Reinvestigation on primary processes of PSII-dimer from Thermosynechococcus vulcanus by femtosecond pump-probe spectroscopy.

Cyanobacterial photosynthetic apparatus efficiently capture sunlight, and the energy is subsequently transferred to photosystem I (PSI) and II (PSII), to produce electrochemical potentials. PSII is a unique membrane protein complex that photo-catalyzes oxidation of water and majorly contains photosynthetic pigments of chlorophyll a and carotenoids. In the present study, the ultrafast energy transfer and charge separation dynamics of PSII from a thermophilic cyanobacterium Thermosynechococcus vulcanus were reinvestigated by femtosecond pump-probe spectroscopic measurements under low temperature and weak intensity excitation condition. The results imply the two possible models of the energy transfers and subsequent charge separation in PSII. One is the previously suggested "transfer-to-trapped limit" model. Another model suggests that the energy transfers from core CP43 and CP47 antennas to the primary electron donor ChlD1 with time-constants of 0.71 ps and 3.28 ps at 140 K (0.17 and 1.33 ps at 296 K), respectively and that the pheophytin anion (PheoD1-) is generated with the time-constant of 43.0 ps at 140 K (14.8 ps at 296 K) upon excitation into the Qy band of chlorophyll a at 670 nm. The secondary electron transfer to quinone QA: PheoD1-QA → PheoD1QA- is observed with the time-constant of 650 ps only at 296 K. On the other hand, an inefficient ß-carotene → chlorophyll a energy transfer (33%) occurred after excitation to the S2 state of ß-carotene at 500 nm. Instead, the carotenoid triplet state appeared in an ultrafast timescale after excitation at 500 nm.

Removal of endocrine disruptor compounds, CO2 fixation, and macromolecules accumulation in Thermosynechococcus sp. CL-1 cultivation.

Recently, concern on several environmental issues including the pollutant discharge and high concentration of CO2 have gained high interest due to its impact on ecosystem and global warming effect, respectively. Implementation of photosynthetic microorganism carries out numerous advantages including high efficiency of CO2 fixation, the great endurance under extreme conditions and generation of valuable bioproducts. Thermosynechococcus sp. CL-1 (TCL-1), a cyanobacterium, has the ability to perform CO2 fixation and accumulation of various byproducts under extreme conditions like high temperature and alkalinity, presence of estrogen, or even using swine wastewater. This study aimed to assess TCL-1 performance under various endocrine disruptor compounds (bisphenol-A, 17-ß-estradiol/E2, and 17-α-ethynilestradiol/EE2), concentrations (0-10 mg/L), light intensities (500-2000 µE/m2/s), and dissolved inorganic carbon/DIC levels (0-113.2 mM). Addition of E2 content even until 10 mg/L carried out insignificant biomass growth interruption along with the improvement in CO2 fixation rate (79.8 ± 0.1 mg/L/h). Besides the influence of E2, application of higher DIC level and light intensity also enhanced the CO2 fixation rate and biomass growth. The highest biodegradation of E2 at 71% was achieved by TCL-1 in the end of 12 h cultivation period. TCL-1 dominantly produced protein (46.7% ± 0.2%), however, production of lipid and carbohydrate (39.5 ± 1.5 and 23.3 ± 0.9%, respectively) also could be considered as the potential source for biofuel production. Thus, this study can provide an efficient strategy in simultaneously dealing with environmental issues with side advantage in production of macromolecules.

Simulating the low-temperature, metastable electrochromism of Photosystem I: Applications to Thermosynechococcus vulcanus and Chroococcidiopsis thermalis.

Low-temperature, metastable electrochromism has been used as a tool to assign pigments in Photosystem I (PS I) from Thermosynechococcus vulcanus and both the white light and far-red light (FRL) forms of Chroococcidiopsis thermalis. We find that a minimum of seven pigments is required to satisfactorily model the electrochromism of PS I. Using our model, we provide a short list of candidates for the chlorophyll f pigment in FRL C. thermalis that absorbs at 756 nm, whose identity, to date, has proven to be controversial. Specifically, we propose the linker pigments A40 and B39 and two antenna pigments A26 and B24 as defined by crystal structure 1JB0. The pros and cons of these assignments are discussed, and we propose further experiments to better understand the functioning of FRL C. thermalis.

