Going around the Kok cycle of the water oxidation reaction with femtosecond X-ray crystallography.

The water oxidation reaction in photosystem II (PS II) produces most of the molecular oxygen in the atmosphere, which sustains life on Earth, and in this process releases four electrons and four protons that drive the downstream process of CO2 fixation in the photosynthetic apparatus. The catalytic center of PS II is an oxygen-bridged Mn4Ca complex (Mn4CaO5) which is progressively oxidized upon the absorption of light by the chlorophyll of the PS II reaction center, and the accumulation of four oxidative equivalents in the catalytic center results in the oxidation of two waters to dioxygen in the last step. The recent emergence of X-ray free-electron lasers (XFELs) with intense femtosecond X-ray pulses has opened up opportunities to visualize this reaction in PS II as it proceeds through the catalytic cycle. In this review, we summarize our recent studies of the catalytic reaction in PS II by following the structural changes along the reaction pathway via room-temperature X-ray crystallography using XFELs. The evolution of the electron density changes at the Mn complex reveals notable structural changes, including the insertion of OX from a new water molecule, which disappears on completion of the reaction, implicating it in the O-O bond formation reaction. We were also able to follow the structural dynamics of the protein coordinating with the catalytic complex and of channels within the protein that are important for substrate and product transport, revealing well orchestrated conformational changes in response to the electronic changes at the Mn4Ca cluster.

Evolutionary diversity of proton and water channels on the oxidizing side of photosystem II and their relevance to function.

One of the reasons for the high efficiency and selectivity of biological catalysts arise from their ability to control the pathways of substrates and products using protein channels, and by modulating the transport in the channels using the interaction with the protein residues and the water/hydrogen-bonding network. This process is clearly demonstrated in Photosystem II (PS II), where its light-driven water oxidation reaction catalyzed by the Mn4CaO5 cluster occurs deep inside the protein complex and thus requires the transport of two water molecules to and four protons from the metal center to the bulk water. Based on the recent advances in structural studies of PS II from X-ray crystallography and cryo-electron microscopy, in this review we compare the channels that have been proposed to facilitate this mass transport in cyanobacteria, red and green algae, diatoms, and higher plants. The three major channels (O1, O4, and Cl1 channels) are present in all species investigated; however, some differences exist in the reported structures that arise from the different composition and arrangement of membrane extrinsic subunits between the species. Among the three channels, the Cl1 channel, including the proton gate, is the most conserved among all photosynthetic species. We also found at least one branch for the O1 channel in all organisms, extending all the way from Ca/O1 via the 'water wheel' to the lumen. However, the extending path after the water wheel varies between most species. The O4 channel is, like the Cl1 channel, highly conserved among all species while having different orientations at the end of the path near the bulk. The comparison suggests that the previously proposed functionality of the channels in T. vestitus (Ibrahim et al., Proc Natl Acad Sci USA 117:12624-12635, 2020; Hussein et al., Nat Commun 12:6531, 2021) is conserved through the species, i.e. the O1-like channel is used for substrate water intake, and the tighter Cl1 and O4 channels for proton release. The comparison does not eliminate the potential role of O4 channel as a water intake channel. However, the highly ordered hydrogen-bonded water wire connected to the Mn4CaO5 cluster via the O4 may strongly suggest that it functions in proton release, especially during the S0 → S1 transition (Saito et al., Nat Commun 6:8488, 2015; Kern et al., Nature 563:421-425, 2018; Ibrahim et al., Proc Natl Acad Sci USA 117:12624-12635, 2020; Sakashita et al., Phys Chem Chem Phys 22:15831-15841, 2020; Hussein et al., Nat Commun 12:6531, 2021).

Structural evidence for intermediates during O2 formation in photosystem II.

In natural photosynthesis, the light-driven splitting of water into electrons, protons and molecular oxygen forms the first step of the solar-to-chemical energy conversion process. The reaction takes place in photosystem II, where the Mn4CaO5 cluster first stores four oxidizing equivalents, the S0 to S4 intermediate states in the Kok cycle, sequentially generated by photochemical charge separations in the reaction center and then catalyzes the O-O bond formation chemistry1-3. Here, we report room temperature snapshots by serial femtosecond X-ray crystallography to provide structural insights into the final reaction step of Kok's photosynthetic water oxidation cycle, the S3→[S4]→S0 transition where O2 is formed and Kok's water oxidation clock is reset. Our data reveal a complex sequence of events, which occur over micro- to milliseconds, comprising changes at the Mn4CaO5 cluster, its ligands and water pathways as well as controlled proton release through the hydrogen-bonding network of the Cl1 channel. Importantly, the extra O atom Ox, which was introduced as a bridging ligand between Ca and Mn1 during the S2→S3 transition4-6, disappears or relocates in parallel with Yz reduction starting at approximately 700 µs after the third flash. The onset of O2 evolution, as indicated by the shortening of the Mn1-Mn4 distance, occurs at around 1,200 µs, signifying the presence of a reduced intermediate, possibly a bound peroxide.

