Concerted Mechanism of Water Insertion and O2 Release during the

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules 2

Concerted Mechanism of Water Insertion and O Release during the S to S Transition of the Oxygen-Evolving Complex in Photosystem II 4

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Mitsuo Shoji, Hiroshi Isobe, Yasuteru Shigeta, Takahito Nakajima, and Kizashi Yamaguchi J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b03465 • Publication Date (Web): 01 Jun 2018 Downloaded from http://pubs.acs.org on June 1, 2018

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The Journal of Physical Chemistry

Concerted Mechanism of Water Insertion and O2 Release during the S4 to S0 Transition of the Oxygen-Evolving Complex in Photosystem II Mitsuo Shoji, *, a Hiroshi Isobe,b Yasuteru Shigeta, a Takahito Nakajima,c and Kizashi Yamaguchi *, d,e

a

Center for Computational Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba 305-8577,

Japan b

Graduate School of Natural Science and Technology, Faculty of Science, Okayama University,

Okayama 700-8530, Japan c

Riken Center for Computational Science, Kobe, Hyogo 650-0047, Japan

d

Institute for NanoScience Design, Osaka University, Toyonaka, Osaka 560-0043, Japan

e

Handairigaku Techno-Research (NPO), Toyonaka, Osaka 560-0043, Japan

* Corresponding authors at: Center for Computational Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8577, Japan (M. Shoji) and Institute for NanoScience Design, Osaka University, Toyonaka, Osaka 560-8531, Japan (K. Yamaguchi) E-mail addresses: [email protected] (M. Shoji) and [email protected] (K. Yamaguchi).

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Abstract: The O2 release of the oxygen-evolving complex (OEC) of the photosystem II (PSII) is one of the essential processes responsible for the highly efficient O2 production. Despite its importance, the detailed molecular mechanism is still unsolved. In the present study, we show the O2 release is directly coupled with a water insertion into the Mn cluster based on the quantum mechanics/molecular mechanics (QM/MM) calculations. In this mechanism, the O2 molecule first dissociates from the Mn sites in order, i.e. the O atom coordinating to the Mn3 (O5a) first dissociates, then the other O atom coordinating to the Mn1 (O5d) dissociates in the next step in the late S4 state (1 -> 2). Next, the O2 migrates to a space surrounded by the Val185 and His332 side chains as one water coordinating to the Ca2+ ion (W3) comes into the O2 bonded site (2 -> 3). Finally, a pre-S0 state (4) is formed after a proton transfer from the inserted water to the other proton acceptor site (W2) (3 -> 4). The highest activation barrier during these reactions was found at the O2 release step (2 -> 3) that only requires E‡ = 12.7 kcal mol–1 (G‡ = 10.4 kcal mol–1). A series of the reactions (2 -> 3) look like a chain crash of billiard balls, because the W3 is inserted into the catalytic center from the water abundant side (Ca2+ ion side), and then the O2 moiety is pushed out to the opposite side (Val185 side). The hydrophobic residue of Val185 convers the active O5 site and forms an O2–specific permeation tunnel. The present sequential reactions clearly demonstrate the efficient removal of the toxic O2 from the catalytic center and implications of the essential roles of Val185, Ca2+ ions and water molecules, which are all present in the active site of PSII as the indispensable constituents.


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1. Introduction The water splitting reaction catalyzed in the photosystem II (PSII) is one of the most important chemical reactions evolved by photosynthetic organisms. In this reaction, water molecules are decomposed into O2, protons and electrons using sunlight energy. PSII is a multi-subunit membrane protein complex and contains a Mn4CaO5 (Mn4Ca) cluster in the catalytic active site called oxygen-evolving complex (OEC).1,2 The water splitting reaction proceeds through five oxidation states labeled by the Si (i = 0-4) states according to the Kok cycle, where the index i refers to the number of stored oxidizing equivalents.3 In 2011, the high-resolution X-ray structure of PSII in the dark-stable S1 state was solved at the 1.9 Å resolution by Umena et al.1 The Mn4Ca cluster was determined to be composed of a dangling Mn (Mn4), µ-oxo atom (O4) and a Mn3CaO4 cubane. It was also determined that two water molecules (W1, W2) directly coordinate to the Mn4 and the other two water molecules (W3, W4) coordinate to the Ca ion in the cubane moiety. Based on the high-resolution X-ray structure,1 extensive experimental and realistic theoretical researches have been performed to investigate more precise molecular-level properties and reactions.1,2,4 On the other hand, in the higher oxidation states, a number of studies based on EXAFS, mass spectrometry, FTIR spectroscopy and theoretical calculations have been performed.5-19 Recently, S3 state structures have been determined by using the femtosecond X-ray free electron laser, 20-22 and an inserted water (oxygen atom named O6) can be observed at the 2.35 Å resolution.22 These remarkable developments have been performed, however, there still remain considerable debate about the detailed molecular structures of Mn4Ca cluster, protonation state and reactions expected during the S state transitions. Among all the S state transitions, the S state transition induced by the third flash (S3 to S0 transition via a transient S4 state) is thought to contain two important chemical reactions i.e., O-O bond formation and O2 release. For the O-O bond formation, a number of reaction mechanisms have been proposed.10-15,17 One potential mechanism for the O-O bond formations is the radical coupling proposed by Siegbahn.10-14 In his mechanism, O-O bond is formed between the two O atoms in the Mn4-µ2-oxo-Mn3 and oxyl-Mn1 moieties. In contrast to the extensive studies for the O-O bond formation step, the subsequent O2 release reaction has not been investigated precisely.14 Siegbahn has performed extensive QM calculations using the QM models composed of 200 atom for all the S state transitions including in the O2 release process,10-14 and elucidated important structural features and the inherent reactions of OEC. However, his QM model seems to be difficult and incomplete for some reactions. In fact, Siegbahn’s QM model14 does not involves the key residue of Val 185, and His residues, His 332 and His 337, and the His side chains are rotated about 90 degree in disagreement with the X-ray structures due to the loss of the hydrogen bonds with surrounding amino acids. As the hydrophobic wall of Val 185 and non-rotating His 332 may play 3 ACS Paragon Plus Environment


