Early Binding of Substrate Oxygen is Responsible for

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Spectroscopy and Photochemistry; General Theory

Early Binding of Substrate Oxygen is Responsible for Spectroscopically Distinct S-State in Photosystem II 2

Yulia N. Pushkar, Alireza Karbakhsh Ravari, Scott Jensen, and Mark C Palenik J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.9b01255 • Publication Date (Web): 16 Aug 2019 Downloaded from pubs.acs.org on August 27, 2019

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The Journal of Physical Chemistry Letters

Early Binding of Substrate Oxygen is Responsible for Spectroscopically Distinct S2-State in Photosystem II Yulia Pushkar1*, Alireza K. Ravari1, Scott C. Jensen1, Mark Palenik2 1

Department of Physics and Astronomy, Purdue University, West Lafayette, Indiana 47907, United States

2

Code 6189, Chemistry Division, Naval Research Laboratory, Washington DC, 20375, United States

AUTHOR INFORMATION *Corresponding Author [email protected]

ACS Paragon Plus Environment

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ABSTRACT

The biological generation of oxygen by the oxygen-evolving complex (OEC) in photosystem II (PS II) is one of nature's most important reactions. The OEC is a Mn4Ca-cluster that has multiple Mn-O-Mn and Mn-O-Ca bridges and binds four water molecules. Previously binding of an additional oxygen was detected in the S2 to S3 transition. Here we demonstrate that early binding of the substrate oxygen to the 5-coordinate Mn1 center in the S2 state is likely responsible for the S2 high-spin EPR signal. Substrate binding in the Mn1-OH form explains the prevalence of the high spin S2-state at higher pH and its low temperature conversion into the S3-state. The given interpretation was confirmed by X-ray absorption spectroscopic measurements, DFT and broken symmetry DFT calculations of structures and magnetic properties. Structural, electronic and spectroscopic properties of the high spin S2 state model are provided and compared with the available S3-state models. New interpretation of the high spin S2 state opens opportunity for analysis of factors controlling the oxygen substrate binding in PS II.

TOC GRAPHICS

KEYWORDS: Photosystem II, Water Oxidation, Metalloprotein


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Oxygenic photosynthesis is a light driven process for energy assimilation via conversion of water and carbon dioxide to carbohydrates and molecular oxygen. The initial steps of photosynthesis takes place in photosystem II (PS II), a multi-subunit metalloenzyme complex embedded in the thylakoid membranes of green plants, algae, and cyanobacteria.1 Inside PS II resides the oxygenevolving complex (OEC) which consists of the Mn4Ca cluster in a finely-tuned protein environment, Figure 1.2-4 The OEC carries out a critical function of photosynthesis via the Kok cycle which is comprised of the light induced succession of states, S0-S4, where molecular oxygen is formed in the final S3 to S0 transition, Figure 1.5 The origin of the substrate oxygen atoms for the O-O bond formation has been proposed based on computational studies6-13, spectroscopic observation14-15 and recent time resolved X-ray diffraction (TR-XRD) analysis2-4. The Mn4Ca cluster contains multiple oxygen atoms in the form of Mn-O-Mn and Mn-O-Ca bridges as well as two waters/OH ligands on a single Mn center and two water ligands on Ca2+. According to the high oxidation state paradigm used in this study, the S1-state features two MnIII and two MnIV ions. The alternative low oxidation state paradigm assigns all MnIII ions in the S1-state.16-18 Earlier proposals of the additional oxygen binding in the S3 state10-12, 19 were subsequently confirmed by recent TR-XRD showing oxygen atom binding to the 5-coordinate Mn center in the S2 to S3 transition.2, 20 The transient presence of this oxygen in the OEC suggests that it is one of the substrate oxygens for O-O bond formation. One of the pathways for this oxygen transfer to initially 5-coordinated Mn1 center was proposed via valence isomers at the S2 state, Figure 2A.21 In each of the valence isomers the MnIII ion remains 5-coordinate but changes its position. Support for such valence isomerism comes entirely from DFT calculations.21 The requirement of a closed cubane formation for advancement to the S3 state, however, has not been supported thus far.22 The valence isomerism hypothesis implies a