CO2 fixation and cultivation of Thermosynechococcus sp. CL-1 for the production of phycocyanin.

Cultivation of Cyanobacteria is preferable for CO2 fixation process due to its efficiency and production of beneficial byproducts like phycocyanin. In this study, Thermosynechococcus sp. CL-1 (TCL-1) was cultivated in a 30 L flat panel photobioreactor using a 3-fold-modified Fitzgerald medium with 113.2 mM dissolved inorganic carbon. The highest CO2 fixation rate of 21.98 ± 1.52 mg/L/h was followed by higher lipid content (49.91 % dry weight content or %dwc) than the generated carbohydrate (24.22 %dwc). TCL-1 also potentially produced phycocyanin that was dominated by C-phycocyanin (98.10 ± 6.67 mg/g) along with a lower amount of allophycocyanin and phycoerythrin under extraction using various types of solvent. Stability of phycocyanin extract was further examined during storage under various temperatures and light illuminations. Extraction with 36 % glucose solvent presented a protective effect to phycocyanin from heat and photo-damage which was proven by the kinetics study of phycocyanin degradation in this study.

Small angle neutron scattering and lipidomic analysis of a native, trimeric PSI-SMALP from a thermophilic cyanobacteria.

The use of styrene-maleic acid copolymers (SMAs) to produce membrane protein-containing nanodiscs without the initial detergent isolation has gained significant interest over the last decade. We have previously shown that a Photosystem I SMALP from the thermophilic cyanobacterium, Thermosynechococcus elongatus (PSI-SMALP), has much more rapid energy transfer and charge separation in vitro than detergent isolated PSI complexes. In this study, we have utilized small-angle neutron scattering (SANS) to better understand the geometry of these SMALPs. These techniques allow us to investigate the size and shape of these particles in their fully solvated state. Further, the particle's proteolipid core and detergent shell or copolymer belt can be interrogated separately using contrast variation, a capability unique to SANS. Here we report the dimensions of the Thermosynechococcus elongatus PSI-SMALP containing a PSI trimer. At ~1.5 MDa, PSI-SMALP is the largest SMALP to be isolated; our lipidomic analysis indicates it contains ~1300 lipids/per trimeric particle, >40-fold more than the PSI-DDM particle and > 100 fold more than identified in the 1JB0 crystal structure. Interestingly, the lipid composition to the PSI trimer in the PSI-SMALP differs significantly from bulk thylakoid composition, being enriched ~50 % in the anionic sulfolipid, SQDG. Finally, utilizing the contrast match point for the SMA 1440 copolymer, we also can observe the ~1 nm SMA copolymer belt surrounding this SMALP for the first time, consistent with most models of SMA organization.

Thermosynechococcus switches the direction of phototaxis by a c-di-GMP-dependent process with high spatial resolution.

Many cyanobacteria, which use light as an energy source via photosynthesis, show directional movement towards or away from a light source. However, the molecular and cell biological mechanisms for switching the direction of movement remain unclear. Here, we visualized type IV pilus-dependent cell movement in the rod-shaped thermophilic cyanobacterium Thermosynechococcus vulcanus using optical microscopy at physiological temperature and light conditions. Positive and negative phototaxis were controlled on a short time scale of 1 min. The cells smoothly moved over solid surfaces towards green light, but the direction was switched to backward movement when we applied additional blue light illumination. The switching was mediated by three photoreceptors, SesA, SesB, and SesC, which have cyanobacteriochrome photosensory domains and synthesis/degradation activity of the bacterial second messenger cyclic dimeric GMP (c-di-GMP). Our results suggest that the decision-making process for directional switching in phototaxis involves light-dependent changes in the cellular concentration of c-di-GMP. Direct visualization of type IV pilus filaments revealed that rod-shaped cells can move perpendicular to the light vector, indicating that the polarity can be controlled not only by pole-to-pole regulation but also within-a-pole regulation. This study provides insights into previously undescribed rapid bacterial polarity regulation via second messenger signalling with high spatial resolution.

