8 15 where is my water

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Close and continue browsing. Niagara Parks is committed to protecting the health and safety of all guests and staff. Face coverings are required at all indoor spaces and attractions within Niagara Parks, and proof of vaccination is required at some locations. The main change is the tilting of the histidine side chain and the backbone, providing the driving force for the D1-E to move away from Ca While the motion of E, ligated to Mn1 and located close to the O1 channel, changes its position, there is not enough space for the direct insertion of water from the O1 channel to occur.

An alternative route for the Ox insertion is via W3, which is a ligand of Ca Ugur et al. If W3 is the entrance of substrate water to the Ox from the O1 channel, it likely needs to come via W4 first, which is refilled from the water wheel-like ring penta cluster of W26, 27, 28, 29, and 30 Fig. In this case, Ca likely plays a pivotal role to shuffle water from W4, W3, and then to Ox.

The gray arrow represents the possible proton pathway, while the blue dashed arrow represents the potential stepwise water insertion pathway. This drop of the electron density is likely related to the loss or weakening of the H-bond to Yz due to Yz oxidation, thereby weakening the H-bond between W25 and the side chain of E, possibly priming it for replacing W3. The Cl1 and O4 channels as well as the Yz network, have been proposed in the literature as a proton release pathway 32 , 34 , 39 , 40 , 54 , 55 , The current structural study provides several indications of the Cl1 channel being the proton exit pathway in the S 2 to S 3 transition.

Below we discuss the possibility of proton transfer for all three pathways and provide support for the hypothesis of the Cl1 channel being the proton pathway in the S 2 to S 3 transition. Theoretical studies suggested that the water chain in the O4 channel provides a downhill proton transfer 39 , In the S 1 to S 2 transition Fig.

The dislodging of W20 disconnects the hydrogen-bonding network of the O4 channel from the OEC in the S 2 -state, and therefore it is unlikely that the O4 channel can serve as a proton release pathway during the S 2 to S 3 transition.

This channel has been proposed as a proton release channel during the S 0 to S 1 transition 36 , 39 , 57 , In the RT structural data, however, one water W , which could be essential for proton transfer via this network Supplementary Fig. If this water does not exist or is highly mobile, the Yz network would require a proton transfer through an asparagine D1-N 27 , 34 residue that is not generally considered suitable for proton relay.

The Yz network ending at the lumen surface residue PsbV-K is also rich in other asparagine residues D1-N , and these require tautomerization or amide rotations to allow proton transferr through them An alternative proton pathway that involves waters would require an interaction between W57 and W These two waters, however, are separated by 6.

Therefore, we concluded that the Yz network, based on the RT structural data, is unlikely to be a proton release pathway. The Cl1 channel has been proposed as a proton release pathway during the S 2 to S 3 transition in many studies in the literature 26 , 54 , 55 , 61 , and our current structural observation prefers this assignment.

We hypothesize this motion might be triggered by the excess positive charge after Yz oxidation, and slower protonation and H-bonding rearrangements, and is related to the opening of the channel to proton transfer and release as illustrated in Fig. The proton transfer from the protonated D1-D61 to a deprotonated D1-E65 or E was recently reported to be exothermic via one or two waters This conformation is stabilized by a newly formed H-bond between E65 and R, preventing the released proton to return.

With the current data, however, we cannot conclude when exactly the proton is released to the bulk from the Cl1 channel. Alternatively, the gate may stay open until the proton is released in an apparent single proton hopping event occurring with Mn oxidation In any case, the gate opening and closing can directly explain the involvement of multiple protonatable side chains as observed in pH-dependent oxygen activity 63 and the reported pH dependency of the proton-coupled electron transfer PCET of Mn oxidation in the S 2 to S 3 transition 56 , 64 : In the open state Fig.

This will slow down the deprotonation of D61 and also the deprotonation of the newly inserted substrate water Ox. In Fig. Upon the S 2 -state formation Figs. This redox change may trigger the structural changes around the area; the WO4 distance is shortened, likely due to equal sharing of the proton between them, leading to the weakening of the hydrogen-bond between W19 and W The W20 density is not visible in the S 2 -state, only reappearing in the S 0 -state.

One plausible explanation for this motion is that W1 releases a proton via D1-D61 and becomes an OH — , that interacts with S Also, the role of D1-E65 and D1-D61 in the proton release has been widely discussed 20 , 27 , 35 , 46 , 54 , 55 , 59 , 66 , 67 , 68 , Since the water is easier to be deprotonated when bound to a metal-like Mn or Ca, we propose water is first bound to Ca or Mn prior to being inserted at the open coordination site of Mn1, possibly via W3.

