Humboldt-Universität zu Berlin - Institut für Biologie

Towards the first atomic movie of water oxidation in Photosystem II

Sfb1078 (2017 – 2020): Protonation Dynamics in Protein Function
Project A5: Zouni/Dobbek
Principal Investigators: Zouni, Dobbek
Prof. A. Zouni: supervision of PhD R. Hussein; TA J. Wersig
Dobbek group: Postdoc Dr. M. Ibrahim


Oxygenic photosynthesis, which is found in plants, algae and cyanobacteria, is considered the basis for aerobic life on earth. It provides us with food and the oxygen present in our atmosphere. The photo-oxidation of water takes place in Photosystem II (PSII) that is situated in the thylakoid membrane and contains the oxygen-evolving complex (OEC), where electrons are extracted from water to release molecular oxygen and protons. Energy is stored in the form of molecules reduced by the electrons taken from water and energy-rich molecules (ATP) generated by using a proton gradient across the thylakoid membrane.

The OEC consists of a cluster having four manganese (Mn) ions and one calcium (Ca) ion connected by μ-oxo bridges (Mn4CaO5 cluster). This metal complex cycles through five different intermediate states, known as S states (S0, S1, S2, S3, and S4), in which S1 is the dark-stable state and S3 is the last semi- stable state before O – O bond formation and O2evolution. A detailed understanding of the O – O bond formation mechanism remains a challenge and will require elucidation of both the structures of the OEC in the different S-states and the binding of the two substrate water molecules to the catalytic site.

Our ultimate goal is to make a complete high-resolution atomic movie of photosynthesis by capturing many structural frames from the entire process, including the elusive transient states S3-S4-S0, where the molecular oxygen is formed after breaking down two water molecules (substrates). To achieve this goal, we apply femtosecond pulses generated from an X-ray Free Electron Laser (XFEL) to obtain damage-free crystal structures at room temperature.

Kok cycle.png


Figure 1a, Schematic overview of the reaction center in the PSII core complex (PSIIcc) with the redox-active cofactors are shown, which are involved in the light-induced electron transfer on the acceptor side. The coupled light-induced water oxidation reaction takes place at the OEC at the catalytic center of the Mn4CaO5 cluster on the donor side. b, Schematic overview of the changes of the bonds in the Mn4CaO5 cluster of the OEC during the oxygen-dependent Kok cycle with the respective schematic structure for each of the measured S-states. The atomic distances within the Mn4CaO5 cluster are given in ångström (Kern et al, Nature , 2018)


Recent results

Recently and for the first time, using SFX crystallography and in a combination with simultaneous X-ray emission spectroscopy, we captured all (meta-) stable states of Kok's cycle as high-resolution structures (2.04–2.08 Å) (Kern et al, Nature , 2018) (Figure 1).

 In addition, we reported structures of two transient states at 150 and 400 µs, revealing notable structural changes including the binding of one additional 'water', Ox, during the S2 → S3 state transition.


Objectives and future plans

  • Currently we are working on elucidating the atomic structures of the transient states between S2 and S3 and between S3 and S0 .
  • Applying Neutron diffraction studies as complementary to X-ray diffraction analysis: neutron diffraction has the ability to visualize protons and can be therefore, help in mapping out water transport channels and revealing details of the hydrogen-bonding network surrounding the OEC, which is difficult to reveal using X-ray diffraction.
    We have been working on optimizing the crystals size and quality of the dPSIIcc crystals to be suitable for neutron diffraction studies. As a preliminary result, a macroseeding protocol was developed to generate a large single dPSIIcc crystal of 0.2-0.3 mm (long axis) (Figure 2). A large single dPSIIcc crystal of 2.0 mm × 0.8 mm × 0.3 mm was measured at the MaNDi instrument of the SNS at ORNL. Upon exposure for 24 h at room temperature, weak diffraction with a maximum resolution of 8.0 Å was observed (Hussein et al, Cryst. Growth Des. , 2017).
  • Very recently, we have grown PSII crystals in D2O for neutron diffraction measurements.

Figure 2 Single macrocrystal of dimeric PSIIcc obtained by macroseeding (Hussein et al, Cryst.Growth Des., 2017).

Figure 2 Single macrocrystal of dimeric PSIIcc obtained by macroseeding (Hussein et al, Cryst. Growth Des., 2017).


