Crystal Structure Of Photosystem II Research Articles

Composition Heterogeneity of Metal Ions Bound at the Oxygen-Evolving Center of Photosystem II in Living Cells.

Recent resolution advancement of in situ cryo-electron tomography (cryo-ET) and cryo-electron microscopy (cryo-EM) enables us to visualize large enzymes-in-action in atomic detail in their native environments inside living cells, such as photosystem II (PSII) and the ribosome. A variety of crystallographic and cryo-EM structures of PSII have been published for the purified PSII dimeric core complexes by itself, in supercomplexes with photosystem I (PSI) and light-harvesting complexes (LHC), and in megacomplexes with phycobilisome (PBS). In the latter case, two or five copies of asymmetric dimeric PSII molecules are present in highly asymmetric environments that differ from other 2-fold symmetric structures. Previous systematic analysis of X-ray free-electron laser (XFEL) crystal structures of PSII has shown different degrees of composition heterogeneity of metal ion cofactor bound at the oxygen-evolving center (OEC), including between two monomers of the same PSII dimer. This study analyzed the metal ions bound at four OECs in two asymmetric dimeric PSII molecules within in situ cryo-ET structures reported for an asymmetric PBS-PSII-PSI-LHC megacomplex determined in a living organism without purification and shows that composition heterogeneity with reduced metal ion occupancies at the OEC of PSII is a general phenomenon. This finding could have profound implications for spectroscopic interpretations of unpurified PSII samples.

Protonation structure of the closed-cubane conformation of the O2-evolving complex in photosystem II.

In photosystem II (PSII), one-electron oxidation of the most stable state of the oxygen-evolving Mn4CaO5 cluster (S1) leads to the S2 state formation, Mn1(III)Mn2(IV)Mn3(IV)Mn4(IV) (open-cubane S2) or Mn1(IV)Mn2(IV)Mn3(IV)Mn4(III) (closed-cubane S2). In electron paramagnetic resonance (EPR) spectroscopy, the g=4.1 signal is not observed in cyanobacterial PSII but in plant PSII, whereas the g=4.8 signal is observed in cyanobacterial PSII and extrinsic-subunit-depleted plant PSII. Here, we investigated the closed-cubane S2 conformation, a candidate for a higher spin configuration that accounts for g>4.1 EPR signal, considering all pairwise exchange couplings in the PSII protein environment (i.e. instead of considering only a single exchange coupling between the [Mn3(CaO4)] cubane region and the dangling Mn4 site). Only when a ligand water molecule that forms an H-bond with D1-Asp61 (W1) is deprotonated at dangling Mn4(IV), the g=4.1 EPR spectra can be reproduced using the cyanobacterial PSII crystal structure. The closed-cubane S2 is less stable than the open-cubane S2 in cyanobacterial PSII, which may explain why the g=4.1 EPR signal is absent in cyanobacterial PSII.

Glycerol binding at the narrow channel of photosystem II stabilizes the low-spin S2 state of the oxygen-evolving complex.

The oxygen-evolving complex (OEC) of photosystem II (PSII) cycles through redox intermediate states Si (i = 0-4) during the photochemical oxidation of water. The S2 state involves an equilibrium of two isomers including the low-spin S2 (LS-S2) state with its characteristic electron paramagnetic resonance (EPR) multiline signal centered at g = 2.0, and a high-spin S2 (HS-S2) state with its g = 4.1 EPR signal. The relative intensities of the two EPR signals change under experimental conditions that shift the HS-S2/LS-S2 state equilibrium. Here, we analyze the effect of glycerol on the relative stability of the LS-S2 and HS-S2 states when bound at the narrow channel of PSII, as reported in an X-ray crystal structure of cyanobacterial PSII. Our quantum mechanics/molecular mechanics (QM/MM) hybrid models of cyanobacterial PSII show that the glycerol molecule perturbs the hydrogen-bond network in the narrow channel, increasing the pKa of D1-Asp61 and stabilizing the LS-S2 state relative to the HS-S2 state. The reported results are consistent with the absence of the HS-S2 state EPR signal in native cyanobacterial PSII EPR spectra and suggest that the narrow water channel hydrogen-bond network regulates the relative stability of OEC catalytic intermediates during water oxidation.

