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.

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.

Mononuclear copper(II)-superoxo complexes 2(X)-OO(*) having triplet (S = 1) ground states were obtained via reaction of O(2) with the copper(I) starting materials 1(X) supported by tridentate ligands L(X) [1-(2-p-X-phenethyl)-5-(2-pyridin-2-ylethyl)-1,5-diazacyclooctane; X = CH(3), H, NO(2)] in various solvents. The superoxo complexes 2(X)-OO(*) mimic the structure [tetrahedral geometry with an end-on (eta(1))-bound O(2)(*-)] and the aliphatic C-H bond activation chemistry of peptidylglycine alpha-hydroxylating monooxygenase and dopamine beta-monooxygenase.

Metal-O 2 adducts, such as metal-superoxo and -peroxo species, are key intermediates often detected in the catalytic cycles of dioxygen activation by metalloenzymes and biomimetic compounds. The synthesis and spectroscopic characterization of an end-on nickel(II)-superoxo complex with a 14-membered macrocyclic ligand was reported previously. Here we report the isolation, spectroscopic characterization, and high-resolution crystal structure of a mononuclear side-on nickel(III)-peroxo complex with a 12-membered macrocyclic ligand, [Ni(12-TMC)(O 2 )] + (1) 4,7,4,7,. Different from the end-on Ni(II)-superoxo complex, the Ni(III)-peroxo complex is not reactive in electrophilic reactions, but is capable of conducting nucleophilic reactions. The Ni(III)-peroxo complex transfers the bound dioxygen to manganese(II) complexes, thus affording the corresponding nickel(II) and manganese(III)-peroxo complexes. The present results demonstrate the significance of supporting ligands in tuning the geometric and electronic structures and reactivities of metal-O 2 intermediates that have been shown to have biological as well as synthetic usefulness in biomimetic reactions.Metalloenzymes activate dioxygen to carry out a variety of biological reactions including biotransformation of naturally occurring molecules, oxidative metabolism of xenobiotics, and oxidative phosphorylation. One goal in biomimetic research is to understand the mechanistic details of dioxygen activation and oxygenation reactions and the structures of reactive intermediates occurring at the active sites of the metalloenzymes 1 . In the unified mechanism of dioxygen activation, dioxygen first binds to a reduced metal center that forms metal-superoxo and -peroxo intermediates, followed by O-O bond cleavage leading to the formation of high-valent metal-oxo species that are believed to carry out substrate oxidations 1 . Among the metal-oxygen intermediates, mononuclear metal-O 2 adducts, such * Corresponding authors: wwnam@ewha.ac.kr, edward.solomon@stanford.edu. Correspondence and requests for materials should be addressed to W.N. Author contributions: J.C., E.I.S., and W.N. conceived and designed the experiments; J.C., R.S., J.A., S.Y.K., and M.K. performed the experiments; J.C., R.S., J.A., M.K., and T.O. analyzed the data; J.C., R.S., E.I.S., and W.N. co-wrote the paper. 25 . We now report for the first time the synthesis, spectroscopic and electronic properties, and crystal structure of a mononuclear side-on (η 2 ) nickel(III)-peroxo complex stabilized by a 12-membered macrocyclic ligand, [Ni(III)(12-TMC)(O 2 )] + (1) (12-TMC = 1,4,7,10-tetramethyl-1,4,7,10-tetraazacyclododecane). The reactivities of the Ni(III)-peroxo complex in electrophilic and nucleophilic reactions and peroxo group transfer to other metal complexes have been discussed as well. Results and discussionThe starting nickel complex, [Ni(12-TMC)(CH 3 CN)] 2+ (3), was synthesized and characterized with UV-vis absorption spectroscopy, electrospray ionization mass spectrometry (ESI MS), and X...

