title: Light-induced structural changes and the site of O=O bond formation in PSII caught by XFEL.
abstract: 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). The structure of PSII has been analysed at 1.9 Å resolution by synchrotron radiation X-rays, which revealed that the OEC is a Mn4CaO5cluster organized in an asymmetric, 'distorted-chair' form. This structure was further analysed with femtosecond X-ray free electron lasers (XFEL), providing the 'radiation damage-free' 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 Mn4CaO5cluster. 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 Mn4CaO5cluster 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 previously.
authors.affiliation: Research Institute for Interdisciplinary Science and Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima Naka, Okayama 700-8530, Japan.
authors.forename: Michihiro
authors.initials: M
authors.lastname: Suga
authors.affiliation: Research Institute for Interdisciplinary Science and Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima Naka, Okayama 700-8530, Japan.|Japan Science and Technology Agency, PRESTO, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan.
authors.forename: Fusamichi
authors.initials: F
authors.lastname: Akita
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
authors.forename: Michihiro
authors.initials: M
authors.lastname: Sugahara
authors.affiliation: Japan Science and Technology Agency, PRESTO, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan.|RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
authors.forename: Minoru
authors.initials: M
authors.lastname: Kubo
authors.affiliation: Research Institute for Interdisciplinary Science and Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima Naka, Okayama 700-8530, Japan.
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Nakajima
authors.affiliation: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
authors.forename: Takanori
authors.initials: T
authors.lastname: Nakane
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
authors.forename: Keitaro
authors.initials: K
authors.lastname: Yamashita
authors.affiliation: Research Institute for Interdisciplinary Science and Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima Naka, Okayama 700-8530, Japan.
authors.forename: Yasufumi
authors.initials: Y
authors.lastname: Umena
authors.affiliation: Research Institute for Interdisciplinary Science and Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima Naka, Okayama 700-8530, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Nakabayashi
authors.affiliation: Research Institute for Interdisciplinary Science and Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima Naka, Okayama 700-8530, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Yamane
authors.affiliation: Research Institute for Interdisciplinary Science and Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima Naka, Okayama 700-8530, Japan.
authors.forename: Takamitsu
authors.initials: T
authors.lastname: Nakano
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.|Institute for Protein Research, Osaka University, Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Mamoru
authors.initials: M
authors.lastname: Suzuki
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.|Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Masuda
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.|Department of Cell Biology and Anatomy, Graduate School of Medicine, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Inoue
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.|Department of Chemistry, Graduate School of Science, Kobe University, 1-1 Rokkodai, Nada-ku, Kobe 657-8501, Japan.
authors.forename: Tetsunari
authors.initials: T
authors.lastname: Kimura
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Nomura
authors.affiliation: Research Institute for Interdisciplinary Science and Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima Naka, Okayama 700-8530, Japan.
authors.forename: Shinichiro
authors.initials: S
authors.lastname: Yonekura
authors.affiliation: Research Institute for Interdisciplinary Science and Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima Naka, Okayama 700-8530, Japan.
authors.forename: Long-Jiang
authors.initials: LJ
authors.lastname: Yu
authors.affiliation: Research Institute for Interdisciplinary Science and Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima Naka, Okayama 700-8530, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Sakamoto
authors.affiliation: Research Institute for Interdisciplinary Science and Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima Naka, Okayama 700-8530, Japan.|Department of Picobiology, Graduate School of Life Science, University of Hyogo, 3-2-1 Kouto, Kamigori-cho, Ako-gun, Hyogo 678-1297, Japan.
authors.forename: Taiki
authors.initials: T
authors.lastname: Motomura
authors.affiliation: Research Institute for Interdisciplinary Science and Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima Naka, Okayama 700-8530, Japan.|Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, No.20 Nanxincun, Xiangshan, Beijing 100093, China.
authors.forename: Jing-Hua
authors.initials: JH
authors.lastname: Chen
authors.affiliation: Division of Material Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Kato
authors.affiliation: Division of Material Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan.
authors.forename: Takumi
authors.initials: T
authors.lastname: Noguchi
authors.affiliation: Japan Synchrotron Radiation Research Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5198, Japan.
authors.forename: Kensuke
authors.initials: K
authors.lastname: Tono
authors.affiliation: Japan Synchrotron Radiation Research Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5198, Japan.
authors.forename: Yasumasa
authors.initials: Y
authors.lastname: Joti
authors.affiliation: Japan Synchrotron Radiation Research Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5198, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kameshima
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
authors.forename: Takaki
authors.initials: T
authors.lastname: Hatsui
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.|Department of Cell Biology, Graduate School of Medicine, Kyoto University, Yoshidakonoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.
authors.forename: Eriko
authors.initials: E
authors.lastname: Nango
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
authors.forename: Rie
authors.initials: R
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authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
authors.forename: Hisashi
authors.initials: H
authors.lastname: Naitow
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Matsuura
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
authors.forename: Ayumi
authors.initials: A
authors.lastname: Yamashita
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Yamamoto
authors.affiliation: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
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authors.initials: O
authors.lastname: Nureki
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.|Japan Synchrotron Radiation Research Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5198, Japan.
authors.forename: Makina
authors.initials: M
authors.lastname: Yabashi
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Ishikawa
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.|Department of Cell Biology, Graduate School of Medicine, Kyoto University, Yoshidakonoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.
authors.forename: So
authors.initials: S
authors.lastname: Iwata
authors.affiliation: Research Institute for Interdisciplinary Science and Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima Naka, Okayama 700-8530, Japan.|Department of Picobiology, Graduate School of Life Science, University of Hyogo, 3-2-1 Kouto, Kamigori-cho, Ako-gun, Hyogo 678-1297, Japan.|Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, No.20 Nanxincun, Xiangshan, Beijing 100093, China.
authors.forename: Jian-Ren
authors.initials: JR
authors.lastname: Shen
mesh_terms: D055162:Biocatalysis / Q000528:radiation effects; D003461:Crystallography / Q000379:methods*; D000458:Cyanobacteria / Q000737:chemistry; D004579:Electron Transport / Q000528:radiation effects; D004583:Electrons; D005583:Fourier Analysis; D007834:Lasers; D008027:Light; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D019159:Nonheme Iron Proteins / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects; D010100:Oxygen / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects*; D045332:Photosystem II Protein Complex / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects*; D011522:Protons; D013696:Temperature; D013997:Time Factors; D014867:Water / Q000737:chemistry / Q000378:metabolism
chemical_list: D019159:Nonheme Iron Proteins; D045332:Photosystem II Protein Complex; D011522:Protons; D014867:Water; D008345:Manganese; D010100:Oxygen