Untangling the sequence of events during the S2 → S3 transition in photosystem II and implications for the water oxidation mechanism Mohamed Ibrahima,1, Thomas Franssonb,1, Ruchira Chatterjeec,1, Mun Hon Cheahd,1, Rana Husseina, Louise Lassallec, Kyle D. Sutherlinc, Iris D. Youngc,2, Franklin D. Fullere, Sheraz Gulc, In-Sik Kimc, Philipp S. Simonc, Casper de Lichtenbergd,f, Petko Chernevd, Isabel Bogaczc, Cindy C. Phamc, Allen M. Orvilleg,h, Nicholas Saicheki, Trent Northeni, Alexander Batyuke, Sergio Carbajoe, Roberto Alonso-Morie, Kensuke Tonoj,k, Shigeki Owadaj,k, Asmit Bhowmickc, Robert Bolotovskyc, Derek Mendezc, Nigel W. Moriartyc, James M. Holtonc,l,m, Holger Dobbeka, Aaron S. Brewsterc, Paul D. Adamsc,n, Nicholas K. Sauterc, Uwe Bergmanno, Athina Zounia,3, Johannes Messingerd,f,3, Jan Kernc, Vittal K. Yachandrac,3, and Junko Yanoc,3 aInstitut für Biologie, Humboldt-Universität zu Berlin, D-10115 Berlin, Germany; bInterdisciplinary Center for Scientific Computing, University of Heidelberg, 69120 Heidelberg, Germany; cMolecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; dDepartment of Chemistry - Ångström, Molecular Biomimetics, Uppsala University, SE 75120 Uppsala, Sweden; eLinac Coherent Light Source, SLAC National Accelerator Laboratory, Menlo Park, CA 94025; fInstitutionen för Kemi, Kemiskt Biologiskt Centrum, Umeå Universitet, SE 90187 Umeå, Sweden; gDiamond Light Source Ltd, Harwell Science and Innovation Campus, OX11 0DE Didcot, United Kingdom; hResearch Complex at Harwell, Rutherford Appleton Laboratory, OX11 0FA Didcot, United Kingdom; iEnvironmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; jJapan Synchrotron Radiation Research Institute, Sayo-cho, Sayo-gun, 679-5198 Hyogo, Japan; kRIKEN SPring-8 Center, Sayo-cho, Sayo-gun, 679-5148 Hyogo, Japan; lStanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, CA 94025; mDepartment of Biochemistry and Biophysics, University of California, San Francisco, CA 94158; nDepartment of Bioengineering, University of California, Berkeley, CA 94720; and oStanford PULSE Institute, SLAC National Accelerator Laboratory, Menlo Park, CA 94025 Edited by Pierre Joliot, Institut de Biologie Physico-Chimique, Paris, France, and approved April 1, 2020 (received for review January 11, 2020) In oxygenic photosynthesis, light-driven oxidation of water to mo- chlorophyll a moiety P680 (most likely consisting of the four exci- lecular oxygen is carried out by the oxygen-evolving complex tonically coupled chlorophylls PD1,PD2,ChlD1,andChlD2)andthe BIOPHYSICS AND COMPUTATIONAL BIOLOGY (OEC) in photosystem II (PS II). Recently, we reported the room- neighboring pheophytin molecules PheoD1/D2 as well as the two temperature structures of PS II in the four (semi)stable S-states, S1, plastoquinones QA and QB. The one-electron photochemistry at S2,S3, and S0, showing that a water molecule is inserted during the → S2 S3 transition, as a new bridging O(H)-ligand between Mn1 Significance and Ca. To understand the sequence of events leading to the for- mation of this last stable intermediate state before O formation, 2 A new bridging oxygen ligand is incorporated between one of we recorded diffraction and Mn X-ray emission spectroscopy (XES) the Mn atoms and Ca in the Mn Ca cluster during the transition data at several time points during the S → S transition. At the 4 2 3 from the one-photon induced S intermediate state to the two- electron acceptor site, changes due to the two-electron redox 2 photon induced S state in the catalytic water oxidation re- chemistry at the quinones, Q and Q , are observed. At the donor 3 A B action in photosystem II. However, the sequence of events leading site, tyrosine Y and His190 H-bonded to it move by 50 μs after the Z to this change is not known. Here we report an X-ray crystallog- second flash, and Glu189 moves away from Ca. This is followed by raphy and spectroscopy study at room temperature using an X-ray Mn1 and Mn4 moving apart, and the insertion of O (H) at the X free electron laser to collect a “molecular movie” of the structural open coordination site of Mn1. This water, possibly a ligand of and oxidation state change steps leading to the insertion of this Ca, could be supplied via a “water wheel”-like arrangement of five new oxygen bridge, in the 50 μs to 200 ms time scales after waters next to the OEC that is connected by a large channel to the photon absorption, which triggers the S → S state transition. bulk solvent. XES spectra show that Mn oxidation (τ of ∼350 μs) 2 3 → during the S2 S3 transition mirrors the appearance of OX elec- Author contributions: U.B., A.Z., J.M., J.K., V.K.Y., and J.Y. designed research; M.I., T.F., tron density. This indicates that the oxidation state change and the R.C., M.H.C., R.H., L.L., K.D.S., I.D.Y., F.D.F., S.G., I.