National and international cooperations:

  • Prof Holger Dau (FU Berlin)
  • Prof. Nicoleta Bondar (FU Berlin)
  • Prof. J. Messinger (Uppsala University (Sweden))
  • Prof. Nicholas Sauter, Lawrence Berkeley National Laboratory, Berkeley, USA
  • Dr. Jan Kern, Lawrence Berkeley National Laboratory, Berkeley, USA
  • Dr. Junko Yano, Lawrence Berkeley National Laboratory, Berkeley, USA
  • Dr. Vittal Yachandra, Lawrence Berkeley National Laboratory, Berkeley, USA
  • 4. XFEL Beamtimes: LCLS (USA), SwissFEL (Switzerland), PAL (Korea); SACLA (Japan)
  • X-ray sychrotrons: BESSY (Berlin), DESY (Hamburg), ALS (Stanford, USA)
  • Neutron diffraction measurements: MaNDi Instrument (Oakridge (USA)

    Mohamed Ibrahim.png

Dr. M. Ibrahim (Postdoc in AG Dobbek group)

Rana Ali.png

R. Hussein (PhD in AG Zouni group)

Recently published research article:


  1. -Kern J, Chatterjee R, Young ID, FD Fuller, Lassalle L, Ibrahim M, Gul S, Fransson T; Brewster AS, Alonso-Mori R, Hussein R, Zhang M, Douthit L, de Lichtenberg C, Cheah MH, Shevela D, Wersig J, Seuffert I, Sokaras D, Pastor E, Weninger Cl, Kroll T,…, Dobbek H, Adams PD, Bergmann U, Sauter NK,  Zouni A , Messinger J, Yano J and Yachandra VK. Structures of the intermediates of Kok's photosynthetic water oxidation clock. Nature volume 563, pages 421-425 ( 2018 ).
  2. Hussein R, Mohamed Ibrahim M, Chatterjee R, Coates L, Müh F, Vittal K. Yachandra VK, Yano J, Jan Kern J, Dobbek H, and  Zouni A. Optimizing Crystal Size of Photosystem II by Macroseeding: Toward Neutron Protein Crystallography. Cryst. Growth Des., Article ASAP DOI: 10.1021 / acs.cgd.7b00878 Publication Date (Web):  November 13, 2017 .
  3. Bommer M, Coates L, Dau H,  Zouni A , Dobbek H. Protein crystallization and initial neutron diffraction studies of the photosystem II subunit PsbO. Acta Crystallogr F Struct Biol Commun. 2017 Sep 1; 73 (Pt 9): 525-531. doi: 10.1107 / S2053230X17012171. Epub  2017 Aug 31

  4. Fuller F.D, Gul S, Chatterjee R, Burgie E.S, Young I.D, Lebrette H, Srinivas V, Brewster A.S, Michels-Clark T, Clinger J.A, Andi B, Ibrahim M, Pastor E, de Lichtenberg C, Hussein R, Pollock C.J, Zhang M, Stan C.A, Kroll T,…,Zouni A, Johannes Messinger, Uwe Bergmann,…,Orville A.M, Kern J, Yachandra V.K & Yano J. Drop-on-demand sample delivery for studying biocatalysts in action at X-ray free-electron lasers. Nature Methods 14, 443–449 (2017) doi:10.1038/nmeth.4195 Published online: 27 February 2017

  5. Young ID, Ibrahim M, Chatterjee R, Gul S, Koroidov S, Brewster AS, Tran R, Alonso-Mori R, Kroll T, Michels-Clark T, Laksmono H, Sierra RG, Stan CA, Hussein R, Zhang M, Douthit L, Kubin M, de Lichtenberg C, Pham LV, Nilsson H, Cheah MH, Shevela D, Saracini C, Bean MA, Seuffert I, Sokaras D, Weng TC. Pastor E,…, Zouni A, Messinger J, Bergmann U, Sauter NK, Kern J, Yanchandra VK, Junko Yano. Structure of photosystem II and substrate binding at room temperature. Nature 540, 453–457 (15 December 2016) doi:10.1038/nature20161
  6. Alonso-Mori R, Asa K, Bergmann U, Brewster AS, Chatterjee R, Cooper JK, Frei HM, Fuller FD, Goggins E, Gul S, Fukuzawa H, Iablonskyi D, Ibrahim M, Katayama T, Kroll T, Kumagai Y, McClure BA, Messinger J, Motomura K, Nagaya K, Nishiyama T, Saracini C, Sato Y, Sauter NK, Sokaras D, Takanashi T, Togashi T, Ueda K, Weare WW, Weng TC, Yabashi M, Yachandra VK, Young ID, Zouni A, Kern JF, Yano J. Towards characterization of photo-excited electron transfer and catalysis in natural and artificial systems using XFELs. Faraday Discuss. 2016 Dec 16;194:621-638. doi: 10.1039/c6fd00084c.