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relatively high flexibility of the Mn4Ca cluster where two forms undergo structural isomerization via low barrier, Figure 2A.21 Earlier we reported the high quality room temperature extended X-ray absorption fine structure (EXAFS) measurement of the S1 state and found it to be essentially identical to the low (20 K) temperature data.23 No visible reduction in the intensity of the kspace oscillations supported a rigid structure of the Mn4Ca cluster at room temperature. Figure 1. Current model of the Kok cycle depicting electron/proton release and DFT optimized atomistic structures of the OEC. The high spin (HS) S2 state with the Mn1-OH fragment is shown in the center. Water molecules are shown in dark blue, hydroxides are light blue.

The S1 and S2 states are only different by the oxidation of one Mn ion from MnIII to MnIV with no structural difference between the two.24-25 Currently there is no experimental evidences

of

an

increased

structural

flexibility of the S2 state. Early spectroscopic insight into the composition and function of the OEC was provided by EPR when the S2-state S=1/2 (LS) multiline signal was discovered in 1980.26-27 Already in 1984 a second EPR signal associated with the S2-state was reported and it was interpreted as a high spin (HS) signal28 which is close to g~4.1 in plants29-30 and is at slightly higher g values in cyanobacterial PSII31-32. Existence of these two different EPR signals was used to support the valence isomers hypothesis.21 However, the origin of g~4 signal remains a subject of debate.8 Here


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at the S2-state featuring the Mn1-OH group with B) showing all atoms used in DFT calculations of Model A (Also shown in Movie S1). Highlighted dashed lines indicate hydrogen bonds. D) Two modes of ammonia binding in the S2 state of PS II: (A) Binding in the place of the W1 and (B) binding in the close proximity to Mn1 with the formation of the H-bond to Mn4-O5-Mn3 bridge.

we propose assignment of the S2 HS EPR signal which is different from the structure derived in the

valence

isomerism

hypothesis.

Our

interpretation explains the large body of currently available experimental data. The model shows that depending on the environment conditions such as pH, the buffer composition and presence of small substrate analogs, the 5coordinate Mn1 center in the S2 state is susceptible to ligand binding, Figure 2B and 2C. Since the hydroxyl (-OH) group is a stronger Figure 2. A) Valence isomers hypothesis proposes a pathway for the oxygen atom transfer

ligand in comparison to water, ligand binding

to initially 5-coordinated Mn1 center in the S2

should be facilitated at increased pH in

state. In valence isomers model MnIII ion remains

agreement with experiment.33 While binding of

5-coordinate but changes its position. Support for such valence isomerism comes entirely from DFT

the additional oxygen at Mn1 is expected for the

calculations.

S2 to S3 transition, early ligand binding to Mn1

B) and C) Models of the early substrate binding

gives rise to the HS S2 state, Figure 2B and 2C.


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Table 1. Energy change of the Ca-S2 and Sr-S2 states of the OEC model associated with addition of the water or ammonia.

S-state, protonation state of two water molecules on dangler Mn and status of the 5-coordinate Mn1

E, meV

Ca-S2 (OH,OH)/Mn1 (S=1/2) + H2O  Ca-S2 (H2O,OH)/Mn1-OH (S=5/2)

-180

Sr-S2 (OH,OH)/Mn1 (S=1/2) + H2O  Sr-S2 (H2O, OH)/Mn1-OH (S=5/2)

-210

Ca-S2 (H2O,OH)/Mn1 (S=1/2) + NH3  Ca-S2 (NH3,OH)/Mn1 (S=1/2) +H2O

-240

Ca-S2 (NH3, OH)/Mn1 (S=1/2) + H2O  Ca-S2 (NH3, H2O)/Mn1-OH (S=5/2)