Cyanobacteria, like plants, grow by capturing energy from sunlight. But they have an advantage over their leafy counterparts: they can explore their environment to find the type of light that best suits their needs. These movements rely on hook-like structures, called type IV pili, which allow the cells to pull themselves forward. The pili are usually located at the opposite poles of a rod-shaped cell, allowing the bacteria to move along their longer axis. Yet, the molecular mechanisms that allow cyanobacteria to react to the light are poorly understood. To explore these processes in more detail, Nakane, Enomoto et al. started by shining coloured lights on the rod-shaped cyanobacteria Thermosynechococcus vulcanus. This revealed that the cells moved towards green light but reversed rapidly when blue light was added. The behaviour was disrupted when the genes for three light-sensing proteins were artificially switched off. These molecular players act by changing the levels of cyclic di-GMP, a signalling molecule that may interact with type IV pili. The experiments also showed that T. vulcanus cells were not only moving along their longer axis, but also at a right-angle. This observation contrasts with how other rod-shaped bacteria can explore their environment. A closer look revealed that the cyanobacteria could perform these movements by making asymmetrical adjustment to the way that pili at each pole were working. Further research is now needed to more finely dissect the molecular mechanisms which control this remarkable type of motion.

Effects of mutations of D1-R323, D1-N322, D1-D319, D1-H304 on the functioning of photosystem II in Thermosynechococcus vulcanus.

Photosystem II (PSII) has a number of hydrogen-bonding networks connecting the manganese cluster with the lumenal bulk solution. The structure of PSII from Thermosynechococcus vulcanus (T. vulcanus) showed that D1-R323, D1-N322, D1-D319 and D1-H304 are involved in one of these hydrogen-bonding networks located in the interfaces between the D1, CP43 and PsbV subunits. In order to investigate the functions of these residues in PSII, we generated seven site-directed mutants D1-R323A, D1-R323E, D1-N322R, D1-D319L, D1-D319R, D1-D319Y and D1-H304D of T. vulcanus and examined the effects of these mutations on the growth and functions of the oxygen-evolving complex. The photoautotrophic growth rates of these mutants were similar to that of the wild type, whereas the oxygen-evolving activities of the mutant cells were decreased differently to 63-91% of that of the wild type at pH 6.5. The mutant cells showed a higher relative activity at higher pH region than the wild type cells, suggesting that higher pH facilitated proton egress in the mutants. In addition, oxygen evolution of thylakoid membranes isolated from these mutants showed an apparent decrease compared to that of the cells. This is due to the loss of PsbU during purification of the thylakoid membranes. Moreover, PsbV was also lost in the PSII core complexes purified from the mutants. Taken together, D1-R323, D1-N322, D1-D319 and D1-H304 are vital for the optimal function of oxygen evolution and functional binding of extrinsic proteins to PSII core, and may be involved in the proton egress pathway mediated by YZ.

Simultaneous 17ß-estradiol degradation, carbon dioxide fixation, and carotenoid accumulation by Thermosynechococcus sp. CL-1.

Thermosynechococcus sp. CL-1 (TCL-1) has a high potency to utilize CO2 under extreme conditions including high temperature, alkaline condition, and the occurrence of 17ß-estradiol (E2). In this study, TCL-1 cultivation with E2 addition in the range of 0-20 mg/L was combined with various growth arrangements (light intensity and dissolved inorganic nitrogen/DIN level). After 120 h cultivation, the 1.0 mg/L E2, 200 µmol photons/m2/s light intensity, and 5.8 mM available nitrogen performed the best growth with 4.58 ± 0.18 mg/L/h biomass productivity, 94.9 ± 3.3% total estrogen removal, and 11.41 ± 0.11 mg/L/h CO2 fixation rate. Estrogen degradation was mainly carried out by biodegradation route which started from E2 conversion into estrone/E1 and with only 4-6% influence from the abiotic factors. Compared with the accumulated zeaxanthin, ß-carotene was dominantly generated with a productivity of 0.043 ± 0.019 mg/L/h. Therefore, TCL-1 cultivation is an efficient strategy for simultaneous CO2 fixation, estrogen removal, and carotenoid accumulation as valuable byproducts.

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.

Electrospinning for building 3D structured photoactive biohybrid electrodes.

