Energetics of proton release on the first oxidation step in the water-oxidizing enzyme

In photosystem II (PSII), the Mn4CaO5 cluster catalyses the water splitting reaction. The crystal structure of PSII shows the presence of a hydrogen-bonded water molecule directly linked to O4. Here we show the detailed properties of the H-bonds associated with the Mn4CaO5 cluster using a quantum mechanical/molecular mechanical approach. When O4 is taken as a μ-hydroxo bridge acting as a hydrogen-bond donor to water539 (W539), the S0 redox state best describes the unusually short O4–OW539 distance (2.5 Å) seen in the crystal structure. We find that in S1, O4 easily releases the proton into a chain of eight strongly hydrogen-bonded water molecules. The corresponding hydrogen-bond network is absent for O5 in S1. The present study suggests that the O4-water chain could facilitate the initial deprotonation event in PSII. This unexpected insight is likely to be of real relevance to mechanistic models for water oxidation.

QM region = small, [the Mn 4 CaO 5 cluster (including the ligands), water molecules shown in Figure 1, and the side chain of CP43-Thr335]. III  III  III  III  III  III   Mn2  IV  IV  IV  IV  IV  III   Mn3  IV  III  III  IV  III  IV   Mn4  III  III  II  III  III  II   O4-

Absence of an H-bond of a putative OHat O5 in the PSII protein environment.
It was suggested that a strong H-bond results in a more downfield 1 Figure S7a in Ref. 8 ). In contrast, they have also reported that they were able to generate an O5-O W2 H-bond using "a small model of the oxidized S 0 ' model" (not "the QM/MM S 0 model", see Figure S7b in Ref. 8 (Figure 7), far from linearity. We also calculated the  H values for OHat O5 and found them to be 4.7 (H 2 O at W2) and 6.1 (OHat W2) ppm (Figure 7), values that are lower than those seen even with weak H-bonds. Thus, it is clear that the hydroxyl O5 has no H-bond partner in the PSII protein environment in the lower S states investigated in the present study, regardless of the W2 protonation state. These results are a further demonstration that characteristics of H-bonds cannot be defined by the distances alone 10 .

Proton release from O5 and a water molecule near Cl -(W446).
One might possibly assume that H 3 O + can be stabilized at W446 due to the presence of Cl -. However, and (III, III, III, III) for the 1.9-Å structure 12 . They also proposed that a single proton relocation between W2 and D1-His337 triggered the change between (III, III, III, III) and (III, IV, III, II) based on the difference in the Mn 4 Ca geometries for the 1.9 Å 9 and 2.9 Å 11 crystal structures 12 . However, recent theoretical studies by Kurashige et al. 13 reported that the Mn oxidation state (III, IV, III, II) is the most relevant state for the 1.9-Å structure 13 . The calculations reported in the present work also find this valence distribution for the S -1 state (Table 1). Thus, it is also possible to have (III, IV, III, II) in the 1.9-Å structure, without altering the protonation state of W2 and D1-His337, and therefore the single proton relocation proposed by Gatt et al 11 is not a unique condition required for switching between the two Mn redox distributions. For further discussions, see Ref. 14 and references therein.
Requirements for long-distance sequential PT. In the O4-water chain, each pair of water molecules is H-bonded with a O water -O water distance of < ~2.8 Å (Table 1). This strongly-coupled chain of water molecules is expected to facilitate the release of protons in a sequential way 15,16 . When O4 is protonated Another possibility might be the involvement of more water molecules that are not seen in the 1.9-Å structure. It is likely that almost all stable water molecules, in particular those involved in H-bond networks are clarified at the resolution of 1.9 Å. On the other hand, it seems likely that water molecules exist that are not seen in the crystal structure irrespective of the resolution simply because they are too mobile. The possible presence of such water molecules may compensate for the slightly long H-bond distance of 3.57 Å between D1-Asn338 and D2-Asn350 and promote the PT.
Mn 4 CaO 5 model. The ground state of the Mn 4 Ca cluster in the S 1 state is considered to be either a singlet or quintet in EPR studies [18][19][20] . On the other hand, as considered in previous theoretical studies (e.g., 21,22 ), the cluster was considered to be in the S 1 state with ferromagnetically coupled Mn atoms; the total spin S = 7 and the resulting Mn oxidation state (Mn1, Mn2, Mn3, M4) = (III, IV, IV, III). The resulting optimized Mn 4 CaO 5 geometry appears not to be crucial to the spin configurations "within the same Mn redox distribution", as demonstrated in previous theoretical studies 21,22 . It is of note that the two S 2 conformers suggested in Refs. [23][24][25] to explain the two spin states seen by EPR 26

Supplementary Methods
The 1 H NMR chemical shifts ( H ) were calculated by using the GIAOs method 27 implemented in the Qsite 28 program. The absolute shielding constant of 1 H of tetramethylsilane (TMS) was calculated to be 31.6 ppm on the basis of the atomic coordinates in Ref. 29 and used as the TMS reference for  H .