Capturing the sequence of events during the water oxidation reaction in photosynthesis using XFELs.

Ever since the discovery that Mn was required for oxygen evolution in plants by Pirson in 1937 and the period-four oscillation in flash-induced oxygen evolution by Joliot and Kok in the 1970s, understanding of this process has advanced enormously using state-of-the-art methods. The most recent in this series of innovative techniques was the introduction of X-ray free-electron lasers (XFELs) a decade ago, which led to another quantum leap in the understanding in this field, by enabling operando X-ray structural and X-ray spectroscopy studies at room temperature. This review summarizes the current understanding of the structure of Photosystem II (PS II) and its catalytic centre, the Mn4 CaO5 complex, in the intermediate Si (i = 0-4)-states of the Kok cycle, obtained using XFELs.

A Survey Assessing Antimicrobial Prescribing at United Nations Relief and Works Agency Primary Health Care Centers in Jordan.

Antimicrobial resistance (AMR) is a public health emergency. There is insufficient information on AMR in the context of humanitarian settings. An understanding of behavioral and institutional-level factors can strengthen antimicrobial stewardship. This study used a semistructured questionnaire to assess both knowledge, attitudes, and practices (KAP) on antimicrobial use, resistance and stewardship, and options to improving prescribing, among prescribers at the Primary Healthcare facilities of the United Nations' Relief and Works Agency Jordan field office. Responses to the KAP questions were evaluated using the Capability, Opportunity, Motivation, Behavior (COM-B) framework and Bloom's cutoffs. For each framework component, Bloom's cutoffs and interpretations were as follows: ≥ 80%, "good"; 60-79%, "moderate"; and < 60%, "poor." Fourteen options to improve prescribing were each assessed using 5-point Likert scales from very unhelpful to very helpful, aggregated by helpful and very helpful and ranked as follows: > 90%, best/most acceptable; > 80-90%, acceptable; and 70-80% as maybe acceptable/good. The questionnaire response rate was 59% (37/63) with a completion rate of 92% (34/37). Aggregate scores for real knowledge on AMR was 97%; opportunity to improve prescribing 88%; and motivation 16%-participants did not believe that there was a connection between their prescribing and AMR or that they had a key role in helping control AMR. Good options (74% aggregate score) to improving prescribing were the availability of guidelines and resistance data. There was good knowledge of AMR and good opportunities, but poor motivation for rational prescribing or behavioral change. There is a clinical need for AMR data to promote rational antibiotic prescribing.

Emotional responses and coping strategies of medical students during the COVID-19 pandemic.

OBJECTIVES: To assess the emotional responses and coping strategies of medical students during the lockdown and social distancing measures implemented during the coronavirus disease -19 (COVID-19) pandemic. METHODS: This cross­sectional study is based on data collected from undergraduate medical students at the College of Medicine, Alfaisal University Riyadh, Saudi Arabia, during the fall semester of academic year 2020-2021. All the participants completed a self-administered online questionnaire consisting of 3 parts: demographic information, emotional response scale, and 14-item, adapted brief coping orientation to problems experienced inventory to determine the use of avoidant or approach coping strategies. Coping and emotional response scores were compared using t-test. Linear regression analysis was also performed. RESULTS: A total of 261 students from all years were included. Overall scores were higher for avoidant coping strategies. The use of avoidant coping strategies was significantly higher in females (p=0.03) and in preclinical students (p<0.001). Preclinical students had a higher mean score for anger (p=0.002). Conversely, students in the clinical phase had higher scores for anxiety (p=0.005) and sadness (p=0.027). The regression analysis of emotional responses and coping strategies suggests that avoidant coping is a predictor of anger (p=0.003) and sadness (p=0.005). CONCLUSION: Interventions to train medical students in the use of more productive and effective coping strategies may reduce negative emotional responses linked to the present COVID-19 pandemic and in the future.

Effects of x-ray free-electron laser pulse intensity on the Mn K ß 1,3 x-ray emission spectrum in photosystem II-A case study for metalloprotein crystals and solutions.