The low-frequency mode activity of metalloporphyrins has been studied for iron porphine-halides (Fe(P)(X), X = Cl, Br) and nitrophorin 4 (NP4) using femtosecond coherence spectroscopy (FCS) in combination with polarized resonance Raman spectroscopy and density functional theory (DFT). It is confirmed that the mode symmetry selection rules for FCS are the same as for Raman scattering and that both Franck-Condon and Jahn-Teller mode activities are observed for Fe(P)(X) under Soret resonance conditions. The DFT-calculated low-frequency (20-400 cm -1 ) modes, and their frequency shifts upon halide substitution, are in good agreement with experimental Raman and coherence data, so that mode assignments can be made. The doming mode is located at ~80 cm -1 for Fe(P)(Cl) and at ~60 cm -1 for Fe(P)(Br). NP4 is also studied with coherence techniques, and the NO-bound species of ferric and ferrous NP4 display a mode at ~30-40 cm -1 that is associated with transient heme doming motion following NO photolysis. The coherence spectra of three ferric derivatives of NP4 with different degrees of heme ruffling distortion are also investigated. We find a mode at ~60 cm -1 whose relative intensity in the coherence spectra depends quadratically on the magnitude of the ruffling distortion. To quantitatively account for this correlation, a new "distortion-induced" Raman enhancement mechanism is presented. This mechanism is unique to low-frequency "soft modes" of the molecular framework that can be distorted by environmental forces. These results demonstrate the potential of FCS as a sensitive probe of dynamic and functionally important nonplanar heme vibrational excitations that are induced by the protein environmental forces or by the chemical reactions in the aqueous phase.

Metal-dioxygen adducts are key intermediates detected in the catalytic cycles of dioxygen activation by metalloenzymes and biomimetic compounds. In this study, mononuclear cobalt(III)-peroxo complexes bearing tetraazamacrocyclic ligands, (17) Å and 1.438(6) Å, respectively. The cobalt(III)-peroxo complexes showed reactivities in the oxidation of aldehydes and O 2 -transfer reactions. In the aldehyde oxidation reactions, the nucleophilic reactivity of the cobalt-peroxo complexes was significantly dependent on the ring size of the macrocyclic ligands, with the reactivity of [Co(13-TMC)(O 2 )] + > [Co(12-TMC)(O 2 )] + . In the O 2 -transfer reactions, the cobalt(III)-peroxo complexes transferred the bound peroxo group to a manganese(II) complex, affording the corresponding cobalt(II) and manganese(III)-peroxo complexes. The reactivity of the cobalt-peroxo complexes in O 2 -transfer was also significantly dependent on the ring size of tetraazamacrocycles, and the reactivity order in the O 2 -transfer reactions was the same as that observed in the aldehyde oxidation reactions.wwnam@ewha.ac.kr. Supporting Information Available. Non-phase shift corrected Fourier transform data, their corresponding EXAFS data, and FEFF best fit parameters for 2 and 4. X-ray crystal structures of 1 and 3, resonance Raman data of 2 and 4, EPR data of 1 -4, COSY NMR spectrum of 2, kinetic data of the reactions of 4 with 2-PPA and para-X-Ph-CHO, UV-vis spectral changes of the O 2 -transfer reactions of 2 and 4, and X-ray crystallographic files of 1 -4 in CIF format. This material is available free of charge via the Internet at

Femtosecond coherence spectroscopy is applied to a series of ferric heme protein samples. The low-frequency vibrational spectra that are revealed show dominant oscillations near 40 cm(-1). MbCN is taken as a typical example of a histidine-ligated, six-coordinate, ferric heme and a comprehensive spectroscopic analysis is carried out. The results of this analysis reveal a new heme photoproduct species, absorbing near 418 nm, which is consistent with the photolysis of the His(93) axial ligand. The photoproduct undergoes subsequent rebinding/recovery with a time constant of approximately 4 ps. The photoproduct lineshapes are consistent with a photolysis quantum yield of 75-100%, although the observation of a relatively strong six-coordinate heme coherence near 252 cm(-1) (assigned to nu(9) in the MbCN Raman spectrum) suggests that the 75% lower limit is much more likely. The phase and amplitude excitation profiles of the low-frequency mode at 40 cm(-1) suggest that this mode is strongly coupled to the MbCN photoproduct species and it is assigned to the doming mode of the transient penta-coordinated material. The absolute phase of the 40 cm(-1) mode is found to be pi/2 on the red side of 418 nm and it jumps to 3pi/2 as excitation is tuned to the blue side of 418 nm. The absolute phase of the 40 cm(-1) signal is not explained by the standard theory for resonant impulsive stimulated Raman scattering. New mechanisms that give a dominant momentum impulse to the resonant wavepacket, rather than a coordinate displacement, are discussed. The possibilities of heme iron atom recoil after photolysis, as well as ultrafast nonradiative decay, are explored as potential ways to generate the strong momentum impulse needed to understand the phase properties of the 40 cm(-1) mode.