PKa of the ligand water molecules in the oxygen-evolving Mn4CaO5 cluster in photosystem II

Release of the protons from the substrate water molecules is prerequisite for O2 evolution in photosystem II (PSII). Proton-releasing water molecules with low pKa values at the catalytic moiety can be the substrate water molecules. In some studies, one of the ligand water molecules, W2, is regarded as OH−. However, the PSII crystal structure shows neither proton acceptor nor proton-transfer pathway for W2, which is not consistent with the assumption of W2 = OH−. Here we report the pKa values of the four ligand water molecules, W1 and W2 at Mn4 and W3 and W4 at Ca2+, of the Mn4CaO5 cluster. pKa(W1) ≈ pKa(W2) << pKa(W3) ≈ pKa(W4) in the Mn4CaO5 cluster in water. However, pKa(W1) ≈ pKa(D1-Asp61) << pKa(W2) in the PSII protein environment. These results suggest that in PSII, deprotonation of W2 is energetically disfavored as far as W1 exists.

Chlorophyll a with a farnesyl tail in thermophilic cyanobacteria

Photosystem II (PSII) of oxygenic photosynthetic organisms normally contains exclusively chlorophyll a (Chl a) as its major light-harvesting pigment. Chl a canonically consists of the chlorin headgroup with a 20-carbon, 4-isoprene unit, phytyl tail. We have examined the 1.9Å crystal structure of PSII from thermophilic cyanobacteria reported by Shen and coworkers in 2012 (PDB accession of 3ARC/3WU2). A newly refined electron density map from this structure, presented here, reveals that some assignments of the cofactors may be different from those modeled in the 3ARC/3WU2 structure, including a specific Chl a that appears to have a truncated tail by one isoprene unit. We provide experimental evidence using high-performance liquid chromatography and mass spectrometry for a small population of Chl a esterified to a 15-carbon farnesyl tail in PSII of thermophilic cyanobacteria.

Origins of Water Molecules in the Photosystem II Crystal Structure.

The cyanobacterial photosystem II (PSII) crystal structure includes more than 1300 water molecules in each monomer unit; however, their precise roles in water oxidation are unclear. To understand the origins of water molecules in the PSII crystal structure, the accessibility of bulk water molecules to channel inner spaces in PSII was investigated using the water-removed PSII structure and molecular dynamics (MD) simulations. The inner space of the channel that proceeds toward the D1-Glu65/D2-Glu312 pair (E65/E312 channel) was entirely filled with water molecules from the bulk region. In the same channel, a diamond-shaped cluster of water molecules formed near redox-active TyrZ in MD simulations. Reorientation of the D2-Leu352 side chain resulted in formation of a hexagonal water network at the Cl-2 binding site. Water molecules could not enter the main region of the O4-water chain, which proceeds from the O4 site of the Mn4CaO5 cluster. However, in the O4-water chain, the two water binding sites that are most distant from the protein bulk surface were occupied by water molecules that approached along the E65/E312 channel, one of which formed an H-bond with the O4 site. These findings provide key insights into the significance of the channel ends, which may utilize water molecules during the PSII photocycle.

Mass spectrometry-based cross-linking study shows that the Psb28 protein binds to cytochrome b559 in Photosystem II

Photosystem II (PSII), a large pigment protein complex, undergoes rapid turnover under natural conditions. During assembly of PSII, oxidative damage to vulnerable assembly intermediate complexes must be prevented. Psb28, the only cytoplasmic extrinsic protein in PSII, protects the RC47 assembly intermediate of PSII and assists its efficient conversion into functional PSII. Its role is particularly important under stress conditions when PSII damage occurs frequently. Psb28 is not found, however, in any PSII crystal structure, and its structural location has remained unknown. In this study, we used chemical cross-linking combined with mass spectrometry to capture the transient interaction of Psb28 with PSII. We detected three cross-links between Psb28 and the α- and β-subunits of cytochrome b559, an essential component of the PSII reaction-center complex. These distance restraints enable us to position Psb28 on the cytosolic surface of PSII directly above cytochrome b559, in close proximity to the QB site. Protein-protein docking results also support Psb28 binding in this region. Determination of the Psb28 binding site and other biochemical evidence allow us to propose a mechanism by which Psb28 exerts its protective effect on the RC47 intermediate. This study also shows that isotope-encoded cross-linking with the "mass tags" selection criteria allows confident identification of more cross-linked peptides in PSII than has been previously reported. This approach thus holds promise to identify other transient protein-protein interactions in membrane protein complexes.

Two Different Structures of the Oxygen-Evolving Complex in the Same Polypeptide Frameworks of Photosystem II.