In the last ten years, x-ray free-electron lasers (XFELs) have been successfully employed to characterize metalloproteins at room temperature using various techniques including x-ray diffraction, scattering, and spectroscopy. The approach has been to outrun the radiation damage by using femtosecond (fs) x-ray pulses. An example of an important and damage sensitive active metal center is the Mn4CaO5 cluster in photosystem II (PS II), the catalytic site of photosynthetic water oxidation. The combination of serial femtosecond x-ray crystallography and Kß x-ray emission spectroscopy (XES) has proven to be a powerful multimodal approach for simultaneously probing the overall protein structure and the electronic state of the Mn4CaO5 cluster throughout the catalytic (Kok) cycle. As the observed spectral changes in the Mn4CaO5 cluster are very subtle, it is critical to consider the potential effects of the intense XFEL pulses on the Kß XES signal. We report here a systematic study of the effects of XFEL peak power, beam focus, and dose on the Mn Kß1,3 XES spectra in PS II over a wide range of pulse parameters collected over seven different experimental runs using both microcrystal and solution PS II samples. Our findings show that for beam intensities ranging from ∼5 × 1015 to 5 × 1017 W/cm2 at a pulse length of ∼35 fs, the spectral effects are small compared to those observed between S-states in the Kok cycle. Our results provide a benchmark for other XFEL-based XES studies on metalloproteins, confirming the viability of this approach.

Structural dynamics in the water and proton channels of photosystem II during the S2 to S3 transition.

Light-driven oxidation of water to molecular oxygen is catalyzed by the oxygen-evolving complex (OEC) in Photosystem II (PS II). This multi-electron, multi-proton catalysis requires the transport of two water molecules to and four protons from the OEC. A high-resolution 1.89 Å structure obtained by averaging all the S states and refining the data of various time points during the S2 to S3 transition has provided better visualization of the potential pathways for substrate water insertion and proton release. Our results indicate that the O1 channel is the likely water intake pathway, and the Cl1 channel is the likely proton release pathway based on the structural rearrangements of water molecules and amino acid side chains along these channels. In particular in the Cl1 channel, we suggest that residue D1-E65 serves as a gate for proton transport by minimizing the back reaction. The results show that the water oxidation reaction at the OEC is well coordinated with the amino acid side chains and the H-bonding network over the entire length of the channels, which is essential in shuttling substrate waters and protons.

Room temperature XFEL crystallography reveals asymmetry in the vicinity of the two phylloquinones in photosystem I.

Photosystem I (PS I) has a symmetric structure with two highly similar branches of pigments at the center that are involved in electron transfer, but shows very different efficiency along the two branches. We have determined the structure of cyanobacterial PS I at room temperature (RT) using femtosecond X-ray pulses from an X-ray free electron laser (XFEL) that shows a clear expansion of the entire protein complex in the direction of the membrane plane, when compared to previous cryogenic structures. This trend was observed by complementary datasets taken at multiple XFEL beamlines. In the RT structure of PS I, we also observe conformational differences between the two branches in the reaction center around the secondary electron acceptors A1A and A1B. The π-stacked Phe residues are rotated with a more parallel orientation in the A-branch and an almost perpendicular confirmation in the B-branch, and the symmetry breaking PsaB-Trp673 is tilted and further away from A1A. These changes increase the asymmetry between the branches and may provide insights into the preferential directionality of electron transfer.

A mathematical model to estimate the incidence of child wasting in Yemen.

