Photosystem II (PSII) is a huge membrane-protein complex consisting of 20 different subunits with a total molecular mass of 350 kDa for a monomer. It catalyses light-driven water oxidation at its catalytic centre, the oxygen-evolving complex (OEC)1,2,3. The structure of PSII has been analysed at 1.9 Å resolution by synchrotron radiation X-rays, which revealed that the OEC is a Mn4CaO5 cluster organized in an asymmetric, ‘distorted-chair’ form4. This structure was further analysed 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 owing to the lack of intermediate-state structures. Here we describe the structural changes in PSII induced by two-flash illumination at room temperature at a resolution of 2.35 Å using time-resolved serial femtosecond crystallography with an XFEL provided by the SPring-8 ångström compact free-electron laser. An isomorphous difference Fourier map between the two-flash and dark-adapted states revealed two areas of apparent changes: around the QB/non-haem iron and the Mn4CaO5 cluster. The changes around the QB/non-haem iron region reflected the electron and proton transfers induced by the two-flash illumination. In the region around the OEC, a water molecule located 3.5 Å from the Mn4CaO5 cluster disappeared from the map upon two-flash illumination. This reduced the distance between another water molecule and the oxygen atom O4, suggesting that proton transfer also occurred. Importantly, the two-flash-minus-dark isomorphous difference Fourier map showed an apparent positive peak around O5, a unique μ4-oxo-bridge located in the quasi-centre of Mn1 and Mn4 (refs 4,5). This suggests the 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 consistent with that proposed previously6,7.
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Protein Data Bank
We thank K. Kawakami and N. Kamiya for sharing unpublished information, H. Mino for information on the flash illumination conditions and H. Ago and K. Yamaguchi for discussions. This work was supported by a program for promoting the enhancement of research universities at Okayama University, JSPS KAKENHI Grant Nos JP15H01642, JP16H06162, JP16H06296 (M. Suga), JP16K21181 (F.A.), JP15H05588 (Y.U.), JP15H03841, JP15H01055 (M.K.) and JP24000018 (J.-R.S.), an X-ray Free Electron Laser Priority Strategy Program (J.-R.S., S.Iw.) from MEXT, Japan, an Asahi Glass Foundation (F.A.), a Kato Memorial Bioscience Foundation (F.A.), an Inamori Foundation (M. Suga), the Research Acceleration Program from Japan Science and Technology agency (JST) (S.Iw.), PRESTO from JST (M.K. and F.A.), a grant from Pioneering Project ‘Dynamic Structural Biology’ of RIKEN (M.K.), and the Strategic Priority Research Program of CAS (XDB17030100) (J.-R.S.). The XFEL experiments were performed at beamline 3 of SACLA with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (proposal nos 2013B1259, 2014A1243, 2014A6927, 2014B1281, 2014B6927, 2014B8048, 2015A1108, 2015A6522, 2015B2108, 2015B6522, 2015B8044, 2016A2542, 2016A6621 and 2016A8033), and we thank the staff at SACLA for their help. We also acknowledge computational support from the SACLA HPC system and the Mini-K supercomputer system.
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Nature Structural & Molecular Biology (2019)