important role especially in the O2 release pathways. The O2 release process contains a sequence of micro-chemical reactions, therefore, the detailed mechanisms in the O2 release step still remain to be elucidated and meaningful as the essential OEC reactions. For the O2 migration process, Vassiliev and co-workers showed two distinct O2 exit channels using molecular dynamics (MD) approaches.23,24 In the present study, “O2 release” is used to indicate the initial O2 dissociation process from the Mn4Ca cluster and “O2 migration” is used to indicate the following long range O2 diffusion process. From their MD results, the released O2 near the Mn4Ca cluster diffuses through the hydrophilic transmembrane region of PSII, not through the hydrophobic area. The O2 channels coincide with the water channels, while the permeation energy profile is different between O2 and water due to their different molecular properties.23 One channel originates from a space near the W2 (site s2) to the bulk solvent (called channel 1)25 and the other channel does from W4 (site s1) to the bulk solvent at two distant exit sites on the lumenal surface (branches of channel 4).25 These permeation barriers of O2 were calculated to be in the range of 2.8–6.9 kcal mol-1, therefore, O2 can be easily and fast removed from the active site.23–25 Although these MD simulations clearly showed the O2 permeation pathways in PSII, it is not clear how the O2 is dissociated from the Mn4Ca cluster and what conditions such as protonation states, presence/absence of water molecules, and surrounding amino acid residues, are required for the smooth O2 release. In the present study, reaction mechanisms of PSII-OEC for the O2 release process during the S4 to S0 state transition were investigated. We utilized the quantum mechanics/molecular mechanics (QM/MM) method to fully evaluate the reaction energy profile. We searched the potential reaction pathways by assuming that (I) the O-O bond formation is already performed between the Mn1 and Mn4 (Mn3)14,27 and (II) one additional water (W) is already present near the hydrogen-bonded water molecules (W5-W7) located near the Ca2+ ion and the side chains of Asp170 and Tyr161. The second assumption is considered reasonable, because in the S3 to S0 state transition, at least one water molecule comes close to the Mn4Ca cluster to complement a space generated by the O2 dissociation and the provided water position is full of water molecules (W5-W7) only linked in the hydrogen-bond network. The computational model and methodology used in the present study are described in the next section 2. Calculated results are described in section 3; all the reaction pathways examined using the QM/MM method are described in the subsections 3.1–3.7; free energy profile is discussed in subsection 3.8; basis set effects and van der Waals corrections to the energy profile are discussed in subsection 3.9. In the subsections 3.1-3.7, each reaction step in the most favorable reaction pathway is discussed in detail especially on the structural, spin densities and energetic changes. In discussion section (section 4), the calculated results are compared with available experimental data to validate the present O2 release mechanism. Comparisons with the previous Siegbahn’s theoretical result14 4 ACS Paragon Plus Environment

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are described in detail. Roles of Val185 and Ca2+ ion in the O2 release process are discussed. 2. Computational details As the QM/MM setup procedures used in the present study are the same as in our previous studies,26,27 only the most important or different parts are described here. Detailed computational conditions are described in the Supporting Information. The original atomic coordinates of the QM/MM atoms were taken from the 1.9 Å resolution X-ray structure (PDB ID 3ARC(3WU2)). 1 All the QM/MM calculations were performed using the NWChem 6.3 program package.28 The molecular structure visualizations were performed using the visual molecular dynamics (VMD).29 The applied theoretical level is (UB3LYP/DZVP)|AMBER-ff99, where the DZVP basis sets indicate LANL-2DZ for the Mn and Ca atoms and 6-31G* for the other atoms.30–34 In order to explore the O2 release pathways, the amino acid side chains near the His332 (Ala188) and Val185 were included in the QM region, and stationary amino acid residues of Tyr161 and His190 were excluded from the QM region. The QM region contains about 110 atoms (Figure 1) and the total QM charge is zero. Amino acid residues included in the QM region are summarized in the Supporting Information (Table S1). For the protonation state of W2, we assumed that W2 = OH in S4. As this assumption should be carefully treated as this assumption directly lowers the redox potential of Mn4 as well as the Mn4Ca cluater. The full QM/MM system is a spherical protein model composed of about 17,000 atoms, which cover all the residues within 30 Å from the original O5 position of the OEC active site. QM/MM optimizations were performed for the atoms within 18 Å around the center (original O5 position) while all the remaining atoms are fixed. An electronic embedding scheme was adapted for the QM-MM non-bonded interactions. For the total energy calculations, all the QM-MM non-bonded interactions were calculated without cutoff. The reaction pathways were searched using the nudged elastic band (NEB) method, and the determined transition states were further refined and checked to be at the first-order saddle points. In the first stage, the highest spin state (2S+1 = 14) was assumed in order to search for the potential stable intermediate states. The most stable spin alignments were then investigated by evaluating all the effective exchange interactions among the spin sites (Mn and O2 atoms).35,36 It was found that the antiferromagnetic coupled spin states with 2S+1 = 2 were the most stable in the initial and the following states (1, 2, 3). Therefore, the low spin states were adapted for all the calculated states in the present study. The two oxygen atoms coordinating to Mn1 and Mn3 (Mn4) are termed “O5d” and “O5a” in the present study, respectively. They come from the original O5 notation and their coordinating Mn1 and Mn4 atom names, which are also termed MnD and MnA, respectively. The intermediate states are numbered in order from 1 to 4, and the states in different conformations are labeled by the second alphabet letter. For example, in state 1, two different 5 ACS Paragon Plus Environment


conformations for the O2 bonding are labeled 1a and 1b. The energy profile was further evaluated by adapting the triple-zeta valence polarized (TZVP) level basis and Grimme’s DFT-D3 dispersion corrections.37 For the TZVP basis set, we selected LANL-2TZ(f) for Mn atoms, LANL08 for Ca atom, and 6-311G** for other atoms.30-34 3. Results 3.1 Possible O2 release pathways Four different O2 release pathways with and without an incoming water molecule (W) were examined (Figure 1 illustrates the model with W). The four pathways are (A) O2 slightly dissociates from the Mn4Ca cluster (O atoms of the O2 are apart from the coordinated Mn atoms by about 4 Å), (B) O2 moves toward the Ca2+ coordination site (W3) and are replaced by W3, (C) O2 moves toward the Val185 direction, and (D) O2 moves toward the His332 direction. Without adding the water molecule (W), all the routes require high activation barriers (∆E‡ > 20 kcal mol-1) and the products states are not stabilized. The relative energies for the states are calculated to be ∆E(ANoW) = 20.3, ∆E(BNoW) = 12.5, ∆E(CNoW) = 19.3, and ∆E(DNoW) = 8.3 kcal mol-1. More details for the energy profiles and the molecular structures are summarized in the Supporting Information. In particular, the pathway to exchange W3 with O2 (Pathway B) is impossible owing to the limited room near the Mn4Ca cluster for passing through each other. These results indicate that the freely mobile space for O2 is strongly restricted due to the tight hydrogen-bonding network comprised by surrounding molecules. It was also suggested that the dissociation energy of O2 from the Mn4Ca cluster must be complemented by some other reactions. On the other hand for the pathways with W, only C, the O2 release pathway towards the Val185, possesses an activation barrier low enough to be overcome thermally at room temperature. The activation barriers in other pathways are not so low compared to the pathway C (∆E‡(A) > 14.3, ∆E‡(B) = 59.8, ∆E‡(C) = 12.7, ∆E‡(D) = 20.3 kcal mol-1), though the product states become much stabilized by adding W (∆E(A) = 14.3, ∆E(B) = -2.2, ∆E(C) = -2.2, and ∆E(D) = -9.0 kcal mol-1). It should be noted that the state A is not a stable state, as the state A is obtained by restrained optimizations for the long Mn-O distances (L(Mn3-O) = 4.1 Å and L(Mn4-O) = 4.1 Å) and it still does not arrive to the transition state. It is also noted that the position of the dissociated O2 in state A becomes close to the O2 in state C as the Mn-O restrain distances increase in pathway A, though the complete difference between the states A and C are the position of W, i.e., in state C, W moves to close to the vacant space generated after the O2 dissociation, but W does not in state A. Therefore in the following subsections, energetically favorable reaction steps of the pathway C are discussed in detail. Schematic illustrations for all the intermediate states along the pathway C, the 6 ACS Paragon Plus Environment

