently observed in photothermal beam deflection experiments.^13,70 This accumulated charge (and contraction) is not released until the S₀ state is reformed after O₂ release.⁷⁰ As a result, we hypothesize that the Ca²⁺–W3 electrostatic interaction, as well as the surrounding hydrogen-bonding network, is stronger in S₂ and S₃ compared to S₀ and S₁. This effect of the increased charge would have to be dramatic to explain the 10⁶-fold decrease in W3 exchange compared to the Ca²⁺-aqua ion in bulk water (Table 2).

When Ca²⁺ is substituted with Sr²⁺, only W_s is significantly affected: k_s is three- to four-fold faster in S₁, S₂, and S₃ when Sr²⁺ is present.⁷¹ Both O5 and W3 are adjacent to Ca²⁺/Sr²⁺ and are, therefore, reasonable candidates for W_s. Because Ca²⁺ and Sr²⁺ have very similar Lewis acidities (similar pK_as of the aqua ion, Table 2), W3 is expected to be fully protonated throughout the Kok cycle providing an adequate nucleophile in the water-nucleophile attack mechanism.³⁶ However, Sr²⁺ is considerably larger than Ca²⁺ (ionic radii are 1.12 Å and 0.99 Å, respectively⁷²) resulting in a weaker electrostatic interaction between Sr²⁺ and its ligands. This difference is reflected in the faster exchange rate of the water ligands bound to [Sr(H₂O)₆]²⁺ compared to [Ca(H₂O)₆]²⁺ (Table 2), and is also consistent with the faster exchange of W_s when Sr²⁺ is substituted for Ca²⁺ in the OEC.

In our model for OEC turnover and substrate water exchange shown in Fig. 4, the observed W_s kinetics reflect O5 exchanging with W2 in S₀ and S₁, and W3 exchange in S₂ and S₃. The W_f kinetic reflects both W2 and W3 exchange in S₀ and S₁, and only W2 in S₂ and S₃. This new substrate exchange model provides a complete description of all of the kinetic phases of substrate exchange throughout the S-state cycle and is consistent with data from model high-valent oxomanganese complexes. It, thus, provides a framework from which to design additional experimental measurements to test and validate this hypothesis.

3. Mechanism of O–O bond formation

The structure of the S₄ intermediate will determine if the mechanism of photosynthetic water oxidation involves water-nucleophile attack or oxo–oxyl radical coupling. Unfortunately, few experimental clues about S₄ are available. Given that the Mn oxidation states in S₃ are (IV,IV,IV,IV), the S₄ state is predicted to contain either a Mn^IV–oxyl radical or Mn^V–oxo species.

A Mn^IV–oxyl radical species has been suggested through computational studies by Siegbahn and supports an oxo–oxyl radical coupling mechanism. On the other hand, a high-spin Mn^V–oxo is predicted to serve as the electrophile in a water-nucleophile attack mechanism. Borovik and coworkers have recently reported experimental support for the latter option through studies of [Mn^VH₃buea(O)] (where buea = tris[(N′-tert-butylureaylato)-N-ethylene]aminato).^73,74 The buea ligand imposes C₃ symmetry around Mn and stabilizes the Mn–oxo group through intramolecular hydrogen bonding. [Mn^VH₃buea(O)] is a high-spin S = 1 system as confirmed by EPR spectroscopy and supported by DFT calculations.⁷³ Therefore, the d_xz and d_yz orbitals in [Mn^VH₃buea(O)] are degenerate and the Mn–oxo bond is likely to be weaker (and more reactive) than corresponding Mn^V–oxo species with tetragonal symmetry (S = 0). By quantifying the hyperfine contributions from ¹⁷O-labeled [Mn^VH₃buea(O)] using EPR spectroscopy, Borovik and coworkers determined that 0.45 spins reside on the oxo group. Therefore, the Mn^V–oxo group is strongly covalent, but does not contain an oxyl radical. For [Mn^IIIH₃buea(O)]²⁻, only 0.30 spins reside on the oxo group, suggesting that the Mn–O bond becomes more strongly covalent (and the unit more electrophilic) with increasing Mn oxidation state.⁷⁴

To date, no experimental evidence of a Mn^IV–oxyl radical species has been found, calling into question the accuracy of the oxo–oxyl radical coupling mechanism. However, if a reactive high-spin Mn^V=O species is present in the S₄ state, analogous to that found in [Mn^VH₃buea(O)], a water-nucleophile attack mechanism would lead to O–O bond formation.