INTRODUCTION: The ongoing civil war in Yemen has severely restricted imports of food and fuel, disrupted livelihoods and displaced millions, worsening already high pre-war levels of food insecurity. Paired with frequent outbreaks of disease and a collapsed health system, this has brought rates of wasting in children under five to the country's highest recorded levels, which continue to increase as the crisis worsens and aid becomes increasingly limited. In their planning of services to treat and prevent wasting in children, humanitarian agencies rely on a standard calculation to estimate the expected number of cases for the coming year, where incidence is estimated from prevalence and the average duration of an episode of wasting. The average duration of an episode of moderate and severe wasting is currently estimated at 7.5 months-a globally-used value derived from historical cohort studies. Given that incidence varies considerably by context-where food production and availability, treatment coverage and disease rates all vary-a single estimate cannot be applied to all contexts, and especially not a highly unstable crisis setting such as Yemen. While recent studies have aimed to derive context-specific incidence estimates in several countries, little has been done to estimate the incidence of both moderate and severe wasting in Yemen. METHODS: In order to provide context-specific estimates of the average duration of an episode, and resultingly, incidence correction factors for moderate and severe wasting, we have developed a Markov model. Model inputs were estimated using a combination of treatment admission and outcome records compiled by the Yemen Nutrition Cluster, 2018 and 2019 SMART surveys, and other estimates from the literature. The model derived estimates for the governorate of Lahj, Yemen; it was initialized using August 2018 SMART survey prevalence data and run until October 2019-the date of the subsequent SMART survey. Using a process of repeated model calibration, the incidence correction factors for severe wasting and moderate wasting were found, validating the resulting prevalence against the recorded value from the 2019 SMART survey. RESULTS: The average durations of an episode of moderate and severe wasting were estimated at 4.86 months, for an incidence correction factor k of 2.59, and 3.86 months, for an incidence correction factor k of 3.11, respectively. It was found that the annual caseload of moderate wasting was 36% higher and the annual caseload of severe wasting 58% higher than the originally-assumed values, estimated with k = 1.6. CONCLUSION: The model-derived incidence rates, consistent with findings from other contexts that a global incidence correction factor cannot be sufficient, allow for improved, context-specific estimates of the burden of wasting in Yemen. In crisis settings such as Yemen where funding and resources are extremely limited, the model's outputs holistically capture the burden of wasting in a way that may guide effective decision-making and may help ensure that limited resources are allocated most effectively.

Urinary Diversion and Metabolic Acidosis: A Case Report.

Urinary diversion, a surgical technique to redirect urine usually after cystectomy, and its complications are potential challenges to physicians and early recognition decreases mortality and morbidity. A 45-year-old male with a history of type 2 diabetes mellitus and hypertension for over three years underwent urinary diversion as a treatment for invasive bladder cancer and was discharged ambulatory with an indwelling Foleys catheter. The patient returned to the emergency room with a primary complaint of hematuria. The patient was noted to be frail-looking, dehydrated and tachycardic. Laboratory results yielded an acute kidney injury reflected by the elevated creatinine, hyperkalemia and a venous blood gas demonstrating a hyperchloremic metabolic acidosis. The patient had bilateral distended renal calyces by ultrasound and irrigation of bladder through a three-way Foley showed to have large thick clots. The patient was admitted under the surgical intensive care unit after involving appropriate sub-specialties. The patient was started on sodium bicarbonate infusion, broad-spectrum antibiotics and a urinary catheter in place to monitor urine output. The patient's acidosis steadily improved with correction of his laboratory parameters, transferred out of ICU and the remaining stay in the medical ward was uneventful. The purpose of this case report is to help demonstrate the association between urinary diversion and the type of acidosis that subsequently develops in relation to this surgical procedure.

Solution Structure of the Detergent-Photosystem II Core Complex Investigated by Small-Angle Scattering Techniques.

Albeit achieving the X-ray diffraction structure of dimeric photosystem II core complexes (dPSIIcc) at the atomic resolution, the nature of the detergent belt surrounding dPSIIcc remains ambiguous. Therefore, the solution structure of the whole detergent-protein complex of dPSIIcc of Thermosynechococcus elongatus (T. elongatus) solubilized in n-dodecyl-ß-d-maltoside (ßDM) was investigated by a combination of small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS) with contrast variation. First, the structure of dPSIIcc was studied separately in SANS experiments using a contrast of 5% D2O. Guinier analysis reveals that the dPSIIcc solution is virtually free of aggregation in the studied concentration range of 2-10 mg/mL dPSIIcc, and characterized by a radius of gyration of 62 Å. A structure reconstitution shows that dPSIIcc in buffer solution widely retains the crystal structure reported by X-ray free electron laser studies at room temperature with a slight expansion of the entire protein. Additional SANS experiments on dPSIIcc samples in a buffer solution containing 75% D2O provide information about the size and shape of the whole detergent-dPSIIcc. The maximum position of P(r) function increases to 68 Å, i.e., it is about 6 Å larger than that of dPSIIcc only, thus indicating the presence of an additional structure. Thus, it can be concluded that dPSIIcc is surrounded by a monomolecular belt of detergent molecules under appropriate solubilization conditions. The homogeneity of the ßDM-dPSIIcc solutions was also verified using dynamic light scattering. Complementary SAXS experiments indicate the presence of unbound detergent micelles by a separate peak consistent with a spherical shape possessing a radius of about 40 Å. The latter structure also contributes to the SANS data but rather broadens the SANS curve artificially. Without the simultaneous inspection of SANS and SAXS data, this effect may lead to an apparent underestimation of the size of the PS II-detergent complex. The formation of larger unbound detergent aggregates in solution prior to crystallization may have a significant effect on the crystal formation or quality of the ßDM-dPSIIcc.