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energy profile and the molecular structures in the QM region are shown in Figures 2, 3 and 4, respectively. Among the four reaction steps, complicated three reaction steps, 1b -> 2, 2 -> 3 = (C) and 3 -> 4 are precisely characterized by the key atomic distances, atomic spin densities with the total energy changes in the next subsections. 3.2 Conformational change (twisting) of the O2 moiety (1a -> 1b) The first reaction is a conformational change in the coordination of the O22- moiety toward the remaining Mn4Ca cluster. The O22- moiety coordinates to the Mn1 and Mn3 atoms in a zig-zag conformation in the most stable state (1a), and it changes the coordination in another zig-zag metastable conformation (1b) in this first reaction step. In 1a, the Ca-O5d distance (L1a(Ca-O5d)) is longer than the Ca-O5a distance (L1a(Ca-O5a)) by ∆L1a(Ca-O5) = L1a(Ca-O5d) – L1a(Ca-O5a) = 0.54 Å, and in 1b, the difference decreases to ∆L1b(Ca-O5) = –0.25 Å. The O2 conformational change of the 1a -> 1b transition requires a small activation barrier of 4.5 kcal/mol. The electronic structures (charge and spin distributions) for the Mn4Ca and O2 moieties remain almost unchanged during this reaction (see supporting information, Table S2 and Figure S1). 3.3 Mn3-O5a bond cleavage and Mn3-O5d bond formation (1b -> 2) In the 1b -> 2 reaction step, the Ca-O5a distance becomes longer, and the O5a dissociates from Mn3. As the Mn-O5d distances change from (L1b(Mn1-O5d)=1.87 and L1b(Mn3-O5d)=2.76 Å) to (L2(Mn1-O5d)=2.39 and L2(Mn3-O5d)=2.38 Å), the O5d becomes closer from Mn1 to Mn3 (Figure 5). The 1b -> 2 reaction step is similar to the 1a -> 1b reaction in that the O2 moiety rotates in a counterclockwise direction around the axis passing through the Ca2+ ion and the O3 atom. During this reaction step, the O2 moiety is partly oxidized from 1O22- to 3O2 judging from the total atomic spin density of the O2 moiety (from 0.28 to 1.67). As the total atomic spin density of the O2 moiety is not 2.0 in 2, the O2 moiety is not yet completely oxidized to 3O2 due to some interactions with the Mn1 and Mn3. The O5a-O5d distance becomes shorter from L1b(O5a–O5d) = 1.39 Å to L2(O5a–O5d) = 1.24 Å, though it is still slightly longer than that of O2 at a gas phase, i.e. Lgas(O–O) = 1.21 Å. Mn1 and Mn3 increased the negative spin densities from (-3.00, -2.87) in 1b to (-3.65, -3.57) in 2, whereas the spin densities of Mn4 and Mn2 are unchanged (Figure 5). Therefore, the formal Mn charges (Mn1, Mn2, Mn3, Mn4) could be assigned as (IV, IV, IV, III) and (III, IV, III, III) for 1b and 2, respectively. Note here that O2 still weakly coordinates both to the Mn1 and Mn3 atoms with the O5d atom in 2. The activation energy barrier (E(TS1b,2) = 10.1 kcal mol–1) is relatively higher than that of the 1a -> 1b reaction, and the intermediate state 2 is higher in energy than that of 1a by 7.6 kcal mol–1. Thus, the backward reactions from 2 to 1a and 1b easily 7 ACS Paragon Plus Environment


occur unless a reaction from 2 to 3 undergoes. 3.4 O2 release pathway through the Val185 gate (2 -> 3) In the 2 -> 3 reaction step, the O–O bond length is further shortened from L2(O5a–O5d) = 1.24 to L3(O5a–O5d) = 1.21 Å, and their atomic spin densities (O5a, O5d) changed from (0.95, 0.72) in 2 to (0.96, 1.00) in 3 (Figure 6). Therefore, in 3, the O2 moiety is considered to be completely dissociated from the Mn sites (Mn1 and Mn3) and becomes a free 3O2. Since the O2 leaves from the Mn sites, the nearest water (W3), a water molecule coordinating to Ca2+ ion, closes to an open space surrounded by three Mn sites (Mn1, Mn3 and Mn4), where O5a was located at the same position in 1. Although the additional activation energy barrier is not high (E(TS2,3) – E(2) = 4.7 kcal mol–1), the transition state of the 2 –> 3 reaction step (TS2,3) is the most unstable (E(TS2,3) = 12.7 kcal mol–1) in the whole O2 release steps (all the reaction steps discussed in the present study). The O2 release pathway of the 2 -> 3 reaction step corresponds to an initial O2 migration pathway, and is surrounded by the water molecule coordinating to Mn4 (W2) and the side chains of His332 and Val185. The O2 permeates through the narrow hole along the molecular surfaces formed by these surrounding molecules. Because of the nonpolar and small molecular size of O2, and an immediate insertion of W3 into the open space, the O2 release can occur with the low activation barrier. As mentioned in the section 3.1, without W, the O2 release cannot occur due to the high activation energy (E(TS2,3, –W) – E(2–W) = 10 kcal mol–1) and 3–W is not stabilized enough (E(3–W) – E(2–W) = 8 kcal mol–1), in which X–W means the state X without W. Therefore, the presence of W is essential in the present O2 release process. The resultant product 3, is stable and energetically comparable to the initial state 1 (E(3) = –0.9 kcal mol–1). The O2 in 3 is located near a water molecule connecting to the water channel to the bulk water, suggesting that the O2 is opened through the O2 channel in PSII. Moreover, by the significant stabilization from TS2,3 to 3, the backward reaction will be prohibited due to the higher energy barrier than that for the next reaction (3 -> 4). If the O2 is only dissociated from the Mn4Ca cluster without any stabilization, O2 is easily recombined with the active site. Therefore, the moderately stable intermediate 3 is rather crucial in the energy profile to achieve an effective O2 release. 3.5 Proton transfer from W3 to W2 via W5 (3 -> 4) The final reaction is a proton transfer from W3 to W2 (Figure 7). We found a proton transfer pathway via W5 which can mediate between W3 and W2 and totally carries a proton for a long distance (2.4 Å in direct distance). As a general proton relay mechanism, one proton is transferred 8 ACS Paragon Plus Environment