The oxygen-evolving complex (OEC) forms the heart of photosystem II (PSII) in photosynthesis. The crystal structure of PSII from Thermosynechococcus vulcanus has been reported at a resolution of 1.9 Å and at an averaged X-ray dose of 0.43 MGy. The OEC structure is suggested to be partially reduced to Mn(II) by EXAFS and DFT computational studies. Recently, the "radiation-damage-free" structures have been published at 1.95 Å resolution using XFEL, but reports continued to appear that the OEC is reduced to the S0-state of the Kok cycle. To elucidate much more precise structure of the OEC, in this study two structures were determined at extremely low X-ray doses of 0.03 and 0.12 MGy using conventional synchrotron radiation source. The results indicated that the X-ray reduction effects on the OEC were very small in the low dose region below 0.12 MGy, that is, a threshold existed for the OEC structural changes caused by X-ray exposure. The OEC structures of the two identical monomers in the crystal were clearly different under the threshold of the radiation dose, although the surrounding polypeptide frameworks of PSII were the same. The assumption that the OECs in the crystal were in the dark-stable S1-state of the Kok cycle should be re-evaluated.

Atomic Structure and Water-splitting Reaction Mechanism of Photosystem II Revealed by X-ray Free Electron Laser

Photosystem II (PSII) is a huge membrane-protein complex which catalyzes light-driven water-oxidation reaction at its catalytic center, the oxygen evolving complex (OEC). The crystal structure of PSII has been determined at 1.9 Å resolution using synchrotron radiation which revealed that the OEC is a Mn4CaO5 cluster. Some of the manganese atoms of the OEC are, however, rapidly reduced by X-ray irradiation which results in slight elongation of the distances between manganese cations. Furthermore, high-resolution 3D structural information is only limited to the dark-stable S1 state and the structures in the other intermediate states are missing. X-ray free electron laser (XFEL) has the potential to address these unsolved problems, and unveil the water-splitting reaction mechanism of PSII. In this review, we will introduce the analyses of the damage-free structure and an intermediate structure of PSII using XFEL, demonstrating the potential to obtain high spatial resolution of biological samples by XFEL.

The protonation state around TyrD/TyrD• in photosystem II is reflected in its biphasic oxidation kinetics

The tyrosine residue D2-Tyr160 (TyrD) in photosystem II (PSII) can be oxidized through charge equilibrium with the oxygen evolving complex in PSII. The kinetics of the electron transfer from TyrD has been followed using time-resolved EPR spectroscopy after triggering the oxidation of pre-reduced TyrD by a short laser flash. After its oxidation TyrD is observed as a neutral radical (TyrD•) indicating that the oxidation is coupled to a deprotonation event. The redox state of TyrD was reported to be determined by the two water positions identified in the crystal structure of PSII [Saito et al. (2013) Proc. Natl. Acad. Sci. USA 110, 7690]. To assess the mechanism of the proton coupled electron transfer of TyrD the oxidation kinetics has been followed in the presence of deuterated buffers, thereby resolving the kinetic isotope effect (KIE) of TyrD oxidation at different H/D concentrations. Two kinetic phases of TyrD oxidation – the fast phase (msec-sec time range) and the slow phase (tens of seconds time range) were resolved as was previously reported [Vass and Styring (1991) Biochemistry 30, 830]. In the presence of deuterated buffers the kinetics was significantly slower compared to normal buffers. Furthermore, although the kinetics were faster at both high pH and pD values the observed KIE was found to be similar (~2.4) over the whole pL range investigated. We assign the fast and slow oxidation phases to two populations of PSII centers with different water positions, proximal and distal respectively, and discuss possible deprotonation events in the vicinity of TyrD.

Novel Features of Eukaryotic Photosystem II Revealed by Its Crystal Structure Analysis from a Red Alga

Photosystem II (PSII) catalyzes light-induced water splitting, leading to the evolution of molecular oxygen indispensible for life on the earth. The crystal structure of PSII from cyanobacteria has been solved at an atomic level, but the structure of eukaryotic PSII has not been analyzed. Because eukaryotic PSII possesses additional subunits not found in cyanobacterial PSII, it is important to solve the structure of eukaryotic PSII to elucidate their detailed functions, as well as evolutionary relationships. Here we report the structure of PSII from a red alga Cyanidium caldarium at 2.76 Å resolution, which revealed the structure and interaction sites of PsbQ', a unique, fourth extrinsic protein required for stabilizing the oxygen-evolving complex in the lumenal surface of PSII. The PsbQ' subunit was found to be located underneath CP43 in the vicinity of PsbV, and its structure is characterized by a bundle of four up-down helices arranged in a similar way to those of cyanobacterial and higher plant PsbQ, although helices I and II of PsbQ' were kinked relative to its higher plant counterpart because of its interactions with CP43. Furthermore, two novel transmembrane helices were found in the red algal PSII that are not present in cyanobacterial PSII; one of these helices may correspond to PsbW found only in eukaryotic PSII. The present results represent the first crystal structure of PSII from eukaryotic oxygenic organisms, which were discussed in comparison with the structure of cyanobacterial PSII.