Therefore, the reprotonation of W1 can proceed from the newly inserted Ox. In summary, we investigated the water and proton channels that connect the OEC to the lumenal bulk water. Based on the RT structures, we propose that the O1 channel with mobile waters is suitable for water intake from the bulk to the OEC, while the more rigid network of the Cl1 channel branch A, formed with amino acids and waters extending through W1, D1-D61, to D1-E65, is suitable for proton relay during the S 2 to S 3 transition.

Based on the observed structural changes, we hypothesize that D1-E65, D2-E, and D1-R form a proton gate by minimizing the back reaction, thus regulating proton release from the OEC to the bulk. We also note that different proton release pathways may be used during different S-state transitions. PS II crystals are highly active in O 2 evolution, show no Mn II contamination 72 , and turnover parameters and S-state populations under our experimental conditions were determined by membrane inlet mass spectroscopy 73 as described previously The O1 channel was mapped by Caver 3.

The channels start near the Ca side and extend through the D1 subunit Supplementary Figure 1. The sample was delivered to the X-ray interaction region using the previously described Drop-on-Tape setup Illumination conditions for populating different S-states are described in the following references 12 , From the data collected at LCLS for different illumination states, as described previously 71 , a total of , integrated lattices were obtained using psana 78 to read the images and dials.

Signal was integrated to the edges of the detector and subsequently, a per-image resolution cutoff was used during the merging step. Integrated intensities were corrected for absorption by the kapton conveyor belt to match the position of the belt and crystals relative to the X-ray beam Ensemble refinement of the crystal and detector parameters was then performed on the data using cctbx. The remaining integrated images were merged using cxi.

A combined dataset at 1. This included all the stable intermediate states of the Kok cycle S 1 , S 2 , S 3 , and S 0 as well as the timepoints between those intermediates. In total, , images were merged to yield a combined dataset cut at 1. The R merge for the dataset is This combined mtz file was used to generate a refined pdb model of the combined dataset as described below, and this pdb model was used as the reference model for merging the separate illumination states. The unit cell outlier rejection option in cxi.

Please note that the merged datasets for the individual illumination conditions are the same as those used in ref. Initial structure refinement against this combined dataset at 1. The R free set was 0.

As a result, the R free reflections for the combined dataset contain all the R free reflections used for the individual datasets. B-factors were reset to a value of 30 and waters were removed. After an initial rigid body refinement step, xyz coordinates and isotropic B-factors were refined for tens of cycles with automatic water-placement enabled.

Custom coordination restraints overrode van der Waals repulsion for coordinated chlorophyll Mg atoms, the non-heme iron, and the OEC. Following real-space refinement in Coot 82 of selected individual sidechains and the PsbO loop region and placement of additional water molecules, the model was refined for several additional cycles with occupancy refinement enabled, then as before without automatic water-placement, and then as before with hydrogen atoms.

NHQ flips and automatic linking were disabled throughout. In the final steps of refinement, Phenix-Auto-water-placement was used to model the waters. The water positions were manually inspected in COOT. The waters positioned within the discussed channels were moved to a different chain and renamed OOO. With reset B-factors to 30 and removed waters, the above model was subsequently refined against the illuminated datasets with the lattermost refinement settings and different OEC bonding restraints.

Using the 1. Bonding restraints for the other datasets loosely restrained the models to metal-metal distances matching spectroscopic data and metal-oxygen distances matching the most likely proposed models 83 , 84 , 85 , 86 , A number of ordered water positions were excluded from subsequent automatic water-placement rounds by renaming the residue names to OOO and the waters coordinating the OEC were incorporated into the OEC restraint CIF file directly. The main conformer was set at 0.

Depending on the resolution and the fraction of S 3 present, metal-metal distances at the OEC had standard deviations between 0. To ensure the reliability of the modeling of waters in the channels, individual polder omit maps were generated, using phenix. To investigate the B-factors of waters in the 1. To investigate the influence of occupancy on the refinement, a parallel refinement in which both occupancy and B-factors were allowed to refine was performed.

Both strategies showed similar trends Data not shown. Similar strategies were used to track the changes in the water B-factors for the different time-points models. Since we are comparing the B factors from different models at different resolutions, it was necessary to standardize them. First, the water B-factors were extracted from each model. Then, the normalized B-factors norm B of each model were calculated by applying a Z-score Eq.

To estimate the peak-height of water omit densities, changes in the electron density at the water W i position were obtained from the omit maps of W i using the FFT program from the CCP4 package 90 , We utilized a water numbering scheme for all waters in the vicinity of the OEC that is consistent with the numbering used in refs. Waters are numbered with increasing numbers indicative of their distance to the OEC along the channels starting with W As the PDB does not allow to retain identifiers for waters the numbering in each deposited coordinate file is different.

Hence, we are providing a table Supplementary Table 3 that correlates the numbering used in this work with the numbering in each of the deposited coordinate files.

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