4. Conclusions

Determining the molecular mechanism of O–O bond formation in photosynthetic water oxidation remains one of the great challenges in bioinorganic chemistry. In the absence of direct experimental evidence of the S₄ intermediate, we can predict its structure and, therefore, the mechanism of O–O bond formation by comparing the OEC to model Mn complexes and by determining where the substrate waters bind throughout the catalytic cycle.

In our proposed model of OEC turnover shown in Fig. 4, the substrate waters are W2 (terminal water on Mn4) and W3 (terminal water on Ca²⁺). The substrate water exchange kinetics in S₀ and S₁ are complicated by the dynamic equilibrium between W2 and the slowly exchanging μ-oxo/hydroxo bridge, O5. This assignment of substrates dictates that O–O bond formation occurs via a water-nucleophilic attack of W3 (as water) on W2 (as a high-spin Mn^V=O species).

Acknowledgements

Funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences (Grant no. DE-FG02-05ER15646). We thank Mikhail Askerka and Dr. Leslie Vogt for helpful discussions.

References

1. S. A. Crowe, L. N. Dossing, N. J. Beukes, M. Bau, S. J. Kruger, R. Frei and D. E. Canfield, Nature, 2013, 501, 535–538.
2. J. P. McEvoy and G. W. Brudvig, Chem. Rev., 2006, 106, 4455–4483.
3. D. J. Vinyard, G. M. Ananyev and G. C. Dismukes, Annu. Rev. Biochem., 2013, 82, 577–606.
4. R. E. Blankenship, Molecular Mechanisms of Photosynthesis, Wiley-Blackwell, 2nd edn, 2014.
5. R. Pal, C. F. A. Negre, L. Vogt, R. Pokhrel, M. Z. Ertem, G. W. Brudvig and V. S. Batista, Biochemistry, 2013, 52, 7703–7706.
6. Y. Umena, K. Kawakami, J.-R. Shen and N. Kamiya, Nature, 2011, 473, 55–60.
7. B. Kok, B. Forbush and M. McGloin, Photochem. Photobiol., 1970, 11, 457–475.
8. B. Forbush, B. Kok and M. P. McGloin, Photochem. Photobiol., 1971, 14, 307–321.
9. V. Krewald, M. Retegan, N. Cox, J. Messinger, W. Lubitz, S. DeBeer, F. Neese and D. A. Pantazis, Chem. Sci., 2015, 6, 1676–1695.
10. D. A. Pantazis, W. Ames, N. Cox, W. Lubitz and F. Neese, Angew. Chem., Int. Ed., 2012, 51, 9935–9940.
11. A. Boussac, M. Sugiura, A. W. Rutherford and P. Dorlet, J. Am. Chem. Soc., 2009, 131, 5050–5051.
12. N. Cox, M. Retegan, F. Neese, D. A. Pantazis, A. Boussac and W. Lubitz, Science, 2014, 345, 804–808.
13. A. Klauss, M. Haumann and H. Dau, Proc. Natl. Acad. Sci. U. S. A., 2012, 109, 16035–16040.
14. N. Kamiya and J. R. Shen, Proc. Natl. Acad. Sci. U. S. A., 2003, 100, 98–103.
15. K. N. Ferreira, T. M. Iverson, K. Maghlaoui, J. Barber and S. Iwata, Science, 2004, 303, 1831–1838.
16. B. Loll, J. Kern, W. Saenger, A. Zouni and J. Biesiadka, Nature, 2005, 438, 1040–1044.
17. A. Guskov, J. Kern, A. Gabdulkhakov, M. Broser, A. Zouni and W. Saenger, Nat. Struct. Mol. Biol., 2009, 16, 334–342.
18. J. Yano, J. Kern, K. D. Irrgang, M. J. Latimer, U. Bergmann, P. Glatzel, Y. Pushkar, J. Biesiadka, B. Loll, K. Sauer, J. Messinger, A. Zouni and V. K. Yachandra, Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 12047–12052.
