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During photosynthetic water oxidation, the Mn4Ca cluster in Photosystem II progresses through five intermediate S-i (i = 0-4) states. X-ray crystallography studies have reported the insertion of one new O ligand during the formation of the S-3 state, but recent studies question the presence of this additional ligand based on cryo-EM and earlier room-temperature crystallography data. There is also controversy about whether the O-O bond interaction already occurs in the S-3 state or in the subsequent S-3 to S-0 transition. Here we report conventional high-resolution data for the S-1, S-2, and S-3 states to a resolution of similar to 1.9 & Aring;, and anomalous diffraction data at two energies (9.5 keV and 7 keV), that was used to model the Mn positions, followed by determination of oxygen positions using the high-resolution maps. We show that the new oxygen atom, O-X (or O6), in the S-3 state is observable as a distinct peak without any restraints, confirming its ligation to Mn1 and Ca. The O-X-O5 distance is similar to 2.1 & Aring;, supporting no strong interaction between them in the S-3 state, suggesting that if this is the O-O bond formation site, it is formed during the S-3 to S-0 transition initiated by the final oxidation of the cluster.

Photosystem II (PS II) is a large membrane-bound protein complex that catalyses light-driven water oxidation in plants and cyanobacteria. The structure of PS II is well studied in cyanobacteria; however, there are very few PS II structures from plants. The currently available plant PS II structures are comparatively low resolution and are frequently incomplete, that is, missing subunits or cofactors.

We optimized the procedure for isolating PS II from Arabidopsis thaliana and employed cryo-electron microscopy to generate a high-resolution structure of an intact and oxygen-evolving PS II from Arabidopsis thaliana at 2.44 Å resolution, which to date represents the highest resolution structure of PS II from higher plants.

At this resolution, many water molecules within the PS II structure can be detected, including waters around the water-splitting manganese cluster, the nonheme iron, and within the water/proton channels connecting these active sites to the protein exterior, allowing for the first detailed description of the water networks in Arabidopsis thaliana and comparison with the highly resolved cyanobacterial PS II.

Our findings further the understanding of design principles of protein-water-cofactor interactions in photosynthetic water splitting, quinone reduction/exchange, and about the role of lipids at the interface between PS II and the light-harvesting proteins.

Light-driven water oxidation by photosystem II sustains life on Earth by providing the electrons and protons for the reduction of CO₂ to carbohydrates and the molecular oxygen we breathe. The inorganic core of the oxygen evolving complex is made of the earth-abundant elements manganese, calcium and oxygen (Mn₄CaO₅ cluster), and is situated in a binding pocket that is connected to the aqueous surrounding via water-filled channels that allow water intake and proton egress. Recent serial crystallography and infrared spectroscopy studies performed with PSII isolated from Thermosynechococcus vestitus (T. vestitus) support that one of these channels, the O1 channel, facilitates water access to the Mn₄CaO₅ cluster during its S₂→S₃ and S₃→S₄→S₀ state transitions, while a subsequent CryoEM study concluded that this channel is blocked in the cyanobacterium Synechocystis sp. PCC 6803, questioning the role of the O1 channel in water delivery. Employing site-directed mutagenesis we modified the two O1 channel bottleneck residues D1-E329 and CP43-V410 (T. vestitus numbering) and probed water access and substrate exchange via time resolved membrane inlet mass spectrometry. Our data demonstrates that water reaches the Mn₄CaO₅ cluster via the O1 channel in both wildtype and mutant PSII. In addition, the detailed analysis provides functional insight into the intricate protein-water-cofactor network near the Mn₄CaO₅ cluster that includes the pentameric, near planar ‘water wheel’ of the O1 channel.

X-ray absorption spectroscopy (XAS) of 3d transition metals provides important electronic structure information for many fields. However, X-ray-induced radiation damage under physiological temperature has prevented using this method to study dilute aqueous systems, such as metalloenzymes, as the catalytic reaction proceeds. Here we present a new approach to enable operando XAS of dilute biological samples and demonstrate its feasibility with K-edge XAS spectra from the Mn cluster in photosystem II and the Fe–S centers in photosystem I. This approach combines highly efficient sample delivery strategies and a robust signal normalization method with high-transmission Bragg diffraction-based spectrometers at X-ray free-electron lasers (XFELs) in a damage-free, shot-by-shot mode. These photon-out spectrometers have been optimized for discriminating the metal Mn/Fe Kα fluorescence signals from the overwhelming scattering background present on currently available detectors for XFELs that lack suitable energy discrimination. We quantify the enhanced performance metrics of the spectrometer and discuss its potential applications for acquiring time-resolved XAS spectra of biological samples during their reactions at XFELs.