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from W3 to W5 and another proton is transferred from W5 to W2 at the same time. Consequently, W3 is deprotonated to OH– and W2 is protonated to H2O in 4. The product 4 is very stable with E(4) = –10.5 kcal mol–1. The activation barrier (E(TS3,4) = 8.3 kcal mol–1) is not high compared to TS1b,2 and TS2,3. At the transition state TS3,4, W5 temporarily becomes H3O+ to accept a proton from W3 and donate one to W2 simultaneously. This reaction pathway required many NEB beads (22) due to the long proton transfer and a large W5 conformational change in the later stage, which show a small activation barrier at the 14th NEB bead. The atomic spin densities for both 3O2 and Mn atoms are unchanged during this proton transfer, meaning that they do not participate in the proton transfer reaction. 3.6 O2 exit channel In the 2 -> 3 reaction step, O2 is pushed out from the Mn4Ca cluster to a space surrounded by the W2 and side chains of Val185 and His332, where the space locates opposite to the Ca2+ ion. Favorable O2 exit channels revealed by MD simulations are channels 1 and 4,23,24 which connect the spaces near the W2 and W4 of OEC to lumenal surfaces, respectively. It was also revealed by Vassiliev et. al that the two O2 exit channels completely overlap to the water channels in PSII.23 The channel 1 is located at an interface formed by the D1, D2 and PsbO proteins involving D1-Asp61, D2-Lys317, D2-Glu312, and D2-Glu310 amino acids and the channel length is short (24 Å). The channel 4 is larger than the channel 1 (~ 30 Å long) and it branches into two pathways near the side chains of the CP43-Val410 and PsbV-Lys47 amino acids at the interface between the D1, PsbV and CP43 subunits. The common region is formed by CP43-Thr412, D1-Glu329, and D1-Asp342, and the one branch (channel 4a) continues to CP43-Glu413, CP43-Val417 and CP43-Gln418, while the other branch (channel 4c) connects to PsbU-Lys104, PsbV-Gly132 and PsbV-Val135. The channels 4 correspond to the “large channel system” suggested by Ho and Styring.38 For the water permeation pathways, Vassiliev et. al reported that all of the channels except channel 3, i.e., channels 1, 2, 4, 5 and X, are valid.25 The channels 2, 3 and 5 matches to the previously identified ”Narrow”, “broad” and “back” channels by Ho and Styring, respectively.38 The channel X is a newly proposed water permeation pathway proposed by Vassiliev et. al. 25 Therefore, PSII has multiple water permeation pathways which can access to the Mn4Ca cluster. 25 Topologies of the channels, structure of the Mn4Ca cluster and trajectories of O2 moieties in states 1a-4 are superimposed in Figure 8. As the added water W in state 1a is neighbor to the W4, and the formed O2 in states 3 and 4 locate next to the W2 (Figure 8), it is expected that the added water W is entered through the channel 4, and the formed O2 is released through the channel 1. Therefore, each channel is used as a one-way traffic for each molecule. This may be a reason why the PSII possesses two different O2 exit channels connecting the Mn4Ca cluster and bulk waters in the 9 ACS Paragon Plus Environment


lumen. The channel 1 has been suggested for a possible proton exit pathway.39 The titratable residues in channel 1 increase from the Mn-cluster to the lumenal bulk side, and these residues are suitable for releasing protons. Moreover, D2-Lys317, CP43-Arg357 and D1-Asp59 residues may be deprotonated in the S-state transitions (in the S1 -> S2, S2 -> S3, and S3 -> S0 transitions, respectively). Therefore, the channel 1 may provide multiple functions and change the role depending on the S states in the PSII catalytic reactions. 3.7 Other reaction branches Here, other reaction branches, which unfortunately found to be higher in energy than the above pathway, are discussed. Another branch from 1a to 2 is a direct pathway, in which the O2 moiety rotates in a clockwise direction around the axis passing through the Ca2+ ion and the O3 atom. The resultant structure is the same as that of 2 in Figures 4 except for the replacement of O5d and O5a atoms. In this pathway, the O5d–Mn1 bond is first cleaved. The activation barrier is calculated to be about 15 kcal mol–1, which is higher than that of TS1b,2. Another branch from 3 to 4 is a direct pathway, in which the proton of W3 transfers to the oxygen atom of W2 without a help of W5. The relative energy of the transition state (TS3,4,direct) along this direct pathway is calculated to be E(TS 3,4,direct

)=19.3 kcal mol–1 due to the loss of the hydrogen bond with W5 during the proton transfer

(L(OW3-OW2) = 2.46 Å, L(HW3-OW3) = 1.34 Å, L(OW2-HW3) = 1.18 Å in the TS3,4,direct). Although this type proton transfer pathway is reported to be preferable by Siegbahn,14 this pathway does not show a low activation barrier in the present QM/MM calculation. More detailed results (the transition state structure, the energy profile and key distances are summarized in the SI.) Messinger and co-workers have suggested two different reaction schemes for the S3 –> S0 transition on the basis of their experiments. One scheme partially resembles the present reaction mechanism.6,40 In the other scheme, an incoming water coordinates to Mn4, and W2=OH– rotates to become O5=OH–. The latter mechanism is quite different from the present scheme, and there exist uncertainty for the incoming water. This water insertion scheme may be similar to the water insertion reactions proposed in the S2 -> S3 transition,41,42 and the side chains of D1-Asp61 and water molecules near the O4 would be necessary to be included in the QM region to describe their proton transfer reactions. In the present study, we assumed W2 = OH in the S4 state. W2 extracts one proton from the inserted water in the 3 -> 4 step; i.e., W2 = OH and W3 = H2O is apparently converted into W2 = H2O + O5 = OH. Therefore, W2 acts as a base. However if one adapts an alternative model with W1 = W2 = H2O, other amino acid residues which can alternatively act as the temporal base should be required. In this case, D1-Asp61 is one candidate. Therefore, further theoretical investigations in these reaction schemes are still required. 10 ACS Paragon Plus Environment