We describe the development of biohybrid electrodes constructed via combination of electrospun (e-spun) 3D indium tin oxide (ITO) with the trimeric supercomplex photosystem I and the small electrochemically active protein cytochrome c (cyt c). The developed 3D surface of ITO has been created by electrospinning of a mixture of polyelthylene oxide (PEO) and ITO nanoparticles onto ITO glass slides followed by a subsequent elimination of PEO by sintering the composite. Whereas the photosystem I alone shows only small photocurrents at these 3D electrodes, the co-immobilization of cyt c to the e-spun 3D ITO results in well-defined photoelectrochemical signals. The scaling of thickness of the 3D ITO layers by controlling the time (10 min and 60 min) of electrospinning results in enhancement of the photocurrent. Several performance parameters of the electrode have been analyzed for different illumination intensities.

Structural insights into cyanobacterial photosystem II intermediates associated with Psb28 and Tsl0063.

Photosystem II (PSII) is a multisubunit pigment-protein complex and catalyses light-induced water oxidation, leading to the conversion of light energy into chemical energy and the release of dioxygen. We analysed the structures of two Psb28-bound PSII intermediates, Psb28-RC47 and Psb28-PSII, purified from a psbV-deletion strain of the thermophilic cyanobacterium Thermosynechococcus vulcanus, using cryo-electron microscopy. Both Psb28-RC47 and Psb28-PSII bind one Psb28, one Tsl0063 and an unknown subunit. Psb28 is located at the cytoplasmic surface of PSII and interacts with D1, D2 and CP47, whereas Tsl0063 is a transmembrane subunit and binds at the side of CP47/PsbH. Substantial structural perturbations are observed at the acceptor side, which result in conformational changes of the quinone (QB) and non-haem iron binding sites and thus may protect PSII from photodamage during assembly. These results provide a solid structural basis for understanding the assembly process of native PSII.

Cryo-EM structure of monomeric photosystem II at 2.78 Å resolution reveals factors important for the formation of dimer.

Photosystem II (PSII) functions mainly as a dimer to catalyze the light energy conversion and water oxidation reactions. However, monomeric PSII also exists and functions in vivo in some cases. The crystal structure of monomeric PSII has been solved at 3.6 Å resolution, but it is still not clear which factors contribute to the formation of the dimer. Here, we solved the structure of PSII monomer at a resolution of 2.78 Å using cryo-electron microscopy (cryo-EM). From our cryo-EM density map, we observed apparent differences in pigments and lipids in the monomer-monomer interface between the PSII monomer and dimer. One ß-carotene and two sulfoquinovosyl diacylglycerol (SQDG) molecules are found in the monomer-monomer interface of the dimer structure but not in the present monomer structure, although some SQDG and other lipid molecules are found in the analogous region of the low-resolution crystal structure of the monomer, or cryo-EM structure of an apo-PSII monomer lacking the extrinsic proteins from Synechocystis sp. PCC 6803. In the current monomer structure, a large part of the PsbO subunit was also found to be disordered. These results indicate the importance of the ß-carotene, SQDG and PsbO in formation of the PSII dimer.

Structural implications for a phycobilisome complex from the thermophilic cyanobacterium Thermosynechococcus vulcanus.

Phycobilisomes (PBSs) are huge, water-soluble light-harvesting complexes used by oxygenic photosynthetic organisms. The structures of some subunits of the PBSs, including allophycocyanin (APC) and phycocyanin (PC), have been solved by X-ray crystallography previously. However, there are few reports on the overall structures of PBS complexes in photosynthetic organisms. Here, we report the overall structure of the PBS complex isolated from the cyanobacterium Thermosynechococcus vulcanus, determined by negative-staining electron microscopy (EM). Intact PBS complexes were purified by trehalose density gradient centrifugation with a high-concentration phosphate buffer and then subjected to a gradient-fixation preparation using glutaraldehyde. The final map constructed by the single-particle analysis of EM images showed a hemidiscoidal structure of the PBS, consisting of APC cores and peripheral PC rods. The APC cores are composed of five cylinders: A1, A2, B, C1, and C2. Each of the cylinders is composed of three (A1 and A2), four (B), or two (C1 and C2) APC trimers. In addition, there are eight PC rods in the PBS: one bottom pair (Rb and Rb'), one top pair (Rt and Rt'), and two side pairs (Rs1/Rs1' and Rs2/Rs2'). Comparison with the overall structures of PBSs from other organisms revealed structural characteristics of T. vulcanus PBS.