Identifying the two substrate water sites of nature’s water-splitting cofactor (Mn₄CaO₅ cluster) provides important information toward resolving the mechanism of O-O bond formation in Photosystem II (PSII). To this end, we have performed parallel substrate water exchange experiments in the S₁ state of native Ca-PSII and biosynthetically substituted Sr-PSII employing Time-Resolved Membrane Inlet Mass Spectrometry (TR-MIMS) and a Time-Resolved ¹⁷O-Electron-electron Double resonance detected NMR (TR-¹⁷O-EDNMR) approach. TR-MIMS resolves the kinetics for incorporation of the oxygen-isotope label into the substrate sites after addition of H₂¹⁸O to the medium, while the magnetic resonance technique allows, in principle, the characterization of all exchangeable oxygen ligands of the Mn₄CaO₅ cofactor after mixing with H₂¹⁷O. This unique combination shows i) that the central oxygen bridge (O5) of Ca-PSII core complexes isolated from Thermosynechococcus vestitus has, within experimental conditions, the same rate of exchange as the slowly exchanging substrate water (W_S) in the TR-MIMS experiments and ii) that the exchange rates of O5 and W_S are both enhanced by Ca²⁺→Sr²⁺ substitution in a similar manner. In the context of previous TR-MIMS results, this shows that only O5 fulfills all criteria for being W_S. This strongly restricts options for the mechanism of water oxidation.

The Mn₄CaO₅₍₆₎ cluster in photosystem II catalyzes water splitting through the S_i state cycle (i = 0-4). Molecular O₂ is formed and the natural catalyst is reset during the final S₃ → (S₄) → S₀ transition. Only recently experimental breakthroughs have emerged for this transition but without explicit information on the S₀-state reconstitution, thus the progression after O₂ release remains elusive. In this report, our molecular dynamics simulations combined with density functional calculations suggest a likely missing link for closing the cycle, i.e., restoring the first catalytic state. Specifically, the formation of closed-cubane intermediates with all hexa-coordinate Mn is observed, which would undergo proton release, water dissociation, and ligand transfer to produce the open-cubane structure of the S₀ state. Thereby, we theoretically identify the previously unknown structural isomerism in the S₀ state that acts as the origin of the proposed structural flexibility prevailing in the cycle, which may be functionally important for nature's water oxidation catalysis.

Photosystem II starts the photosynthetic electron transport chain that converts solar energy into chemical energy and thus sustains life on Earth. It catalyzes two chemical reactions: water oxidation to molecular oxygen and plastoquinone reduction. Coupling of electron and proton transfer is crucial for efficiency; however, the molecular basis of these processes remains speculative owing to uncertain water binding sites and the lack of experimentally determined hydrogen positions. We thus collected high-resolution cryo-electron microscopy data of fully hydrated photosystem II from the thermophilic cyanobacterium Thermosynechococcus vestitus to a final resolution of 1.71 angstroms. The structure reveals several previously undetected partially occupied water binding sites and more than half of the hydrogen and proton positions. This clarifies the pathways of substrate water binding and plastoquinone B protonation.

This chapter compares two different techniques for monitoring photosynthetic O₂ production; the wide-spread Clark-type O₂ electrode and the more sophisticated membrane inlet mass spectrometry (MIMS) technique. We describe how a simple membrane inlet for MIMS can be made out of a commercial Clark-type cell and outline the advantages and drawbacks of the two techniques to guide researchers in deciding which method to use. Protocols and examples are given for measuring O₂ evolution rates and for determining the number of chlorophyll molecules per active photosystem II reaction center.

Water oxidation by photosystem II (PSII) sustains most life on Earth, but the molecular mechanism of this unique process remains controversial. The ongoing identification of the binding sites and modes of the two water-derived substrate oxygens (‘substrate waters’) in the various intermediates (Si states, i = 0, 1, 2, 3, 4) that the water-splitting tetra-manganese calcium penta-oxygen (Mn4CaO5) cluster attains during the reaction cycle provides central information towards resolving the unique chemistry of biological water oxidation. Mass spectrometric measurements of single- and double-labeled dioxygen species after various incubation times of PSII with H218O provide insight into the substrate binding modes and sites via determination of exchange rates. Such experiments have revealed that the two substrate waters exchange with different rates that vary independently with the Si state and are hence referred to as the fast (Wf) and the slow (WS) substrate waters. New insight for the molecular interpretation of these rates arises from our recent finding that in the S2 state, under special experimental conditions, two different rates of WS exchange are observed that appear to correlate with the high spin and low spin conformations of the Mn4CaO5 cluster. Here, we reexamine and unite various proposed methods for extracting and assigning rate constants from this recent data set. The analysis results in a molecular model for substrate-water binding and exchange that reconciles the expected non-exchangeability of the central oxo bridge O5 when located between two Mn(IV) ions with the experimental and theoretical assignment of O5 as WS in all S states. The analysis also excludes other published proposals for explaining the water exchange kinetics.