-550

Ca-S2 (OH,OH)/Mn1 (S=1/2) + NH3  Ca-S2 (OH,OH)/Mn1(NH3) (S=1/2)

-170

Small substrate analogs can potentially block the HS S2-state formation if they interfere with the substrate incorporation pathway. Such refined understanding of the HS S2 state opens new opportunities for experimental analysis of the substrate binding mechanism. Energetics of the substrate binding in the S2 state. For analysis of energies and geometries of PSII intermediates, DFT calculations of molecular models were done with Gaussian09 unrestricted BP86 Becke’s 1988 functional34 with the gradient corrections of Perdew35 and the def2tzvp basis set used for all atoms, Table 1, Table S1. The previously discussed model of the low spin (LS) S2 state (S=1/2) was used.6, 23 His337 was assumed to be in a neutral form as expected at higher pH where contribution of HS EPR signal is more pronounced. The size of our model is maximized to the level when large basis set calculations can be carried out over a practical time period. In the past we documented smaller models results in Davis et al.10 to be very consistent with later enlarged models19, 23, 36 yielding essentially the same spin configurations, Mn-Mn bond geometries and energetics. At the level of O-O bond formation / spin configuration analysis, much smaller models have proven to be very insightful, see earlier works of Siegbahn37. Most of his conclusions


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Table 2. DFT derived structural changes*. S2 low spin, S=1/2 MLS, g~2

S2 – A high spin, S=5/2, g~4

S3 –A, S=3

Mn-Mn:

2.73, 2.75, 2.78, 3.33 Å

2.72; 2.77; 2.84; 3.63 Å

2.73; 2.79; 2.82; 3.59

Mn-Ca:

3.30; 3.35; 3.45 Å

3.22; 3.40; 3.40 Å

3.28; 3.43; 3.43; 3.86

Sr-Mn:

3.43, 3.49, 3.59, 3.84 Å

3.37; 3.51; 3.55; 4.0 Å

*Noticeable differences in bond distances are indicated in bold font.

were drawn from much smaller sized models and were subsequently shown to hold when the protein component was expanded38. Larger models may be needed when proton transfer pathways are studied (which is not the topic of our analysis) as these should include multiple proton shuttling residues. Distances of the initial LS, S=1/2 S2 model (Figure 1, Table 2) are in good agreement with MnMn distances found in EXAFS24-25 (two at 2.73 Å, one at 2.81 Å and one at 3.3 Å) and with SrMn distances reported in Sr EXAFS39 (three at 3.56 Å and one at 4.03 Å). For evaluation of the energetics of substrate binding, the LS S2 model was reacted with H2O, Table 1. In this way, the ambiguity in the estimation of OH- energy was avoided. The product containing the Mn1-OH fragment and a proton added to one of the Mn4–OH ligands was optimized in the S=5/2 spin state, Figure 2C. The newly formed Mn1-OH group was found to have two possible conformations with: A) a hydrogen bond between the Mn1-OH group and bridging oxygen of the Mn4-O-Mn3 bridge (Figure 2B and Movie S1) which is similar to a model reported in Siegbahn8 or B) a hydrogen bond between the Mn1-OH group and –COO- group of the Glu189. The latter model (B) was found to be slightly higher in energy by ~40 meV. Binding of the substrate produced structural changes summarized in Table 2, including a notable elongation of one Mn-Mn distance from 2.78 to 2.84 Å. Protonation of the Mn=O-Ca bridge in the S3 model6, 10, 19 resulted in the structure (C) which,