19. A. Grundmeier and H. Dau, Biochim. Biophys. Acta, 2012, 1817, 88–105.
20. K. Sauer, J. Yano and V. K. Yachandra, Coord. Chem. Rev., 2008, 252, 318–335.
21. P. E. M. Siegbahn, Chem.–Eur. J., 2008, 14, 8290–8302.
22. R. Neutze, R. Wouts, D. van der Spoel, E. Weckert and J. Hajdu, Nature, 2000, 406, 752–757.
23. H. N. Chapman, P. Fromme, A. Barty, T. A. White, R. A. Kirian, A. Aquila, M. S. Hunter, J. Schulz, D. P. DePonte, U. Weierstall, R. B. Doak, F. R. N. C. Maia, A. V. Martin, I. Schlichting, L. Lomb, N. Coppola, R. L. Shoeman, S. W. Epp, R. Hartmann, D. Rolles, A. Rudenko, L. Foucar, N. Kimmel, G. Weidenspointner, P. Holl, M. Liang, M. Barthelmess, C. Caleman, S. Boutet, M. J. Bogan, J. Krzywinski, C. Bostedt, S. Bajt, L. Gumprecht, B. Rudek, B. Erk, C. Schmidt, A. Homke, C. Reich, D. Pietschner, L. Struder, G. Hauser, H. Gorke, J. Ullrich, S. Herrmann, G. Schaller, F. Schopper, H. Soltau, K.-U. Kuhnel, M. Messerschmidt, J. D. Bozek, S. P. Hau-Riege, M. Frank, C. Y. Hampton, R. G. Sierra, D. Starodub, G. J. Williams, J. Hajdu, N. Timneanu, M. M. Seibert, J. Andreasson, A. Rocker, O. Jonsson, M. Svenda, S. Stern, K. Nass, R. Andritschke, C.-D. Schroter, F. Krasniqi, M. Bott, K. E. Schmidt, X. Wang, I. Grotjohann, J. M. Holton, T. R. M. Barends, R. Neutze, S. Marchesini, R. Fromme, S. Schorb, D. Rupp, M. Adolph, T. Gorkhover, I. Andersson, H. Hirsemann, G. Potdevin, H. Graafsma, B. Nilsson and J. C. H. Spence, Nature, 2011, 470, 73–77.
24. J. Kern, R. Alonso-Mori, J. Hellmich, R. Tran, J. Hattne, H. Laksmono, C. Glöckner, N. Echols, R. G. Sierra, J. Sellberg, B. Lassalle-Kaiser, R. J. Gildea, P. Glatzel, R. W. Grosse-Kunstleve, M. J. Latimer, T. A. McQueen, D. DiFiore, A. R. Fry, M. Messerschmidt, A. Miahnahri, D. W. Schafer, M. M. Seibert, D. Sokaras, T.-C. Weng, P. H. Zwart, W. E. White, P. D. Adams, M. J. Bogan, S. Boutet, G. J. Williams, J. Messinger, N. K. Sauter, A. Zouni, U. Bergmann, J. Yano and V. K. Yachandra, Proc. Natl. Acad. Sci. U. S. A., 2012, 109, 9721–9726.
25. J. Kern, R. Alonso-Mori, R. Tran, J. Hattne, R. J. Gildea, N. Echols, C. Glöckner, J. Hellmich, H. Laksmono, R. G. Sierra, B. Lassalle-Kaiser, S. Koroidov, A. Lampe, G. Han, S. Gul, D. DiFiore, D. Milathianaki, A. R. Fry, A. Miahnahri, D. W. Schafer, M. Messerschmidt, M. M. Seibert, J. E. Koglin, D. Sokaras, T.-C. Weng, J. Sellberg, M. J. Latimer, R. W. Grosse-Kunstleve, P. H. Zwart, W. E. White, P. Glatzel, P. D. Adams, M. J. Bogan, G. J. Williams, S. Boutet, J. Messinger, A. Zouni, N. K. Sauter, V. K. Yachandra, U. Bergmann and J. Yano, Science, 2013, 340, 491–495.
26. J. Kern, R. Tran, R. Alonso-Mori, S. Koroidov, N. Echols, J. Hattne, M. Ibrahim, S. Gul, H. Laksmono, R. G. Sierra, R. J. Gildea, G. Han, J. Hellmich, B. Lassalle-Kaiser, R. Chatterjee, A. S. Brewster, C. A. Stan, C. Glöckner, A. Lampe, D. DiFiore, D. Milathianaki, A. R. Fry, M. M. Seibert, J. E. Koglin, E. Gallo, J. Uhlig, D. Sokaras, T.-C. Weng, P. H. Zwart, D. E. Skinner, M. J. Bogan, M. Messerschmidt, P. Glatzel, G. J. Williams, S. Boutet, P. D. Adams, A. Zouni, J. Messinger, N. K. Sauter, U. Bergmann, J. Yano and V. K. Yachandra, Nat. Commun., 2014, 5, 4371.