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3.8 3.8 Free energy profile The discussed energies are not considering the entropy contributions. The free energy (∆G) changes are evaluated by performing the frequency calculations at the same QM/MM level. In Figure 1, free energy profile is shown in comparison with the relative energy profile. In Table 1, energy contributions for the enthalpy (∆H) and entropy (∆S) terms are listed. All the states after TS1b,2 are more stabilized by 2-4 kcal mol-1 at the free energy, however, the entropy (∆H) contributions play dominant contributions for these early O2 dissociation steps. In state 3, O2 is completely dissociated from the Mn site and O2 arrives at the O2 exit channel which connects to the outside of PSII. At the same time, the water molecule (W) is inserted to the Mn site of the Mn4Ca cluster. Therefore, their entropy contributions are largely countered. Large stabilizations for TS3,4 and 4 in the free energy profile substantially contribute to the irreversibility of the O2 release. Nilsson et al. have measured the oxygen-water isotope exchange in the S4 state by using the membrane-inlet mass spectrometry (MIMS), and reported that the equilibrium constant K of the S4 -> S0 transition is greater than 1.0x107.43 This indicates that the S4 -> S0 step is highly exergonic and the overall free energy difference can be estimated to be ∆G0 > 430 meV. In the S4 -> S0 transition, the energy contributions for the O2 release and the H+ production are performed outside the PSII. Thus, their external contribution (∆Gex), which mainly contains the entropic contribution of the product releases, can be divided from the intra enzyme contribution (∆Gin). Dependence on pH as well as the O2 concentration directly affect to ∆Gex. Consequently, overall free energy difference (∆G0) is influenced by the external and internal energy contributions (∆Gex = ∆Gin + ∆G0). This requirement contributes to valance the ∆Gin and ∆G0 values, because if ∆G0 is decreased, the positive ∆Gin value are increased for the unchangeable ∆Gex. This makes to maximize the overall catalytic reaction rates, however, the activation barrier of the reaction (∆Gex) is increased, which means that the S4 -> S0 transition is slowed. Nilsson et al. estimated ∆Gin ~ 400 meV (9.2 kcal mol-1) and ∆Gex ~ 830 meV (19.1 kcal mol-1). In the present study, we evaluated that ∆G (TS2,3) = 10.4 kcal mol-1 (~ 451meV) independently by using the QM/MM calculations, which corresponds to the ∆Gin ~ 400 meV (9.2 kcal mol-1). Therefore, our calculated free energy profile is consistent with the recent experimental kinetics results. 3.9 3.9 Basis set and van der Waals effects 11 ACS Paragon Plus Environment


Other concerns about the energy profile are basis set and van der Waals effects. For the basis set, triple-zeta valence polarized (TZVP) level basis set is required for reliable energies. Here, the TZVP level basis set means that LANL-2TZ(f) for Mn atoms, LANL08 for Ca atom, and 6-311G** for other atoms. Additionally, dispersion effects are expected to be non-negligible especially for the formed O2 molecule. Therefore, we have re-evaluated the energy profile with the TZVP basis set and Grimme’s DFT-D3 dispersion correction. All the results without and with employing full geometry optimizations are summarized in Table 2. Without geometry optimization, TZVP basis set tends to destabilize the states after TS1b,2 by 2-3 kcal mol-1. On the other hand, the vdW correction stabilizes the states after the state TS1b,2 by 2-8 kcal mol-1. The vdW stabilization seems to be reasonable, because O2 molecule is formed in TS2,3 and the O2 moved close to the hydrophobic residue such as Val185 in state 3. The energy changes by full geometry optimizations are found to be small less than ~2 kcal mol-1. And the basis set and vdW effects contribute to the opposite directions and the total changes in the energy profile are limited. Nevertheless, the transition states of TS1b,2 and TS2,3 are more stabilized by ~2 kcal mol-1, and the states 3 is significantly stabilized by 4.1 kcal mol-1. As the state 2 is not similarly stabilized, the energy difference between TS1b,2 and 2 becomes very small. These results indicate that the state 2 is a very unstable and local intermediate state located in the middle of the O2 dissociation pathway.

4. Discussion In the S3 –> S0 transition, not only the O2 release but also the ejections of two protons and the supply of one water molecule are carried out in the Mn4Ca catalytic center. Therefore, how the presently studied O2 release is related to other processes is discussed in comparison with the previous experimental and theoretical results. The S3 –> S0 transition is thought to occur in sequential order upon the initial Yz oxidation.3,44 First, one proton is released in the lag phase with

τlag ~ 50-250 µs. After the Yz is reduced by the Mn4Ca cluster, O2 and the second proton are released in the longer time range of τS ~1.2 ms (referred as the slow phase). One water molecule is expected to be supplied at any stage during the S3 –> S0 transition.45 In the present work, we have theoretically shown the supply of one water molecule near the Ca2+ ion is essential in the O2 release step. Therefore, the present concerted mechanism of the water insertion and the O2 release, water insertion coupled O2 release, is one of the probable reaction pathways in the S3 -> S0 transition. Li and Siegbahn have performed DFT calculations for the O2 release process in the S3 -> S0 transition.14 They reported that the barrier of the O2 release step was calculated 12 ACS Paragon Plus Environment