Critical Assessment of Protein Cross-Linking and Molecular Docking: An Updated Model for the Interaction Between Photosystem II and Psb27.

Photosystem II (PSII) is a large membrane-protein complex composed of about 20 subunits and various cofactors, which mediates the light-driven oxidation of water and reduction of plastoquinone, and is part of the photosynthetic electron transfer chain that is localized in the thylakoid membrane of cyanobacteria, algae, and plants. The stepwise assembly of PSII is guided and facilitated by numerous auxiliary proteins that play specific roles in this spatiotemporal process. Psb27, a small protein localized in the thylakoid lumen, appears to associate with an intermediate PSII complex that is involved in assembly of the Mn4CaO5 cluster. Its precise binding position on the PSII intermediate remains elusive, as previous approaches to the localization of Psb27 on PSII have yielded contradictory results. This was our motivation for a critical assessment of previously used methods and the development of an improved analysis pipeline. The combination of chemical cross-linking and mass spectrometry (CX-MS) with isotope-coded cross-linkers was refined and validated with reference to the PSII crystal structure. Psb27 was localized on the PSII surface adjacent to the large lumenal domain of CP43 on the basis of a cross-link connecting Psb27-K91 to CP43-K381. Additional contacts associating Psb27 with CP47 and the C-termini of D1 and D2 were detected by surface plasmon resonance (SPR) spectroscopy. This information was used to model the binding of Psb27 to the PSII surface in a region that is occupied by PsbV in the mature complex.

High-resolution and low-dose X-ray crystal structure of Photosytem II

Photosystem II (PSII) is a large multi-subunit membrane protein embedded in thylakoid membranes as a dimeric form with molecular weight of 700 kDa. The oxygen-evolving complex (OEC) of PSII is the heart of photosynthesis to split water molecules and to produce electrons and protons. The X-ray crystal structure of PSII was reported recently at a resolution of 1.9 Å with an averaged coordinate error (DPI) of 0.11 Å [1]. The chemical composition of OEC was fixed to as Mn4CaO5(H2O)4, and the structure was unambiguously determined for the first time including all amino-acid residues and oxo-bridging oxygen atoms ligated to the metal atoms.After the structure determination, two problems are newly showed up. One is the resolution problem. The resolution of 1.9 Å is extremely high in comparison with that of crystal structure previously reported [2], however, it is not enough to obtain precise information for bond lengths between metals and oxo-bridging oxygen atoms in OEC. The other is the X-ray reduction problem on Mn atoms. The reflection intensities were measured by a slide-oscillation method at a low X-ray dose of 0.85 MGy. According to the EXAFS studies reported by Glöckner et al. [3], the dose value corresponded to that 25% of OEC in crystal was damaged into Mn(II) aqua complexes. In order to overcome these problems, we have succeeded to get high quality crystals of PSII, which show much higher resolution and isomorphism. The high isomorphism is very important to obtain low-dose data using multiple crystals. Sixteen partial datasets were reduced using XDS and merged to a resolution of 1.77 Å. The calculated X-ray dose was 0.11 MGy, only 13 % of that for the 1.9 Å resolution data. Based on the merged data, structure determination is underway.

Reprint of PSII Manganese Cluster: Protonation of W2, O5, O4 and His337 in the S1 state explored by combined quantum chemical and electrostatic energy computations

Photosystem II (PSII) is a membrane-bound protein complex that oxidizes water to produce energized protons, which are used to built up a proton gradient across the thylakoidal membrane in the leafs of plants. This light-driven reaction is catalyzed by withdrawing electrons from the Mn4CaO5-cluster (Mn-cluster) in four discrete oxidation steps [S1−(S4/S0)] characterized in the Kok-cycle. In order to understand in detail the proton release events and the subsequent translocation of such energized protons, the protonation pattern of the Mn-cluster need to be elucidated. The new high-resolution PSII crystal structure from Umena, Kawakami, Shen, and Kamiya is an excellent basis to make progress in solving this problem. Following our previous work on oxidation and protonation states of the Mn-cluster, in this work, quantum chemical/electrostatic calculations were performed in order to estimate the pKa of different protons of relevant groups and atoms of the Mn-cluster such as W2, O4, O5 and His337. In broad agreement with previous experimental and theoretical work, our data suggest that W2 and His337 are likely to be in hydroxyl and neutral form, respectively, O5 and O4 to be unprotonated. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.