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after geometry optimization in the S=5/2 state, was ~200 meV higher in energy. Model (C) preserves coordination of the Glu189 to Ca2+ ion, Figure 2C. EXAFS analysis. EXAFS analysis has been conducted previously on samples enriched with the g~4 S2 state.40-41 While both studies report the same X-ray Absorption Near Edge Structure (XANES) results, the original study by Liang et al.40 reported that the g~ 4 S2 state has a different structural geometry from the LS S2 state with an elongation of one of the Mn-Mn distances, corresponding to a di-µ-oxo bridge unit, up to 2.85 Å40. However, more recent data were fit with Mn-Mn di-µ-oxo bridge elongating up to 3.30 Å.41 These discrepancies are likely due to the limited k-range recorded in EXAFS measurements at the Mn K-edge of the PS II due to the presence of iron.24-25 Original results are in good agreement with our DFT structural modeling which predicts elongation of one Mn-Mn di-µ-oxo bridge up to 2.84 Å upon –OH group binding to the Mn1, Table 2. Here we repeated the X-ray absorption spectroscopy (XAS) measurements, Figure 3. EXAFS measurements of the samples generated by illumination at 140 K with the inclusion of near-IR light (a method used previously for generation of the g~4 S2 state) are consistent with elongation of at least one Mn-Mn di-µ-oxo bridge, Figure 3B. EXAFS fits improved with splitting the ~2.7 Å Mn-Mn shell into two vectors, Table S2, Figure S1. Typical fits deliver two Mn-Mn interactions at 2.72 Å and one at 2.89 Å in agreement with earlier results40 and proposed DFT models. Simulations of EXAFS oscillations based on DFT derived Models A-C, Figure 2C are described in SI and shown in Figures S2 and S3. It was previously noted that use of the sucrose buffer decreases the intensity of the multiline S=1/2 S2 EPR signal.42 Generation of the g~4 S2 state in the sucrose buffer resulted in the similar EXAFS fits, Table S2. Analysis of spin coupling. To further validate proposed DFT models for the HS S2 state we conducted broken symmetry calculations, Table 3, where the spin on each Mn was allowed to point


8

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Figure 3. Photosystem II Mn K-edge XANES (A) and EXAFS (B) recorded at 20 K. S2 samples were produced by continuous illumination at 200 K or 140 K (with near-IR component). Both samples display oxidative shift in XANES. While low spin S2 state produced by illumination at 200 K is known to have structure indistinguishable from the S1 state, S2 state generated by illumination at 140 K shows visual differences in EXAFS such as shift of the 2nd peak corresponding to Mn-Mn interactions (insert) to longer distances.

either entirely up or down along the Z axis. Properties of the spin orbitals and Kohn-Sham equations make these energies equivalent to eigenstates of total spin, as listed in Table 3. The HS S2 structural Models A, B and C were optimized in highest possible state, S=13/2, and were compared to S=5/2 optimized models. Models A and C show no significant structural changes between these two (S=5/2 and S=13/2) optimizations. Mn3 and Mn2 were found to be MnIII ions in models A and C respectively, Figure 2C, Table 3. Additional verification via optimization using M11-L functionals gave the same result. For HS S2-B model optimized with BP86 at S=13/2, elongation of the Mn2-Mn1 bridge extended to 3.09 Å and the MnIII ion was no longer Mn2 but Mn1. Due to significant structural perturbations in the S=13/2 state we did not pursue this model further. Because a limited number of spin states are accessible to DFT which utilizes single Slaterdeterminants, energies for the OEC models were used to parameterize a Heisenberg− Dirac−Van Vleck (HDVV) Hamiltonian. In the HDVV model the total energy is equal to the dot product of


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Table 3. Energies (in Hartree) for S2-state OEC models from broken symmetry calculations. Model /functional Mn spins