Untangling the sequence of events during the S2 → S3 transition in photosystem II and implications for the water oxidation mechanism.

In oxygenic photosynthesis, light-driven oxidation of water to molecular oxygen is carried out by the oxygen-evolving complex (OEC) in photosystem II (PS II). Recently, we reported the room-temperature structures of PS II in the four (semi)stable S-states, S1, S2, S3, and S0, showing that a water molecule is inserted during the S2 → S3 transition, as a new bridging O(H)-ligand between Mn1 and Ca. To understand the sequence of events leading to the formation of this last stable intermediate state before O2 formation, we recorded diffraction and Mn X-ray emission spectroscopy (XES) data at several time points during the S2 → S3 transition. At the electron acceptor site, changes due to the two-electron redox chemistry at the quinones, QA and QB, are observed. At the donor site, tyrosine YZ and His190 H-bonded to it move by 50 µs after the second flash, and Glu189 moves away from Ca. This is followed by Mn1 and Mn4 moving apart, and the insertion of OX(H) at the open coordination site of Mn1. This water, possibly a ligand of Ca, could be supplied via a "water wheel"-like arrangement of five waters next to the OEC that is connected by a large channel to the bulk solvent. XES spectra show that Mn oxidation (τ of ∼350 µs) during the S2 → S3 transition mirrors the appearance of OX electron density. This indicates that the oxidation state change and the insertion of water as a bridging atom between Mn1 and Ca are highly correlated.

Extending electron paramagnetic resonance to nanoliter volume protein single crystals using a self-resonant microhelix.

Electron paramagnetic resonance (EPR) spectroscopy on protein single crystals is the ultimate method for determining the electronic structure of paramagnetic intermediates at the active site of an enzyme and relating the magnetic tensor to a molecular structure. However, crystals of dimensions typical for protein crystallography (0.05 to 0.3mm) provide insufficient signal intensity. In this work, we present a microwave self-resonant microhelix for nanoliter samples that can be implemented in a commercial X-band (9.5 GHz) EPR spectrometer. The self-resonant microhelix provides a measured signal-to-noise improvement up to a factor of 28 with respect to commercial EPR resonators. This work opens up the possibility to use advanced EPR techniques for studying protein single crystals of dimensions typical for x-ray crystallography. The technique is demonstrated by EPR experiments on single crystal [FeFe]-hydrogenase (Clostridium pasteurianum; CpI) with dimensions of 0.3 mm by 0.1 mm by 0.1 mm, yielding a proposed g-tensor orientation of the Hox state.

Structures of the intermediates of Kok's photosynthetic water oxidation clock.

Inspired by the period-four oscillation in flash-induced oxygen evolution of photosystem II discovered by Joliot in 1969, Kok performed additional experiments and proposed a five-state kinetic model for photosynthetic oxygen evolution, known as Kok's S-state clock or cycle1,2. The model comprises four (meta)stable intermediates (S0, S1, S2 and S3) and one transient S4 state, which precedes dioxygen formation occurring in a concerted reaction from two water-derived oxygens bound at an oxo-bridged tetra manganese calcium (Mn4CaO5) cluster in the oxygen-evolving complex3-7. This reaction is coupled to the two-step reduction and protonation of the mobile plastoquinone QB at the acceptor side of PSII. Here, using serial femtosecond X-ray crystallography and simultaneous X-ray emission spectroscopy with multi-flash visible laser excitation at room temperature, we visualize all (meta)stable states of Kok's cycle as high-resolution structures (2.04-2.08 Å). In addition, we report structures of two transient states at 150 and 400 µs, revealing notable structural changes including the binding of one additional 'water', Ox, during the S2→S3 state transition. Our results suggest that one water ligand to calcium (W3) is directly involved in substrate delivery. The binding of the additional oxygen Ox in the S3 state between Ca and Mn1 supports O-O bond formation mechanisms involving O5 as one substrate, where Ox is either the other substrate oxygen or is perfectly positioned to refill the O5 position during O2 release. Thus, our results exclude peroxo-bond formation in the S3 state, and the nucleophilic attack of W3 onto W2 is unlikely.

Optimizing Crystal Size of Photosystem II by Macroseeding: Toward Neutron Protein Crystallography.