-S.K., P.S.S., C.d.L., P.C., I.B., C.C.P., insertion of water as a bridging atom between Mn1 and Ca are A.M.O., N.S., T.N., A. Batyuk, S.C., R.A.-M., K.T., S.O., A.B., U.B., A.Z., J.M., J.K., V.K.Y., highly correlated. and J.Y. performed research; T.F., R.C., M.H.C., R.H., N.S., T.N., J.M.H., A.S.B., P.D.A., N.K.S., A.Z., and J.M. contributed new reagents/analytic tools; M.I., T.F., R.C., M.H.C., L.L., K.D.S., A. Bhowmick, R.B., D.M., N.W.M., J.M.H., H.D., A.S.B., P.D.A., N.K.S., J.K., and J.Y. analyzed photosynthesis | photosystem II | water oxidation | data; and M.I., T.F., R.C., U.B., A.Z., J.M., J.K., V.K.Y., and J.Y. wrote the paper. oxygen-evolving complex | X-ray free electron laser The authors declare no competing interest. This article is a PNAS Direct Submission. ioxygen, which supports all aerobic life, is abundant in the This open access article is distributed under Creative Commons Attribution-NonCommercial- Datmosphere because of its constant regeneration via photo- NoDerivatives License 4.0 (CC BY-NC-ND). synthetic water oxidation in plants, algae, and cyanobacteria. The Data deposition: X-ray diffraction datasets and associated models have been deposited in water-splitting chemistry occurs in the oxygen-evolving complex the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (https://www.rcsb.org) under PDB ID codes 6W1O for the 0F, 6W1P for the 1F, 6W1Q (OEC) of photosystem II (PS II; Fig. 1A), which contains a het- for the 2F(50 μs), 6W1R for the 2F(150 μs), 6W1T for the 2F(250 μs), 6W1U for the eronuclear, oxo-bridged Mn4Ca cluster that catalyzes the reaction: 2F(400 μs), and 6W1V for the 2F(200 ms) data. 1M.I., T.F., R.C., and M.H.C. contributed equally to this work. → + − + + [1] 2 H2O O2 4 e 4 H . 2Present address: Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA 94158. To allow this reaction to take place, the OEC accumulates four 3To whom correspondence may be addressed. Email: [email protected], oxidizing equivalents, stepping through intermediates that are [email protected], [email protected], or [email protected]. referred to as the S-states (Si,i= 0 through 4) (Fig. 1B) (1, 2). This article contains supporting information online at https://www.pnas.org/lookup/suppl/ These oxidation reactions are driven by the light-induced charge doi:10.1073/pnas.2000529117/-/DCSupplemental. separations in the reaction center of PS II that comprises the www.pnas.org/cgi/doi/10.1073/pnas.2000529117 PNAS Latest Articles | 1of12 Downloaded by guest on September 26, 2021 ABC Acceptor site Cytoplasm YZ - •− •− + Q Q E189 2H e A B Q Q H PQ A B 2 QB W4 e- S QB Q 4F QBH2 2 A W3 •− QAQB •+ PQH2 P680 P680 Ca Q Q + Dark State 1F A B W2 1 PheoD1 Yz• Yz 1 e- Mn4(III2,IV2) 5 Mn4(III3,IV) P680 PheoD2 0 1 •+ PD2 PD1 O2 S S Yz H+ e- •− 1 4 2 2 QA QB W1 3 e- + 3 ChlD2 Donor site • Yz P680 4 ChlD1 H+ H2O P +H2O 680 TyrT Z •− S4 H+ H2O S2 Mn4(III,IV3) + - Lumen QAQB - (H , e ) •+ H+ Yz YZ OEC e- Yz e- 2F E189 4 photons 3 Yz P680 + - •+ S W4 2HO O +4H +4e •+ + W3 2 2 P680 Yz Mn4(IV4) P680• e- S3 - X Q•−Q e P680 Ca A B Q•−Q•− A B W2 1 3F 5 1 QAQBH2 Q Q A B Q W1 4 2 B 2 3 QBH2 3 4 Fig. 1. The S2 → S3 transition in PS II. (A) The structure of PS II with the membrane-embedded helices and the large membrane extrinsic regions on the luminal side of PS II are shown as cartoon in gray and in color the main electron transfer components involved in charge transfer (P680, Pheo) and stabilization with the acceptor quinones, QA and QB, toward the cytoplasm, and YZ and OEC with catalytic Mn4Ca cluster on the donor side at the interface between the membrane and the lumen-bound part of PS II. (B) Kok cycle of the water oxidation reaction that is triggered by the absorption of photons (nanosecond light flashes, 1F to 4F) in the antenna and the reaction center P680. The white and gray areas show the redox changes of the OEC (S1 to S0) and YZ together with the charge separation reactions, while the outer blue circle shows the concomitant acceptor side chemistry of the quinones.