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to be 11.0 kcal mol-1, which corresponds to the rate-limiting step during the S3 -> S0 transition. The activation barrier is similar to our present result of G‡(TS2,3) = 10.4 kcal mol–1, however, some notable differences exist. Regarding the theoretical methodology, their model is based on a QM system, though the size of the QM model is larger with ~200 atoms. It is highly debatable whether our present QM/MM models are better than QM models, however, we have experienced that that such QM models are very difficult for appropriately maintaining the structures in the outer region. In addition to the difference in the theoretical approach, differences especially noteworthy for the transition state structure are listed as follows; (i) The W5 water, which is used for the proton transfer in the present 3 -> 4 step, was missing. (ii) Imidazole rings of His 332 and His337 are rotated about 90 degree, which are completely different both to the present QM/MM model and the crystal structures in the S3 state as well as S1 state.20,22 (iii) Side chain of Val 185 is not included in their QM model. (iv) In their transition state in the O2 release step, the distance between the Mn and O2 was still very short (L(Mn3-O) = 2.00 Å). (v) In their O2 release process, one water was inserted after the O2 release, which is completely different with our concerted mechanism. (vi) Calculations for the route of the early O2 dissociation process were not performed. It should be noted that as their QM model processes larger numbers of QM atoms, structural relaxations over the QM atoms were much induced. For example, motions of Cl- atom and side chain of Lys 317 were observed in the reactions. Therefore, the QM model by Siegbahn and Li seems to be more flexible compared to our present QM/MM model. We emphasize again that the OEC of PSII is full of water molecules and hydrogen-bonds, therefore, non-negligible energy is required if the hydrogen bonds are reconstructed at least based on the present QM/MM model. Based on the kinetics experiments using Val185 mutants, Asp61 mutants and Cl– ion replacement, the lag phase is significantly affected by their substitutions.46 The V185N mutant showed a substantial delay both in the lag (τlag = 800 µs) and slow (τS = 26 ms) phases. The D61N mutant showed a similar slow kinetics with τlag = 600 µs and τS = 24 ms, which are slightly faster than those of the V185N mutant.47 The replacement of Cl– ion with I– ion also results in delay of the lag and slow phases. Since the first proton release is thought to undergo in the lag phase, these results indicate that Val185, Asp61 and the Cl– ion are expected to play directly or indirectly significant roles in the proton pathway.48 The Yz reduction and O-O bond formation are supposed to be accomplished near the time scale of the lag phase, which correspond to the reactions prior to the formation of our initial state (0 in Figure 2). The O2 release reactions calculated in the present study correspond to the early O2 release process during the slow phase. The theoretically estimated activation barriers for the O-O 13 ACS Paragon Plus Environment


bond formation, the water and O2 permutations are 9, 6.8 and 2.6 kcal mol–1, respectively,14,23 which are lower than the barrier evaluated in this study (E(TS2,3) = 12.7 kcal mol–1). Therefore, the initial O2 dissociation from the Mn4Ca cluster (2 -> 3) might be the rate determining process and dominantly influence the efficiency over the PSII catalytic reaction as well as the S3 -> S0 transition. Among the Val185 mutants, threonine (V185T) and phenylalanine (V185F) mutants exhibit different O2 evolving activities.46 The V185T mutant is nearly identical to the wild type in the O2 release kinetics and the miss factor. On the other hand, the V185F mutant could not evolve any oxygen, though a kind of Mn cluster is assembled judging from a fact that the S2 state is reached by forming a stable charge separation. Thr possesses a side chain with similar size to that of Val, while the side chain of Phe is bulkier than that of Val. These results clearly indicate that the size of the hydrophobic group of Val185 must be suitable for the catalytic reaction. The Val185 locates close to the O5 atom of Mn4CaO5 in the S1 state, where the O5 position is almost the same as the place of the O-O bond formation. During the first proton transfer reaction in the S3 -> S0 transition, the proton binding to O5d is transferred to the W2 = OH– in the lag phase, and the O-O bond formation between O5a and O5d is expected with the Yz reduction according to the Siegbahn’s radical coupling scheme.14 Indeed, as the Val185 locates very close to the catalytic center, V185 is probably controlling the catalytic reaction by forming a specific space by preventing other polar substrates from approaching there. Our speculation based on the present theoretical results is that Val185 plays a key role in the initial O2 release process acting as a hydrophobic one-way gate to transfer only O2 from the active site to the O2 channel. We also expect that the hydrophobic wall of Val185 is also essential for activating the O5 = (O, OH) reactivity by isolating it from the hydrogen bonding interactions. As the O2 is a nonpolar and small molecule, only O2 can pass through the small pore formed by the side chains of the Val185 and His332. The present O2 release is favorable in terms of the O2 selectivity and the restraint of the backward flow. It should be emphasized again that the side chain of Val185 directly plays a key role in the initial O2 release. Both a K-edge absorption spectroscopy (XAS) and a membrane-inlet mass spectrometry (MIMS) showed that the O2 release kinetics is unaffected by an elevated O2 partial pressure, indicating that the O2 release is a highly exothermic process (at most 160 meV (3.6 kcal mol-1)).49,50 In the present study, the two reaction steps after the O2 release are O2 migration (2 -> 3) and proton transfer (3 -> 4), and they are significantly exothermic by 8.5 and 9.6 kcalmol-1, respectively. In order to compared to the experimental ∆GS4, S0 value, entropic contributions 43 and the next proton transfer reaction (4 -> 5 in Figure 2) should be explicitly considered. However, the low activation barrier of the O2 release (2 -> 3) by the concerted mechanism and the exothermic reaction steps match to an important feature to enhance the fast removal of O2. Furthermore, as the activation barriers of their backward reactions (4 -> 3 -> 2) become so high that the O2 recombination will be 14 ACS Paragon Plus Environment

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highly suppressed for the Mn4Ca cluster. The plausible role of the Ca2+ ion in the O2 release is now discussed. Although the Ca2+ ion is constituent of the Mn4Ca cluster, the Mn cluster can be constructed and expected to be maintaining the Mn4O5 cluster structure even if the Ca2+ ion is removed.51 Kinetic studies showed that the slow phase of the S3 –> S0 transition delays four-fold (from τS = 1.1 to τS = 4.8 ms) upon the Ca2+/Sr2+ ion exchange.52 The Cl–/I– exchange similarly increases the transition almost five-fold. Although the Ca2+ and Cl- ions are spatially separated, these exchanges synergistically occur. These results are interpreted as an indication that the Ca2+ ion plays an important role in constructing the hydrogen bond network around the Mn4Ca cluster and the proton transfer as the Cl– ion is expected to be deeply related to the hydrogen bond formation.48,51 The FTIR spectrum shows that if the catalytic reaction of the S2 -> S3 transition is inhibited upon the Ca2+ ion substitution, the hydrogen bond network formed by the water molecules around the Mn4Ca cluster is broken.52 Therefore, the Ca2+ ion plays an essential functional role in the catalytic reactions. In the O2 release mechanism evaluated in the present study, the Ca2+ ion supports the insertion of a water molecule (W3), which will be O5 and used in the next catalytic cycle. This process is similar to one of the water insertion reactions (L reaction pathway) examined in the S2 –> S3 transition,26 though there exists the O5 atom and the water inserted into the vacant Mn4 coordination site in the S2 state. It is noteworthy that the water commonly inserts into the vacant coordinates site with the Mn(III) atom in both the S2 –> S3 and S3 –>S0 transitions. In both these reactions, the Ca2+ ion acts as a guide to lead the substrate water into the catalytic center (O5 position). On the other hand, from the synthetic models of iron-peroxide complexes with redox-inactive metal ions, Bang et al. showed that both Ca2+ and Sr2+ ions are suitable in the O2 release step compared to other stronger Lewis acid metal ions (Zn2+, Lu3+, Y3+ and Sc3+).53 Since this complex without the Ca2+ ion can be oxidized to release O2, the Ca2+ ion is expected to be inactively important as not to interfere with the O2 release step. Although the basic molecular frameworks of the synthetic model and native Mn4Ca cluster are significantly different, i.e., in the former, the peroxide is directly coordinated by Ca2+ and Fe3+ ions in the side-on fashion, and in the latter (see 1a), the peroxide is coordinated by two Mn atoms (L1a(Mn1-O5d) = 1.84 Å, L1a(Mn3-O5a) = 1.89 Å) in the end-on fashion, the experimental fact that Ca2+ and Sr2+ ions are only allowed for its constituent for the OEC in the PS II is quite similar to the Bang’s synthetic models. In the native OEC, the O2 moiety is more distant from the Ca2+ ion and their interactions will be more weakened compared with the synthetic model, because the Ca-O lengths (L1a(Ca-O5a) = 2.54 and L1a(Ca-O5d) = 3.07 Å)) are much longer compared to the Mn-O lengths. Since the O2 release step is the highest activation barrier processes in the S3 –> S0 transition in the native OEC, the separation of the Ca2+ ion and O2 moiety may improve the catalytic reaction. On the basis of this idea, it is quite natural 15 ACS Paragon Plus Environment