Elimination of inorganic carbon and nitrogen resided in swine wastewater using Thermosynechococcus sp. CL-1 enriched culture.

Nutrient pollution released from highly accumulated swine wastewater is getting concerned due to global warming and waterbody harmful. Traditional combination of nitrification and denitrification is commonly applied to remove carbon and nitrogen compounds resided in various wastewater with disadvantages of high cost and energy requirements. This study applied the thermophilic flat panel photobioreactor (tFPBR) with high growth rate of TCL-1 culture to evaluate the efficiency of inorganic carbon and nitrogen transformation. This 12-h operation resulted that TCL-1 enriched batch, grown in 50 °C and alkaline environment with 1,000 µE/m2/s light intensity, had high potential for CO2 fixation rate of 122.29 ± 9.93 mg/L/h and nitrogen removal rate of 7.76 mg-N/L/h treating swine wastewater, in comparison with comprehensive community involved in carbon and nitrogen cycles in the field-scale anoxic tank. This study provided the Rapid-growing photosynthetic cyanobacteria in place of slow-growing autotrophic microbes for of carbon and nitrogen transformation in the wastewater system.

The Thermosynechococcus Genus: Wide Environmental Distribution, but a Highly Conserved Genomic Core.

Cyanobacteria thrive in diverse environments. However, questions remain about possible growth limitations in ancient environmental conditions. As a single genus, the Thermosynechococcus are cosmopolitan and live in chemically diverse habitats. To understand the genetic basis for this, we compared the protein coding component of Thermosynechococcus genomes. Supplementing the known genetic diversity of Thermosynechococcus, we report draft metagenome-assembled genomes of two Thermosynechococcus recovered from ferrous carbonate hot springs in Japan. We find that as a genus, Thermosynechococcus is genomically conserved, having a small pan-genome with few accessory genes per individual strain as well as few genes that are unique to the genus. Furthermore, by comparing orthologous protein groups, including an analysis of genes encoding proteins with an iron related function (uptake, storage or utilization), no clear differences in genetic content, or adaptive mechanisms could be detected between genus members, despite the range of environments they inhabit. Overall, our results highlight a seemingly innate ability for Thermosynechococcus to inhabit diverse habitats without having undergone substantial genomic adaptation to accommodate this. The finding of Thermosynechococcus in both hot and high iron environments without adaptation recognizable from the perspective of the proteome has implications for understanding the basis of thermophily within this clade, and also for understanding the possible genetic basis for high iron tolerance in cyanobacteria on early Earth. The conserved core genome may be indicative of an allopatric lifestyle-or reduced genetic complexity of hot spring habitats relative to other environments.

Nitrogen and 17ß-Estradiol level regulate Thermosynechococcus sp. CL-1 carbon dioxide fixation, monosaccharide production, and estrogen degradation.

Thermosynechococcus sp. CL-1 (TCL-1), a thermophilic cyanobacterium from a hot spring in Taiwan, has been known of its efficiency in CO2 fixation, byproducts production (pigments, macromolecules). This study observed the performance of TCL-1 in CO2 fixation, estrogen degradation, and monosaccharide production under various levels of Dissolved Inorganic Nitrogen (DIN) and 17ß-estradiol (E2) as nitrogen supply and estrogen addition. Under nitrogen starvation, TCL-1 performed similar results on CO2 fixation rate and biomass production but enhanced the monosaccharide production compared to the cases of high nitrogen supply. The highest CO2 fixation rate and glucose productivity reached to 151.8 ± 6.6 and 38.1 ± 0.9 mg/L/h, under DIN level of 0.58 mM and 0.5 mg/L E2. Adding E2 in the system did not inhibit the performance of TCL-1. During the cultivation, TCL-1 converted E2 into E1 and the biodegradation was the main path for estrogen degradation. Total E2 degradation reached to 69.4 ± 2.0%.

Generation and characterization of self-assembled protein nanocages based on ß-carboxysomes in Escherichia coli.