27. C. Kupitz, S. Basu, I. Grotjohann, R. Fromme, N. A. Zatsepin, K. N. Rendek, M. S. Hunter, R. L. Shoeman, T. A. White, D. Wang, D. James, J.-H. Yang, D. E. Cobb, B. Reeder, R. G. Sierra, H. Liu, A. Barty, A. L. Aquila, D. Deponte, R. A. Kirian, S. Bari, J. J. Bergkamp, K. R. Beyerlein, M. J. Bogan, C. Caleman, T.-C. Chao, C. E. Conrad, K. M. Davis, H. Fleckenstein, L. Galli, S. P. Hau-Riege, S. Kassemeyer, H. Laksmono, M. Liang, L. Lomb, S. Marchesini, A. V. Martin, M. Messerschmidt, D. Milathianaki, K. Nass, A. Ros, S. Roy-Chowdhury, K. Schmidt, M. Seibert, J. Steinbrener, F. Stellato, L. Yan, C. Yoon, T. A. Moore, A. L. Moore, Y. Pushkar, G. J. Williams, S. Boutet, R. B. Doak, U. Weierstall, M. Frank, H. N. Chapman, J. C. H. Spence and P. Fromme, Nature, 2014, 513, 261–265.
28. M. Askerka, J. Wang, G. W. Brudvig and V. S. Batista, Biochemistry, 2014, 53, 6860–6862.
29. M. Suga, F. Akita, K. Hirata, G. Ueno, H. Murakami, Y. Nakajima, T. Shimizu, K. Yamashita, M. Yamamoto, H. Ago and J.-R. Shen, Nature, 2014, 517, 99–103.
30. M. Askerka, D. J. Vinyard, J. Wang, G. W. Brudvig and V. S. Batista, Biochemistry, 2015, 54, 1713–1716.
31. V. K. Yachandra, K. Sauer and M. P. Klein, Chem. Rev., 1996, 96, 2927–2950.
32. P. E. M. Siegbahn, Biochim. Biophys. Acta, 2013, 1827, 1003–1019.
33. P. E. M. Siegbahn, Chem.–Eur. J., 2006, 12, 9217–9227.
34. J. Messinger, M. Badger and T. Wydrzynski, Proc. Natl. Acad. Sci. U. S. A., 1995, 92, 3209–3213.
35. V. L. Pecoraro, M. J. Baldwin, M. T. Caudle, W.-Y. Hsieh and N. A. Law, Pure Appl. Chem., 1998, 70, 925–929.
36. G. W. Brudvig, Philos. Trans. R. Soc., B, 2008, 363, 1211–1219.
37. V. A. Szalai, D. A. Stone and G. W. Brudvig, in Photosynthesis: Mechanisms and Effects, vol. 2, ed. G. Garab, Kluwer Academic, Dordrecht, The Netherlands, 1998, pp. 1403–1406.
38. S. Romain, L. Vigara and A. Llobet, Acc. Chem. Res., 2009, 42, 1944–1953.
39. W. Hillier and T. Wydrzynski, Biochim. Biophys. Acta, 2001, 1503, 197–209.
40. W. Hillier and T. Wydrzynski, Coord. Chem. Rev., 2008, 252, 306–317.
41. W. Hillier and T. Wydrzynski, Phys. Chem. Chem. Phys., 2004, 6, 4882–4889.
42. M. Lundberg, M. R. A. Blomberg and P. E. M. Siegbahn, Theor. Chem. Acc., 2003, 110, 130–143.
43. J. A. Dean, Lange's Handbook of Chemistry, McGraw Hill Book Co., New York, 1985.
44. J. Burgess, Ions in Solution: Basic Principles of Chemical Interactions, Halsted Press, New York, 1988.
45. D. T. Richens, The Chemistry of Aqua Ions, Wiley, Chichester, U.K., 1997.
46. Y. Ducommun, K. E. Newman and A. E. Merbach, Inorg. Chem., 1980, 19, 3696–3703.
47. F. P. Rotzinger, J. Am. Chem. Soc., 1997, 119, 5230–5238.
48. D. Kuzek and R. J. Pace, Biochim. Biophys. Acta, 2001, 1503, 123–137.