that the O2 release pathway exists opposite to the Ca2+ ion, and thus the O2 release pathway (2 –> 3) examined in the present study is considered to be appropriate. 5. Conclusions In the present study, a concerted mechanism of the O2 release and water insertion in the late S4 state is proposed using the QM/MM calculations. 3O2 is formed after the Mn3-O5a bond cleavage in 2 by reducing the Mn1 and Mn3 atoms. More precisely, there still remain small interactions (d-π interactions) betweem the O2 moiety and the Mn atoms in 2, as the total atomic spin density of the O2 moiety in 2 is 1.67, not 2.0. Completely isolated 3O2 is generated in the next state 3. The highest energy barrier step is found at the TS2,3 state, in which the O2 dissociates from the Mn4Ca cluster through the side chains of Val185 and His332, and one water (W3) occupies the vacant site generated after the release of O2. After the O2 permeation (3), O2 appears to be an entrance to a water pool connecting to the O2 channel 1 (for the spatial placement, see Figure 8). Judging from the remarkable stabilization in 3 and 4, the backward reaction to the O2 recombination will be strongly suppressed compared to the forward reaction with a lower activation barrier to reach the S0 state. Another significant feature is that the O2 release and the incoming water pathways are in the opposite directions, i.e., O2 is pushed out as the water comes to the vacant site surrounded by the Mn1, Mn3 and Mn4 atoms, where one of the O atoms in released O2 were located, as the chain reaction of billiard balls. We do not exclude other possibilities for other O2 release mechanisms which could not be better evaluated in the present study. However, in any O2 release process, the activation barrier for the O2 dissociation step from the Mn4Ca cluster must be stabilized using some mechanisms, because a simple diffusion of O2 into the water molecules must break the existing hydrogen bonds that increases the activation barrier for the initial O2 release/dissociation. Judging from reasonable activation barrier compared to the previous steps and considering the available experimental knowledge, it is highly expected that the present O2 pathway is one of the most probable mechanisms which matches the fast removal of the reactive 3O2 in the S3 –> S0 transition.


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Electronic supplementary information (ESI) available: Energy profiles, optimized structures for other O2 dissociation pathways, results for the direct proton transfer in the 3 -> 4 reaction step, summary of the Löwdin atomic spin densities and atomic distances, energy profile in the 1a -> 1b reaction step, structures of all the transition states, depiction of the OEC with channels and key amino acid residues, and all the xyz coordinates in the QM region.

Notes The authors declare no competing financial interest.

Acknowledgements This research was supported by JSPS KAKENHI Grant Numbers JP24000018, JP16KT0055, JP17H04866 and JP18H05154. Numerical calculations have been carried out under the supports of (1) HPCI system research project (Project ID: hp160169) using the computational resource of the center for computational sciences (CCS), University of Tsukuba, (2) “Interdisciplinary Computational Science Program” at CCS, (3) the research center for computational Sciences, Okazaki, Japan, and (4) HPCI system research projects (Project ID: hp150128 and hp160170) using the computational resources of the K computer provided by the RIKEN Advanced Institute for Computational Science (AICS).



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Acta, 2014, 1837, 1257-1262. 41 Retegan, M.; Krewald, V.; Mamedov, F.; Neese, F.; Lubitz, W.; Cox, N.; Pantazis, D. A. A five-coordinate Mn(IV) intermediate in biological water oxidation: spectroscopic signature and a pivot mechanism for water binding, Chem. Sci., 2016, 7, 72-84. 42 Capone, M.; Narzi, D.; Bovi D.; Guidoni, L. Mechanism of water delivery to the active site of photosystem II along the S2 to S3 Transition, J. Phys. Chem. Lett., 2016, 7, 592-598. 43 Nilsson, H.; Cournac, L.; Rappaport, F.; Messinger, J.; Lavergne, J. Estimation of the driving force for dioxygen formation in photosynthesis, Biochim. Biophys. Acta Bioenerg., 2016, 1857, 23-33. 44 Rappaport, F.; Blanchard-Desce, M.; Lavergne, J. Kinetics of electron transfer and electrochromic change during the redox transitions of the photosynthetic oxygen-evolving complex, Biochim. Biophys. Acta, 1994, 1184, 178-192. 45 Suzuki, H.; Sugiura M.; Noguchi, T. Monitoring water reactions during the S-state cycle of the photosynthetic water-oxidizing center: detection of the DOD bending vibrations by means of Fourier transform infrared spectroscopy, Biochemistry, 2008, 47, 11024-11030. 46 Dilbeck, P. L.; Bao, H.; Neveu C. L.; Burnap, R. L. Perturbing the water cavity surrounding the manganese cluster by mutating the residues D1-valine 185 has a strong effect on the water oxidation mechanism of photosystem II, Biochemistry, 2013, 52, 6824-6833. 47 Dilbeck, P. L.; Hwang, H. J.; Zaharieva, I.; Gerencser, L.; Dau H.; Burnap, R. L. The D1-D61N mutation in Synechocystis sp. PCC 6803 allows the observation of pH-sensitive intermediates in the formation and release of O2 from photosystem II, Biochemistry, 2012, 51, 1079-1091. 48 Boussac, A.; Ishida, N.; Sugiura, M.; Rappaport, F. Probing the role of chloride in photosystem II from Thermosynechococcus elongatus by exchanging chloride for iodide, Biochim. Biophys. Acta, 2012, 1817, 802-810. 49 Haumann, M.; Grundmeier, A.; Zaharieva, I.; Dau, H. Photosynthetic water oxidation at elevated dioxygen partial pressure monitored by time-resolved X-ray absorption measurements, Proc. Natl. Acad. Sci., 2008, 105(45), 17384-17389. 50 Shevela, D.; Beckmann, K.; Clausen, J.; Junge, W.; Messinger, J. Membrane-inlet mass spectrometry reveals a high driving force for oxygen production by photosystem II, Proc. Natl. Acad. Sci., 2011, 108(9), 3602-3607. 51 Lohmiller, T.; Shelby, M. L.; Long, X.; Yachandora, V. K.; Yano, J. Removal of Ca2+ from the oxygen-evolving complex in photosystem II has minimal effect on the Mn4O5 core structure: a polarized Mn X-ray absorption spectroscopy study, J. Phys. Chem. B, 2015, 119, 13742-13754. 52 Nakamura, S.; Ota, K.; Shibuya, Y.; Noguchi, T. Role of a water network around the Mn4CaO5 cluster in photosynthetic water oxidation: a Fourier transform infrared spectroscopy and quantum mechanics/molecular mechanics calculation study, Biochemistry, 2016, 55, 597-607. 21 ACS Paragon Plus Environment