LS S2 BP86

S2 – model A BP86

S2 – model A M11-L

S2 – model C BP86

S2 – model C M11-L

Mn4 Mn3 Mn2 Mn1

3/2 3/2 3/2 2

3/2 2 3/2 3/2

3/2 2 3/2 3/2

3/2 3/2 2 3/2

3/2 3/2 2 3/2

Spin

Energy

Spin

Energy

Spin

Energy

Spin

Energy

Spin

Energy



13/2

-8309.94370

13/2

-8386.41174

13/2

-8385.03924

13/2

-8386.39097

13/2

-8384.92307



7/2

-8309.94603

7/2

-8386.41673

7/2

-8385.04536

7/2

-8386.39871

7/2

-8384.93390



7/2

-8309.94546

5/2

-8386.41754

5/2

-8385.04807

7/2

-8386.40051

7/2

-8384.93755



7/2

-8309.94592

7/2

-8386.41108

7/2

-8385.04082

5/2

-8386.41213

5/2

-8384.94669



5/2

-8309.94619

7/2

-8386.41306

7/2

-8385.04055

7/2

-8386.40051

7/2

-8384.93479



1/2

-8309.94272

1/2

-8386.4121

1/2

-8385.04219

1/2

-8386.40468

1/2

-8384.94076



1/2

-8309.94829

1/2

-8386.41648

1/2

-8385.04696

1/2

-8386.40839

1/2

-8384.94366



1/2

-8309.94887

1/2

-8386.41785

1/2

-8385.04635

1/2

-8386.40549

1/2

-8384.94036

Heisenberg

1/2

-8309.96045

1/2

-8386.43007

7/2

-8385.06341

5/2

-8838.09539

5/2

-8384.98154

Mn spins times the coupling constants Jij that couple Mn atoms i and j, plus a constant background energy. The coupling constants were chosen to minimize the mean squared error between the DFT energies and equivalent states of the model Hamiltonian. Such a fitting procedure is necessary because the number of states accessible via DFT is greater than the number of free parameters in the HDVV Hamiltonian. Diagonalization of the HDVV Hamiltonian in the full (2SMn1+1)(2SMn2+1)(2SMn3+1)(2SMn4+1) dimensional Hilbert space allowed us to find the groundstate energy and spin of each OEC model, the final row of Table 3. In agreement with experiment, the LS S2 model was found to have lowest spin state (S=1/2), Table 3. Binding of the substrate oxygen (-OH) to Mn1 in general resulted in the change of the OEC spin state to high spin which is clearly shown in trends for lowest energy, Table 3. Note that previous experimental evidence has been reported to support a S5/2 for the HS S2-state.31, 43 Ammonia binding in the S2 state. A series of experiments have been reported where ammonia was introduced into PS II to shed light on the nature of the substrates required for O-O bond


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formation.4, 44-46 It was demonstrated that the presence of ammonia prevents formation of the HS S2 EPR33 with multiple studies proposing ammonia substitution of W1 water in the S2 state.4, 44-45 According to our DFT models, this substitution appears to be energetically favorable (~-240 meV) in agreement with the experiments reported, Table 1, Figure 2D. OECs with ammonia in the W1 position can still bind OH- onto Mn1 with a decrease in energy, Table 1. Thus, binding of the ammonia in the W1 position alone would not explain the absence of the HS S2 signal in the presence of ammonia. We also checked whether ammonia can directly block the access to the 5-coordinate Mn1. A configuration was found with stabilizing (~-170 meV) energy and Mn1---N distance of 2.28 Å which we interpret as a lack of direct binding, likely due to the spatial constrains, Figure 2D. NH3 remains in the proximity of the Mn1 when geometry optimization converges as it forms an H-bond with the Mn4-O5-Mn3 oxo bridge, Figure 2D. This is in agreement with biological studies showing two binding sites for ammonia in PS II.47-48 Thus, ammonia can reversibly block access of the substrate to the Mn1 center in the S2 state explaining absence of the HS S2 EPR signal. An alternative explanation is that ammonia can block the substrate access to the Mn1 site somewhere upstream via interference in the water channel. Similar to the ammonia effect, the presence of small alcohols prevents the formation of the g~4.1 signal in plant PS II.49 Mechanism of water oxidation. Since the g~4 S2 EPR signal discovery in 198429-30 several critical properties of this HS S2 OEC state were firmly established such as: i) it has been shown to arise from an active form of the OEC capable of further advancement into S3 state and oxygen evolution; ii) it is interrelated to S=1/2 form of the S2 state characterized by multiline g=2.0 EPR signal; iii) its formation depends on the environment of the OEC and iv) it seems that the near-IR component of light used for excitation is required to produce this state. More recently it was demonstrated that HS S2 state can convert into the S3 state at a temperature as low as 180 K.50