PSII manganese cluster: Protonation of W2, O5, O4 and His337 in the S1 state explored by combined quantum chemical and electrostatic energy computations

Photosystem II (PSII) is a membrane-bound protein complex that oxidizes water to produce energized protons, which are used to built up a proton gradient across the thylakoidal membrane in the leafs of plants. This light-driven reaction is catalyzed by withdrawing electrons from the Mn4CaO5-cluster (Mn-cluster) in four discrete oxidation steps [S1−(S4/S0)] characterized in the Kok-cycle. In order to understand in detail the proton release events and the subsequent translocation of such energized protons, the protonation pattern of the Mn-cluster need to be elucidated. The new high-resolution PSII crystal structure from Umena, Kawakami, Shen, and Kamiya is an excellent basis to make progress in solving this problem. Following our previous work on oxidation and protonation states of the Mn-cluster, in this work, quantum chemical/electrostatic calculations were performed in order to estimate the pKa of different protons of relevant groups and atoms of the Mn-cluster such as W2, O4, O5 and His337. In broad agreement with previous experimental and theoretical work, our data suggest that W2 and His337 are likely to be in hydroxyl and neutral form, respectively, O5 and O4 to be unprotonated. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.

Mass spectroscopy locates the extrinsic proteins of photosystem II

The photosynthetic oxidation of water is catalyzed by photosystem II (PSII), a multisubunit pigment-protein embedded in the thylakoid membranes of cyanobacteria, algae, and plants. This remarkable photoenzyme is capable of generating the highly oxidizing chemical species required to extract four tightly bound electrons of two substrate water-molecules, yielding biosynthetically useful reductant and by-product O2. PSII is a large homodimeric complex in vivo, with a combined mass of ∼700 kDa. Each PSII monomer comprises more than 20 different proteins collectively coordinating ∼60 cofactors, including 35 chlorophylls, 2 pheophytins, 2 plastoquinone molecules, and the Mn4Ca cluster, responsible for H2O-oxidation (1, 2). Despite much progress, including the 1.9 A crystal structure of cyanobacterial PSII (1, 3), crucial structural information is missing. This lack includes extrinsic proteins affecting the Mn4Ca cluster, not observed in the PSII crystal structure because they are lost during the purification and crystallization process. In PNAS, Liu et al. use complementary technical approaches to construct a model for the binding of one of the lost extrinsic proteins, PsbQ, to the PSII, complex, and in doing so provide an example for the solution of the more general structural problem of defining the 3D structure of separately crystallized proteins that assemble into larger macromolecular complexes (4).

MS-based cross-linking analysis reveals the location of the PsbQ protein in cyanobacterial photosystem II

PsbQ is a luminal extrinsic protein component that regulates the water splitting activity of photosystem II (PSII) in plants, algae, and cyanobacteria. However, PsbQ is not observed in the currently available crystal structures of PSII from thermophilic cyanobacteria. The structural location of PsbQ within the PSII complex has therefore remained unknown. Here, we report chemical cross-linking followed by immunodetection and liquid chromatography/tandem MS analysis of a dimeric PSII complex isolated from the model cyanobacterium, Synechocystis sp. PCC 6803, to determine the binding site of PsbQ within PSII. Our results demonstrate that PsbQ is closely associated with the PsbO and CP47 proteins, as revealed by cross-links detected between (120)K of PsbQ and (180)K and (59)K of PsbO, and between (102)K of PsbQ and (440)D of CP47. We further show that genetic deletion of the psbO gene results in the complete absence of PsbQ in PSII complexes as well as the loss of the dimeric form of PSII. Overall, our data provide a molecular-level description of the enigmatic binding site of PsbQ in PSII in a cyanobacterium. These results also help us understand the sequential incorporation of the PsbQ protein during the PSII assembly process, as well as its stabilizing effect on the oxygen evolution activity of PSII.