49. R. Tagore, H. Y. Chen, R. H. Crabtree and G. W. Brudvig, J. Am. Chem. Soc., 2006, 128, 9457–9465.
50. C. W. Cady, K. E. Shinopoulos, R. H. Crabtree and G. W. Brudvig, Dalton Trans., 2010, 39, 3985–3989.
51. M. T. Caudle and V. L. Pecoraro, J. Am. Chem. Soc., 1997, 119, 3415–3416.
52. E. M. Sproviero, K. Shinopoulos, J. A. Gascón, J. P. McEvoy, G. W. Brudvig and V. S. Batista, Philos. Trans. R. Soc., B, 2008, 363, 1149–1156.
53. R. Tagore, R. H. Crabtree and G. W. Brudvig, Inorg. Chem., 2007, 46, 2193–2203.
54. I. L. McConnell, V. M. Grigoryants, C. P. Scholes, W. K. Myers, P.-Y. Chen, J. W. Whittaker and G. W. Brudvig, J. Am. Chem. Soc., 2012, 134, 1504–1512.
55. M. Amin, L. Vogt, S. Vassiliev, I. Rivalta, M. M. Sultan, D. Bruce, G. W. Brudvig, V. S. Batista and M. R. Gunner, J. Phys. Chem. B, 2013, 117, 6217–6226.
56. W. F. Beck and G. W. Brudvig, Biochemistry, 1986, 25, 6479–6486.
57. P. O. Sandusky and C. F. Yocum, Biochim. Biophys. Acta, 1984, 766, 603–611.
58. P. O. Sandusky and C. F. Yocum, Biochim. Biophys. Acta, 1986, 849, 85–93.
59. W. F. Beck, J. C. de Paula and G. W. Brudvig, J. Am. Chem. Soc., 1986, 108, 4018–4022.
60. M. Pérez Navarro, W. M. Ames, H. Nilsson, T. Lohmiller, D. A. Pantazis, L. Rapatskiy, M. M. Nowaczyk, F. Neese, A. Boussac, J. Messinger, W. Lubitz and N. Cox, Proc. Natl. Acad. Sci. U. S. A., 2013, 110, 15561–15566.
61. R. D. Britt, J. L. Zimmermann, K. Sauer and M. P. Klein, J. Am. Chem. Soc., 1989, 111, 3522–3532.
62. P. H. Oyala, T. A. Stich, R. J. Debus and R. D. Britt, J. Am. Chem. Soc., 2015, 137, 8829–8837.
63. M. Askerka, D. J. Vinyard, G. W. Brudvig and V. S. Batista, Biochemistry, 2015, 54, 5783–5786.
64. T.-A. Ono and Y. Inoue, Arch. Biochem. Biophys., 1988, 264, 82–92.
65. D. J. Vinyard and G. W. Brudvig, Biochemistry, 2015, 54, 622–628.
66. H. Nilsson, F. Rappaport, A. Boussac and J. Messinger, Nat. Commun., 2014, 5, 4305.
67. N. Cox and J. Messinger, Biochim. Biophys. Acta, 2013, 1827, 1020–1030.
68. H. Nilsson, T. Krupnik, J. Kargul and J. Messinger, Biochim. Biophys. Acta, 2014, 1837, 1257–1262.
69. L. Rapatskiy, N. Cox, A. Savitsky, W. M. Ames, J. Sander, M. M. Nowaczyk, M. Rögner, A. Boussac, F. Neese, J. Messinger and W. Lubitz, J. Am. Chem. Soc., 2012, 134, 16619–16634.
70. A. Klauss, M. Haumann and H. Dau, J. Phys. Chem. B, 2015, 119, 2677–2689.
71. G. Hendry and T. Wydrzynski, Biochemistry, 2003, 42, 6209–6217.
72. CRC Handbook of Chemistry and Physics, ed. D. R. Lide, CRC Press/Taylor and Francis, Boca Raton, FL, 88th (Internet Version 2008) edn, 2008.
73. T. Taguchi, R. Gupta, B. Lassalle-Kaiser, D. W. Boyce, V. K. Yachandra, W. B. Tolman, J. Yano, M. P. Hendrich and A. S. Borovik, J. Am. Chem. Soc., 2012, 134, 1996–1999.
74. R. Gupta, T. Taguchi, B. Lassalle-Kaiser, E. L. Bominaar, J. Yano, M. P. Hendrich and A. S. Borovik, Proc. Natl. Acad. Sci. U. S. A., 2015, 112, 5319–5324.

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