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Until recently, no explanation on the nature of the g~4 S2 state was available. Presence of the both g~2.0 and g~4 S2 state EPR was taken as a support for the valence isomerism hypothesis (Figure 2A). Some calculations suggest that the HS state is a necessary intermediate to advance to the S 3 state which involves both a conversion to a closed cubane and the integration of a water, 51 though details remained obscure22. For instance, both forms in the valence isomerism model have the same protonation state, thus it does not provide a straightforward explanation for the pronounced pH dependence found when measuring the ratio of LS to HS populations or the shift in the pH dependence when Ca2+ to Sr2+ substitution is used. In the valence isomerism hypothesis pH dependence had to be assigned to a secondary environment effects.33 Our model of early substrate binding was verified here computationally and is in agreement with all available experiments. Increase in the pH, for instance, should shift the equilibrium from 5coordinate Mn1 to 6-coordinate Mn1-OH resulting in the growth of the HS g~4 EPR signal. Sr substitution of Ca appears to make the transition energetically more favorable (Table 1) and might provide an increased cavity size for substrate binding which is also in agreement with previous results.33 In general, the primary effect of near-IR radiation on aqueous solutions containing biological molecules involves modification of hydrogen bond structures, mainly the global and hydration shell water molecules. As such, these modifications can affect substrate binding. Both MnIII and MnIV ions are also susceptible to interaction with near-IR light via excitation of the d-d transitions. This excitation might promote substrate binding even at low temperatures. It has been know that contrary to the S1 to S2 transition, the OEC undergoes major structural changes in the S2 to S3 transition.2,

20, 39, 52-53

Recent spectroscopic and crystallographic data

identified the addition of an oxygen to the 5-coordinate Mn1 in the S2 to S3 transition.2, 10, 19-20 Despite the high quality data obtained, it is still not readily visible in either of the TR-XRD studies


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where substrate oxygen is coming from.2, 20 One group noted disappearance of a water molecule (W665) in the distant environment of the Mn4-O4-Mn3 bridge,2 while another study suggested that W3 coordinated to Ca2+ could be the source of the oxygen added to Mn1.20 W3 as the source of the oxygen is also supported by computational8, 54 and spectroscopic studies.55 Analysis of the effects of mutations in the vicinity of the W3 on the HS S2 state could shed light on the involvement of this particular water as a substrate to Mn1 center. Two distant mutations were previously reported to affect the conditions of the HS S2 formation: difference between the cyanobacteria and spinach in D1-Asn87 to Ala substitution56 and mutations of Lys317 for Arg57. These mutations likely affected the structure of water channels. Each of the listed HS S2 models (A-C) can transition into corresponding S3 states either via one electron oxidation resulting in Mn1-OH configuration or via proton coupled electron transfer, PCET, producing a deprotonated Mn1=O-Ca2+ fragment. All one electron oxidized S3 states formed from Models A-C provide pathways for O-O bond formation, Table S3. Model A has a OO bond distance ~2.58 Å between the Mn4-O-Mn3 bridge and Mn1-OH connected with the hydrogen bond. Elimination of –OH hydrogen is possible via proton transfer to the carboxyl group of the E333 bridging the Mn4-O-Mn3 unit or via the water connected to Ca2+ ion and further transported on via the network of H-bonds. Mn1-OH and one of the waters coordinated to Ca2+ are also in close proximity, ~2.92 Å. Model C is very similar and features ~2.54 Å and ~3.18 Å distances for the O-O bond distance of Mn1-OH to Mn4-O-Mn3 bridge or Ca2+-H2O respectively. Model B exhibits the same distance from Mn1-OH to either the Mn4-O-Mn3 bridge or Ca2+-H2O, both were found to have a O-O distances of ~2.80 Å. For Model B, proton transfer to the –COOof the Glu189 is possible. One electron oxidation to the S3 state changes DFT calculated distances


