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 ...

Bacteriorhodopsin (bR) is a light-driven proton pump and a model membrane transport protein. We used time-resolved serial femtosecond crystallography at an x-ray free electron laser to visualize conformational changes in bR from nanoseconds to milliseconds following photoactivation. An initially twisted retinal chromophore displaces a conserved tryptophan residue of transmembrane helix F on the cytoplasmic side of the protein while dislodging a key water molecule on the extracellular side. The resulting cascade of structural changes throughout the protein shows how motions are choreographed as bR transports protons uphill against a transmembrane concentration gradient.

Serial femtosecond X-ray crystallography (SFX) has revolutionized atomic-resolution structural investigation by expanding applicability to micrometer-sized protein crystals, even at room temperature, and by enabling dynamics studies. However, reliable crystal-carrying media for SFX are lacking. Here we introduce a grease-matrix carrier for protein microcrystals and obtain the structures of lysozyme, glucose isomerase, thaumatin and fatty acid-binding protein type 3 under ambient conditions at a resolution of or finer than 2 Å.

Reactive oxygen species (ROS) have been identified as central mediators in certain signalling events. In the heart, ROS have important functions in ischaemia/reperfusion-induced cardiac injury and in cytokine-stimulated hypertrophy. Extracellular signal-regulated kinase (ERK) is one of the ROS-responsive serine/threonine kinases. Previous studies showed that tyrosine kinases and small G proteins are involved in the activation of ERK by ROS; however, the initial target protein of ROS that leads to ERK activation remains unknown. Here we show that inhibition of the betagamma-subunit of G protein (G betagamma) attenuates hydrogen peroxide (H2O2)-induced ERK activation in rat neonatal cardiomyocytes. The G betagamma-responsive ERK activation induced by H2O2 is independent of ligands binding to Gi-coupled receptors, but requires phosphatidylinositol-3-kinase and Src activation. In in vitro studies, however, treatment with H2O2 increases [35S]GTP-gammaS binding to cardiac membranes and directly activates purified heterotrimeric Gi and Go but not Gs. Analysis using heterotrimeric Go and its individual subunits indicates that H2O2 modifies G alpha(o) but not G betagamma, which leads to subunit dissociation. We conclude that G alpha(i) and G alpha(o) are critical targets of oxidative stress for activation of ERK.

Serial femtosecond crystallography (SFX) allows structures of proteins to be determined at room temperature with minimal radiation damage. A highly viscous matrix acts as a crystal carrier for serial sample loading at a low flow rate that enables the determination of the structure, while requiring consumption of less than 1 mg of the sample. However, a reliable and versatile carrier matrix for a wide variety of protein samples is still elusive. Here we introduce a hydroxyethyl cellulose-matrix carrier, to determine the structure of three proteins. The de novo structure determination of proteinase K from single-wavelength anomalous diffraction (SAD) by utilizing the anomalous signal of the praseodymium atom was demonstrated using 3,000 diffraction images.