Preliminary X-ray diffraction study of crystals of photosystem II from Thermosynechococcus elongates

Photosystem II (PSII) is a multicomponent enzyme complex that catalyzes the light-induced water splitting to molecular oxygen, protons, and electrons. Photosystem II is located in the membranes of cyanobacteria, green algae, and plants. The crystallization of this complex from the thylakoid membranes poses great difficulties. The high sensitivity of photosystem II to light and radiation has an adverse effect on the crystal quality, as well as on the quality of X-ray diffraction data. This is the reason why the crystal structure of PSII from Thermosynechococcus elongates has as yet not been determined at high resolution. The optimization of the strategy for collecting X-ray diffraction data from PSII crystals has resulted in an increase in the resolution to 2.75 A, which made it possible to determine the positions of ions and some water molecules playing an important role in the functioning of PSII.

Assignment of the μ4-O5 atom in catalytic center for water oxidation in photosystem II

The detailed structure of catalytic center of water oxidation, Mn4Ca-cluster, in photosystem II (PSII) has been reported recently. However, due to the radiation damage induced by X-ray and the complexity of the Mn4Ca-cluster, the assignment of the μ4-O5 atom coordinated by three Mn and one Ca2+ ions is still lack of essential evidences. In this article, we synthesized one Mn complex containing two μ4-O atoms. It is found that the lengths of all μ4-O-Mn bonds in this Mn complex are in the range of 1.89–2.10 Å, which are significantly shorter than 2.40–2.61 Å distance of μ4-O5-Mn bonds in Mn4Ca-cluster observed in the crystal structure of PSII. In addition, DFT calculations have been carried out on the Mn4Ca-cluster. It is found that the O atom of μ4-O or μ4-OH always trends to deviate from the center position of four metal ions, resulting in unequal bond lengths of four μ4-O-M (M=Mn or Ca), which is obviously different with larger and nearly equal distances between μ4-O and four metal ions observed in the crystal structure. Based on these results, we suggest that the μ4-atom in Mn4Ca-cluster of PSII is unlikely to be a μ4-O, μ4-OH or μ4-OH2, and its assignment is still an open question.

Detection of the Water-Binding Sites of the Oxygen-Evolving Complex of Photosystem II Using W-Band17O Electron–Electron Double Resonance-Detected NMR Spectroscopy

Water binding to the Mn(4)O(5)Ca cluster of the oxygen-evolving complex (OEC) of Photosystem II (PSII) poised in the S(2) state was studied via H(2)(17)O- and (2)H(2)O-labeling and high-field electron paramagnetic resonance (EPR) spectroscopy. Hyperfine couplings of coordinating (17)O (I = 5/2) nuclei were detected using W-band (94 GHz) electron-electron double resonance (ELDOR) detected NMR and Davies/Mims electron-nuclear double resonance (ENDOR) techniques. Universal (15)N (I = ½) labeling was employed to clearly discriminate the (17)O hyperfine couplings that overlap with (14)N (I = 1) signals from the D1-His332 ligand of the OEC (Stich Biochemistry 2011, 50 (34), 7390-7404). Three classes of (17)O nuclei were identified: (i) one μ-oxo bridge; (ii) a terminal Mn-OH/OH(2) ligand; and (iii) Mn/Ca-H(2)O ligand(s). These assignments are based on (17)O model complex data, on comparison to the recent 1.9 Å resolution PSII crystal structure (Umena Nature 2011, 473, 55-60), on NH(3) perturbation of the (17)O signal envelope and density functional theory calculations. The relative orientation of the putative (17)O μ-oxo bridge hyperfine tensor to the (14)N((15)N) hyperfine tensor of the D1-His332 ligand suggests that the exchangeable μ-oxo bridge links the outer Mn to the Mn(3)O(3)Ca open-cuboidal unit (O4 and O5 in the Umena et al. structure). Comparison to literature data favors the Ca-linked O5 oxygen over the alternative assignment to O4. All (17)O signals were seen even after very short (≤15 s) incubations in H(2)(17)O suggesting that all exchange sites identified could represent bound substrate in the S(1) state including the μ-oxo bridge. (1)H/(2)H (I = ½, 1) ENDOR data performed at Q- (34 GHz) and W-bands complement the above findings. The relatively small (1)H/(2)H couplings observed require that all the μ-oxo bridges of the Mn(4)O(5)Ca cluster are deprotonated in the S(2) state. Together, these results further limit the possible substrate water-binding sites and modes within the OEC. This information restricts the number of possible reaction pathways for O-O bond formation, supporting an oxo/oxyl coupling mechanism in S(4).