53 Bang, S.; Lee, Y.-M.; Hong, S.; Cho, K.-B.; Nishida, Y.; Seo, M. S.; Sarangi, R.; Fukuzumi, S.; Nam, W. Redox-inactive metal ions modulate the reactivity and oxygen release of mononuclear non-haem iron(III)-peroxo complexes, Nature Chem., 2014, 6, 934-940.


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Figure Captions Figure 1. Schematic illustration of the OEC active site of photosystem II. Atoms in the QM region are shown in black. The atoms in Y161 and H190 shown in gray are included in the MM region. Hydrogen bonds are denoted by the red dashed lines. The state is assumed in the late S4 state after the O-O bond is formed and one water (W) is supplied. The O2 is in a O22- state and the Mn formal valence states are (Mn1, Mn2, Mn3, Mn4)=(III, IV, IV, IV). Figure 2. Schematic illustration of the reaction mechanism for the O2 release examined in the present study (1 -> 4). Oxygen atoms coordinating to the Mn1 and Mn3 atoms are named O5d and O5a, respectively. Figure 3. Free energy profile (∆G) and relative energy profile (∆E) calculated at the QM/MM level. Relative energies with respect to the initial state 1a are taken. Their energy contributions (enthalpy and entropy contributions) are listed in Table 1. The second alphabet character of the state name, such as 1a and 1b, indicates the different conformational state. Figure 4. QM/MM optimized structures in the intermediate states. Only the QM atoms are shown for clarity. Molecular structures in the transition states are shown in the Supporting Information. Figure 5. (a) Löwdin atomic spin densities and (b) relative energy (left-hand y axis) and key atomic distances (right-hand y axis) along the NEB reaction coordinate of the 1b -> 2 reaction. (c) Enlarged views of the Mn4Ca cluster for the intermediate and transition states. Figure 6. (a) Löwdin atomic spin densities and (b) relative energy (left-hand y axis) and key atomic distances (right-hand y axis) along the NEB reaction coordinate of the 2 -> 3 reaction. (c) Enlarged views of the Mn4Ca cluster and water (W3) for the intermediate and transition states. Figure 7. (a) Löwdin atomic spin densities and (b) relative energy (left-hand y axis) and key atomic distances (right-hand y axis) along the NEB reaction coordinate of the 3 -> 4 reaction. For the rapid energy change around the transition state (TS3,4), the region between 2nd and 6th NEB beads are more finely searched using 17 NEB beads. (c) Enlarged views of the Mn4Ca cluster, 3O2 and water molecules (W, W2-W5) for the intermediate and transition states. Figure 8. QM/MM optimized structures of the O2 moiety (1a:gray, 1b:pink, 2:cyan, 3:lime and 4:red) are superimposed onto the initial structure (1a: element color). PSII backbones (polypeptides D1, D2, CP43, PsbO, PsbU and PsbV) and QM region in state 4 are represented in ribbon and tube, 23 ACS Paragon Plus Environment


respectively. Two O2 exit channels 1 and 4, same as the water channels, connecting the areas near the OEC (near W2 and W4) and different lumenal surfaces are shown as green and orange color tubes, respectively. The channels’ positional data are obtained from ref. 23.


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Table 1. Relative energy contributions (kcal mol-1) in states.

a

∆E

∆H a

∆S b

∆ G = ∆ H – T∆ S c

1a

0

0

0

0

TS1a,1b

4.50

-0.86

-4.243

4.90

1b

2.97

-0.40

-0.834

2.81

TS1b,2

10.08

-1.72

0.023

8.35

2

7.59

-0.56

10.037

4.04

TS2,3

12.74

-1.52

2.584

10.44

3

-0.85

0.04

5.77

-2.53

TS3,4

7.48

-4.67

2.592

2.03

4

-10.47

-0.52

11.47

-14.41

Zero-point correction and thermal correction

b Entropy c

correction for the internal vibrations in cal mol-1K-1.

T = 298.15 K



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Table 2. Relative energies (∆E / kcal mol-1) calculated with the different basis sets (DZVP or TZVP) and the vdW energy correction (D3). States | method

DZVPa, Opt

TZVPb

TZVPb, D3c

TZVPb, D3c, Optd

1a

0

0

0

0

TS1a,1b

4.5

4.3

4.0

2.77

1b

3.0

3.5

3.4

2.82

TS

10.1

12.4

9.9

8.4

2

7.6

9.8

9.4

7.9

TS2,3

12.7

15.5

10.9

10.9

3

-0.8

1.9

-5.7

-4.9

7.5

10.2

2.4

1.6

-10.5

-9.0

-12.7

-12.5

1b,2

3,4

TS

4 a

DZVP: LANL-2DZ for Mn and Ca and 6-31G* for the other atoms.

b

TZVP: LANL-2TZ(f) for Mn, LANL08 for Ca and 6-311G** for other atoms.

c

D3: Grimme’s DFT-D3 dispersion correction.

d

Opt: Atoms in the QM regions are fully optimized. Results without the Opt are obtained using the

geometries at the DZVP theoretical level (first column).


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TOC Graphic


Concerted Mechanism of Water Insertion and O2 Release during the

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