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insignificantly to ~2.58/3.08 Å for Model A, ~2.52/3.09 Å for Model C and to 2.79/2.87 Å for Model B, Table S3. PCET transition from the HS S2 state would require electron removal and deprotonation. The Mn1-OH group resides in close proximity to the Ca2+ ion in all models (O…Ca2+ distance ~2.46-2.60 Å). Presence of the Ca2+ positive charge might facilitate the deprotonation of the Mn1-OH recent

and its conversion into MnIV=O-Ca2+

crystallographic S3 state coordinates reported by Kern

bridge. In such a case all HS S2 models (A,

Figure

4.

Alignments

of

the

most

et al. 2018 (shown in color) to DFT models (shown in gray) of S3 featuring Mn1IV=O-Ca2+ bridge proposed by

B and C) will converge to the single S3

our group in 2015 (Davis et al. arXiv:1506.08862, 2015)

state model. Analysis of our time resolved

(A) and re-optimized from the Kern et al. 2018 XRD coordinates (B).(C) Features comparison with DFT optimized structure with MnIV-OH group. The root means squared (RMS) of the positional differences between the DFT optimized structures and the XRD coordinates using the (Mn4CaO6) atoms in the OEC are 0.27 Å, 0.23 Å and 0.24 Å respectively.

X-ray emission spectroscopy data favors the deprotonated form of the Mn1=O group in the S3-state.6, 19, 23, 58 Currently, the chemical identity of Mn1-O oxygen in the S3 state is unknown. At least three

major fragments have been proposed for the oxygen group including: a MnIV–OH hydroxo37 group, a MnIV=O oxo10, 19 group and even an O-O peroxo2, 6 group. O-O distance reported in the most recent TR-XRD20 (~2.0 Å) is still considerably closer than the expected ~2.5 Å. Comparison of the most recent TR-XRD data for the S3 state20 with our S3 state model proposed in 201510 results


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in excellent agreement, Figure 4A. DFT optimization starting from the TR-XRD S3 coordinates gives very similar results for both MnIV–OH hydroxo and MnIV=O oxo configurations (S=3), Figure 4 B, C. Differences in Figure 4A and 4B are rotation of two water group on Ca2+ which is caused by de-coordination of the Glu189 residue in Figure 4B. Glu189 remains coordinated to Ca2+ in MnIV–OH hydroxo DFT optimization, Figure 4C. In summary, the incorporation of a hydroxyl ligand to Mn1 gives a clear explanation to the body of experimental data found when advancing to or from the HS S2 state. In addition, this framework provides additional support for the XRD modeling of the S3 state as it shows the energetically favorable integration of an oxygen based ligand, a process which may occur earlier in the cycle under certain conditions. It also provides evidence that the Mn1 is actually 6 coordinated in the HS S2 state and likely the same in the S3 as argued previously. This mechanism also indicates that multiple pathways are possible to advance to the S3 state which deviates from the more common frameworks which propose a single path Kok cycle. Multiple paths may have implications for other S-state transitions as well, including the final transition, S3-S0.


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ASSOCIATED CONTENT Supporting Information. Materials and Methods, DFT and EXAFS details and Results. This material is available free of charge. ACKNOWLEDGMENT This research was supported by NSF, CHE-1350909. The use of the Advanced Photon Source, an Office of Science User Facility operated by the U.S. Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the U.S. DOE under Contract DEAC02-06CH11357. The PNC/XSD (Sector 20) facilities at the Advanced Photon Source and research at these facilities were supported by the U.S. Department of Energy, Basic Energy Science and the Canadian Light Source. Access to EPR was provided by the Amy Instrumentation Facility, Department of Chemistry under the supervision of Dr. Michael Everly. Additional support came from the Office of Naval Research, through the Naval Research Laboratory’s NRL NISE program. We acknowledge the help of Sailesh Kandula in preparation of PS II samples.


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Early Binding of Substrate Oxygen is Responsible for

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