Photosystem II (PSII) catalyzes the photo-oxidation of water to molecular oxygen and protons. The water splitting reaction occurs inside the oxygen-evolving complex (OEC) via a Mn4CaO5 cluster. To elucidate the reaction mechanism, detailed structural information for each intermediate state of the OEC is required. Despite the current high-resolution crystal structure of PSII at 1.85 Å and other efforts to follow the structural changes of the Mn4CaO5 cluster using X-ray free electron laser (XFEL) crystallography in addition to spectroscopic methods, many details about the reaction mechanism and conformational changes in the catalytic site during water oxidation still remain elusive. In this study, we present a rarely found successful application of the conventional macroseeding method to a large membrane protein like the dimeric PSII core complex (dPSIIcc). Combining microseeding with macroseeding crystallization techniques allowed us to reproducibly grow large dPSIIcc crystals with a size of ~3 mm. These large crystals will help improve the data collected from spectroscopic methods like polarized extended X-ray absorption fine structure (EXAFS) and single crystal electron paramagnetic resonance (EPR) techniques and are a prerequisite for determining a three-dimensional structure using neutron diffraction.

Structural insights into the light-driven auto-assembly process of the water-oxidizing Mn4CaO5-cluster in photosystem II.

In plants, algae and cyanobacteria, Photosystem II (PSII) catalyzes the light-driven splitting of water at a protein-bound Mn4CaO5-cluster, the water-oxidizing complex (WOC). In the photosynthetic organisms, the light-driven formation of the WOC from dissolved metal ions is a key process because it is essential in both initial activation and continuous repair of PSII. Structural information is required for understanding of this chaperone-free metal-cluster assembly. For the first time, we obtained a structure of PSII from Thermosynechococcus elongatus without the Mn4CaO5-cluster. Surprisingly, cluster-removal leaves the positions of all coordinating amino acid residues and most nearby water molecules largely unaffected, resulting in a pre-organized ligand shell for kinetically competent and error-free photo-assembly of the Mn4CaO5-cluster. First experiments initiating (i) partial disassembly and (ii) partial re-assembly after complete depletion of the Mn4CaO5-cluster agree with a specific bi-manganese cluster, likely a di-µ-oxo bridged pair of Mn(III) ions, as an assembly intermediate.

Drop-on-demand sample delivery for studying biocatalysts in action at X-ray free-electron lasers.

X-ray crystallography at X-ray free-electron laser sources is a powerful method for studying macromolecules at biologically relevant temperatures. Moreover, when combined with complementary techniques like X-ray emission spectroscopy, both global structures and chemical properties of metalloenzymes can be obtained concurrently, providing insights into the interplay between the protein structure and dynamics and the chemistry at an active site. The implementation of such a multimodal approach can be compromised by conflicting requirements to optimize each individual method. In particular, the method used for sample delivery greatly affects the data quality. We present here a robust way of delivering controlled sample amounts on demand using acoustic droplet ejection coupled with a conveyor belt drive that is optimized for crystallography and spectroscopy measurements of photochemical and chemical reactions over a wide range of time scales. Studies with photosystem II, the phytochrome photoreceptor, and ribonucleotide reductase R2 illustrate the power and versatility of this method.

Structure of photosystem II and substrate binding at room temperature.

Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has generated most of the dioxygen in the atmosphere. PS II, a membrane-bound multi-subunit pigment protein complex, couples the one-electron photochemistry at the reaction centre with the four-electron redox chemistry of water oxidation at the Mn4CaO5 cluster in the oxygen-evolving complex (OEC). Under illumination, the OEC cycles through five intermediate S-states (S0 to S4), in which S1 is the dark-stable state and S3 is the last semi-stable state before O-O bond formation and O2 evolution. A detailed understanding of the O-O bond formation mechanism remains a challenge, and will require elucidation of both the structures of the OEC in the different S-states and the binding of the two substrate waters to the catalytic site. Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage-free, room temperature structures of dark-adapted (S1), two-flash illuminated (2F; S3-enriched), and ammonia-bound two-flash illuminated (2F-NH3; S3-enriched) PS II. Although the recent 1.95 Å resolution structure of PS II at cryogenic temperature using an XFEL provided a damage-free view of the S1 state, measurements at room temperature are required to study the structural landscape of proteins under functional conditions, and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analogue, has been used as a marker, as it binds to the Mn4CaO5 cluster in the S2 and S3 states. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site. This approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O-O bond formation mechanisms.