of water into dioxygen through an S-state cycle of the oxygen evolving complex (OEC).The structure of PSII has been solved by X-ray diffraction (XRD) at 1.9-ångström (Å) resolution, which revealed that the OEC is a Mn 4 CaO 5 -cluster coordinated by a well-defined protein environment 1 . However, extended X-ray absorption fine structure (EXAFS) studies showed that the manganese cations in the OEC are easily reduced by X-ray irradiation 2 , and slight differences were found in the Mn-Mn distances between the results of XRD 1 , EXAFS 3-7 and theoretical studies 8-14 . Here we report a 'radiation-damage-free' structure of PSII from Thermosynechococcus vulcanus in the S 1 state at a resolution of 1.95 Å using femtosecond X-ray pulses of the SPring-8 ångström compact free-electron laser (SACLA) and a huge number of large, highly isomorphous PSII crystals. Compared with the structure from XRD, the OEC in the X-ray free electron These findings provide a structural basis for the mechanism of oxygen evolution, and we expect that this structure will provide a blueprint for design of artificial catalysts for water oxidation.PSII is a multi-subunit pigment-protein complex embedded in the thylakoid membranes of higher plants, green algae and cyanobacteria, and is the only molecular machine capable of oxidizing water by use of visible light. Water molecules are split into electrons, hydrogen atoms and oxygen molecules at the catalytic centre of PSII, namely, the OEC, through four electron and/or proton removing steps as described in the S i -state cycle (with i = 0-4, where i indicates the number of oxidative equivalents accumulated). Because of its ability to split water, the OEC is considered a promising template for the synthesis of artificial catalysts for water-splitting aimed at obtaining clean and renewable energy from sunlight, which is considered a promising way to supplement the consumption of limited and environmentally unfriendly fossil fuels.In order to elucidate the mechanism of the water-splitting reaction, the structure of PSII has been studied extensively by XRD, with a resolution that has gradually increased from 3.8 to 1.9 Å using synchrotron radiation (SR) X-ray sources 1,[15][16][17][18] . In particular, the SR structure of PSII at atomic resolution revealed that the OEC is a Mn 4 CaO 5 cluster organized into a distorted-chair shape, in which the cuboidal part is composed of Mn 3 CaO 3 and the outer manganese is attached via two -oxo-bridges 1 . The high-resolution structure also revealed that four water molecules are coordinated to the Mn 4 CaO 5 cluster, among which, two are coordinated and XRD studies 1 ). Although these provided an important structural basis for the mechanism of water-splitting, the average Mn-ligand and Mn-Mn distances were found to be slightly longer than those deduced from EXAFS 3-7 and from computational analysis based on the SR structure [8][9][10][11][12][13][14] . This has been suggested to result from radiation damage, as hydrated electrons generated by X-ray irradiation 19 a...

SummaryPhotosystem II (PSII) is a huge membrane-protein complex consisting of 20 different subunits with a total molecular mass of 350 kDa for a monomer, and catalyzes light-driven water oxidation at its catalytic center, the oxygen-evolving complex (OEC) [1][2][3] . The structure of PSII has been analyzed at 1.9 Å resolution by synchrotron radiation X-rays, which revealed that OEC is a Mn4CaO5 cluster organized in an asymmetric, "distorted-chair" form 4 . This structure was further analyzed with femtosecond X-ray free electron lasers (XFEL), providing the "radiation damage-free" 5 structure. The mechanism of O=O bond formation, however, remains obscure due to the lack of intermediate state structures. Here we report the structural changes of PSII induced by 2-flash (2F) illumination at room temperature at a resolution of 2.35 Å using time-resolved serial femtosecond crystallography (TR-SFX) with an XFEL provided by the SPring-8 angstrom compact free-electron laser (SACLA). Isomorphous differenceFourier map between the 2F and dark-adapted states revealed two areas of apparent changes; they are around QB/non-heme iron and the Mn4CaO5 cluster. The changes around the QB/non-heme iron region reflected the electron and proton transfers induced by the 2F-illumination. In the region around the Mn4CaO5 cluster, a water molecule located 3.5 Å from the Mn4CaO5 cluster disappeared from the map upon 2Fillumination, leading to a closer distance between another water molecule and O4, suggesting also the occurrence of proton transfer. Importantly, the 2F-dark isomorphous difference Fourier map showed an apparent positive peak around O5, a unique μ3-oxo-bridge located in the quasi-center of Mn1 and Mn4 4,5 . This suggests an insertion of a new oxygen atom (O6) close to O5, providing an O=O distance of 1.5 Å between these two oxygen atoms. This provides a mechanism for the O=O bond formation 4 consistent with that proposed by Siegbahn 6,7 . Fig. 1a shows organization of the electron transfer chain of PSII in a pseudo-C2 symmetry by two subunits D1 and D2. The water-oxidation reaction proceeds via the Si-state cycle 8 (with i=0-4), where dioxygen is produced in the transition of S3→(S4)→S0 (Fig. 1b). The high-resolution structures of PSII analyzed so far were for the dark-stable S1 state 4,5 , although a few studies on the low-resolution intermediate S-state structures have been reported by TR-SFX [9][10][11] . During the revision of our manuscript, Young et al. reported a 2F-illuminated state structure at 2.25 Å resolution where no apparent changes around O5 were observed 12 , although estimations of the resolution could yield somewhat different values so that small movement of some water molecules may escape the detection. In order to achieve resolution high enough to uncover small structural changes induced by flash illuminations yet allowing Si-state transition to proceed efficiently, we determined the optimal crystal size of PSII with a maximum length of 100 µm, which diffracted up to a resolution of 2.1 Å by a SACLA-XFEL ...

Photosynthetic water oxidation is catalyzed by the Mn4CaO5 cluster of photosystem II (PSII) with linear progression through five S-state intermediates (S0 to S4). To reveal the mechanism of water oxidation, we analyzed structures of PSII in the S1, S2, and S3 states by x-ray free-electron laser serial crystallography. No insertion of water was found in S2, but flipping of D1 Glu189 upon transition to S3 leads to the opening of a water channel and provides a space for incorporation of an additional oxygen ligand, resulting in an open cubane Mn4CaO6 cluster with an oxyl/oxo bridge. Structural changes of PSII between the different S states reveal cooperative action of substrate water access, proton release, and dioxygen formation in photosynthetic water oxidation.

Eco‐friendly and low‐cost cellulose nanofiber paper (nanopaper) is a promising candidate as a novel substrate for flexible electron device applications. Here, a thin transparent nanopaper‐based high‐mobility organic thin‐film transistor (OTFT) array is demonstrated for the first time. Nanopaper made from only native wood cellulose nanofibers has excellent thermal stability (>180 °C) and chemical durability, and a low coefficient of thermal expansion (CTE: 5–10 ppm K‐1). These features make it possible to build an OTFT array on nanopaper using a similar process to that for an array on conventional glass. A short‐channel bottom‐contact OTFT is successfully fabricated on the nanopaper by a lithographic and solution‐based process. Owing to the smoothness of the cast‐coated nanopaper surface, a solution processed organic semiconductor film on the nanopaper comprises large crystalline domains with a size of approximately 50–100 μm, and the corresponding TFT exhibits a high hole mobility of up to 1 cm2V‐1 s‐1 and a small hysteresis of below 0.1 V under ambient conditions. The nanopaper‐based OTFT also had excellent flexibility and can be formed into an arbitrary shape. These combined technologies of low‐cost and eco‐friendly paper substrates and solution‐based organic TFTs are promising for use in future flexible electronics application such as flexible displays and sensors.

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