Self-assembly is a powerful means to create new materials and new catalysts. The advantages of biological self-assembly are based on it being highly programmable and prone to multilevel regulation, which can lead to multiple and complex functions. The self-assembly of carboxysomes in cyanobacteria enables the carboxysomes to enrich carbon dioxide in their interior, resulting in the formation of a highly efficient, multiple-enzyme catalytic system. Here, we show that the construction and coexpression of all genes of the ß-carboxysome from the cyanobacterium Thermosynechococcus elongatus BP-1 can lead to the production of ß-carboxysome-like structures in Escherichia coli. These shell structures were characterized intracellularly and extracellularly by transmission electron microscopy. This work lays a foundation for understanding carboxysome assembly and catalysis and the development of novel carboxysome-based nanomaterials utilizing synthetic biology.

Ultrafast energy transfer dynamics of phycobilisome from Thermosynechococcus vulcanus, as revealed by ps fluorescence and fs pump-probe spectroscopies.

Cyanobacterial photosynthetic systems efficiently capture sunlight using the pigment-protein megacomplexes, phycobilisome (PBS). The energy is subsequently transferred to photosystem I (PSI) and II (PSII), to produce electrochemical potentials. In the present study, we performed picosecond (ps) time-resolved fluorescence and femtosecond (fs) pump-probe spectroscopies on the intact PBS from a thermophilic cyanobacterium, Thermosynechococcus vulcanus, to reveal excitation energy transfer dynamics in PBS. The photophysical properties of the intact PBS were well characterized by spectroscopic measurements covering wide temporal range from femtoseconds to nanoseconds. The ps fluorescence measurements excited at 570 nm, corresponding to the higher energy of the phycocyanin (PC) absorption band, demonstrated the excitation energy transfer from the PC rods to the allophycocyanin (APC) core complex as well as the energy transfer in the APC core complex. Then, the fs pump-probe measurements revealed the detailed energy transfer dynamics in the PC rods taking place in an ultrafast time scale. The results obtained in this study provide the full picture of the funnel-type excitation energy transfer with rate constants of (0.57 ps)-1 → (7.3 ps)-1 → (53 ps)-1 → (180 ps)-1 → (1800 ps)-1.

Structural insights into photosystem II assembly.

Biogenesis of photosystem II (PSII), nature's water-splitting catalyst, is assisted by auxiliary proteins that form transient complexes with PSII components to facilitate stepwise assembly events. Using cryo-electron microscopy, we solved the structure of such a PSII assembly intermediate from Thermosynechococcus elongatus at 2.94 Å resolution. It contains three assembly factors (Psb27, Psb28 and Psb34) and provides detailed insights into their molecular function. Binding of Psb28 induces large conformational changes at the PSII acceptor side, which distort the binding pocket of the mobile quinone (QB) and replace the bicarbonate ligand of non-haem iron with glutamate, a structural motif found in reaction centres of non-oxygenic photosynthetic bacteria. These results reveal mechanisms that protect PSII from damage during biogenesis until water splitting is activated. Our structure further demonstrates how the PSII active site is prepared for the incorporation of the Mn4CaO5 cluster, which performs the unique water-splitting reaction.

Two Distinct Oxygen-Radical Conformations in the X-ray Free Electron Laser Structures of Photosystem II.

We report the existence of two distinct oxygen-radical-containing Mn4CaO5/6 conformations with short O···O bonds in the crystal structures of the oxygen-evolving enzyme photosystem II (PSII), obtained using an X-ray free electron laser (XFEL). A short O···O distance of <2.3 Å between the O4 site of the Mn4CaO5 complex and the adjacent water molecule (W539) in the proton-conducting O4-water chain was observed in the second flash-induced (2F) XFEL structure (2F-XFEL), which may correspond to S3. By use of a quantum mechanical/molecular mechanical approach, the OH• formation at W539 and the short O4···OW539 distance (<2.3 Å) were reproduced in S2 and S3 with reduced Mn1(III), which lacks the additional sixth water molecule O6. As the O•- formation at O6 and the short O5···O6 distance (1.9 Å) have been reported in another 2F-XFEL structure with reduced Mn4(III), two distinct oxygen-radical conformations exist in the 2F-XFEL crystals.