  7. Bommer M, Bondar AN, Zouni A, Dobbek H, Dau H. Crystallographic and Computational Analysis of the Barrel Part of the PsbO Protein of Photosystem II: Carboxylate–Water Clusters as Putative Proton Transfer Relays and Structural Switches. Biochemistry201655 (33), pp 4626–4635 DOI: 10.1021/acs.biochem.6b00441
  8. Sauter NK, Echols N, Adams PD, Zwart PH, Kern J, Brewster AS, Koroidov S, Alonso-Mori R, Zouni A, Messinger J, Bergmann U, Yano J, Yachandra V, No observable conformational changes in PSII. Nature 533, E1–E2(19 May 2016) doi:10.1038/nature17983
  9. Roessler CG, Agarwal R, Allaire M, Alonso-Mori R, Andi B, Bachega JF, Bommer M, Brewster AS, Browne MC, Chatterjee R, Cho E, Cohen AE, Cowan M, Datwani S, Davidson VL, Defever J, Eaton B, Ellson R, Feng Y, Ghislain LP, Glownia JM, Han G, Hattne J, Hellmich J, Héroux A, Ibrahim M, Kern J, Kuczewski A, Lemke HT, Liu P, Majlof L, McClintock WM, Myers S, Nelsen S, Olechno J, Orville AM, Sauter NK, Soares AS, Soltis SM, Song H, Stearns RG, Tran R, Tsai Y, Uervirojnangkoorn M, Wilmot CM, Yachandra V, Yano J, Yukl ET, Zhu D, Zouni A. Acoustic Injectors for Drop-On-Demand Serial Femtosecond CrystallographyStructure. 2016 Apr 5;24(4):631-40. doi: 10.1016/j.str.2016.02.007. Epub 2016 Mar 17
  10. Baxter E.L, Aquila L, Alonso-Mori R, Barnes C.O, Bonagura C.A, Brehmer W, Brunger A.T, Calero G, Caradoc-Davies T.T, Chatterjee R, Degrado W.F, Fraser J.S, Ibrahim M, Kern J, Kobilka B.K, Kruse A.C, Larsson K.M, Lemke, H.T, Lyubimov A.Y, Manglik A, McPhillips S.E, Norgren E, Pang S.S, Soltis S.M, Song J, Thomaston J, Tsai Y, Weis W.I, Woldeyes R.A, Yachandra V, Yano J, Zouni A, and Cohen A.E, High-density grids for efficient data collection from multiple crystals, Acta Crystallographica Section D Structural Biology 01/2016; 72(1). DOI: 10.1107/S2059798315020847

  11. Sierra R.G, Gati C, Laksmono H, Han Dao E, Gul S, Fuller F, Kern J, Chatterjee R, Ibrahim M, Brewster A.S, Young I.D, Michels-Clark T, Aquila A, Liang M, Hunter M.S, Koglin J.E, Boutet S, Junco E.A, Hayes B, Bogan M.J, Hampton C.Y, Puglisi E.V, Sauter N.K, Stan C.A, Zouni A, Yano J, Yachandra V.K, Soltis S.M, Puglisi J.D, and DeMirci H, Concentric-flow electrokinetic injector enables serial crystallography of ribosome and photosystem II, Nature Methods.11/2015; DOI: 10.1038/nmeth.3667
  12. Ibrahim M, Chatterjee R, Hellmich J, Tran R, Bommer M, Yachandra V.K, Yano J, Kern J and Zouni A, Improvements in serial femtosecond crystallography of photosystem II by optimizing crystal uniformity using microseeding procedures, Structural Dynamics 07/2015; 2(4):041705. DOI: 10.1063/1.4919741
  13. Kern, J., Tran, R., Alonso-Mori, R., Koroidov, S., Echols, N., Hattne, J., Ibrahim M., Gul, S, Laksmono, H., Sierra, R. G., Gildea, R. J., Han, G., Hellmich, J., Lassalle-Kaiser, B., Chatterjee, R., Brewster, A. S., Stan, C. A., Glöckner, C., Lampe, A., DiFiore, D.,    Milathianaki, D., Fry, A. R., Seibert, M. M., Koglin, J. E., Gallo, E., Uhlig, J., Sokaras, D.,   Weng, T.-C., Zwart, P. H., Skinner, D. E., Bogan, M. J., Messerschmidt, M., Glatzel, P., Williams, G. J., Boutet, S., Adams, P. D., Zouni, A., Messinger, J., Sauter, N. K., Bergmann, U., Yano, J., and Yachandra, V. K., Taking snapshots of photosynthetic water oxidation using femtosecond X-ray diffraction and spectroscopy Nat. Commun. 20145, DOI: 10.1038/ncomms5371
  14. Hellmich J, Bommer M, Burkhardt A, Ibrahim M, Kern J, Meents A, Müh F, Dobbek H, Zouni A, Native-like Photosystem II Superstructure at 2.44 Å Resolution through Detergent Extraction from the Protein Crystal, Structure 201422, 1607-1615
  15. Tran R., Kern J., Hattne J., Kroidov S., Hellmich J., Alonso-Mori R., Sauter NK, Bergmann U., Messinger J.,  Zouni A. , Yano J., Yachandra VK, The Mn 4 Ca photosynthetic water-oxidation catalyst studied by simultaneous X-ray spectroscopy and crystallography using an X-ray free-electron laser,  Phil. Trans. R. Soc. (2014) : 2014369 20130324, DOI: 10.1098 / rstb.2013.0324