CRC 1078/1: Structural Basis of Proton Release in Water Oxidation by Photosystem II (SP A05)

In oxygenic photosynthesis, water is oxidized and dioxygen is formed at the Mn4Ca complex of photosystem II (PSII), a 750 kDa protein-cofactor complex embedded in the thylakoid membrane of plants and cyanobacteria. Reliable structural models of PSII are essential for an understanding of functionally relevant proton dynamics. In 2001, the first crystallographic model has been presented for PSII from the thermophilic cyanobacterium Thermosynechococcus elongatus, and in 2009, we reached a resolution of 2.9 Å. Very recently, in a striking breakthrough, the crystal structure of dimeric PSII from a related thermophilic cyanobacterium (T. vulcanus) was solved at 1.9 Å resolution by Umena and coworkers. The new structure provides a solid basis for the analysis of the molecular mechanisms of water oxidation. However, there remains the problem of modification of the active site by X-ray induced Mn-reduction. In the CRC, we focus on the coupling between electron and proton transfer in photosynthetic water oxidation. For this purpose, the structural basis of the protonation dynamics in PSII, specifically the proton release from the water-oxidizing Mn4Ca complex, will be analysed by X-ray crystallography of chloride-modified variants and of site-directed mutants. In addition, the new technique of room temperature femtosecond X-ray diffraction of PSII microcrystals will be employed to overcome the Mn-reduction problem and to gain time-resolved structure information. Moreover, the necessary pre-conditions for neutron diffraction analysis will be established. The chloride-modified PSII variants and site-directed mutants will be studied by theoretical and spectroscopic techniques in collaboration with other groups.

The Photosystem II core complex (PSIIcc) catalyzes a key step in photosynthesis, the oxidation of water. Despite excellent static structures of PSIIcc, the catalytic mechanism of water-oxidization at the Mn4CaO5 cluster is not sufficiently understood. During the last three years, we made significant progress: we published a new crystal structure of PSIIcc at 2.44 Å resolution based on a new protocol involving detergent extraction from the crystal, resulting in a new crystal packing resembling the native arrangement of PSIIcc in cyanobacteria. We initiated femtosecond (fs) X-ray crystallographic measurements to unravel structural changes at the water-oxidizing complex (WOC) in the catalytic cycle. Residues structuring the water-cluster at the WOC were targeted to obtain a modified PSIIcc, where the oxygen-evolution transition (S3-S0) is affected. As a first result, we obtained microcrystals of the PsbA3 D1-variant of PSIIcc. Crystal uniformity was improved using a new microseeding protocol, yielding diffraction up to 2.5 Å. Insight into the role of the extrinsic subunit PsbO in proton translocation was gained by determining (near) atomic resolution crystal structures of a heterologously expressed PsbO-beta under conditions allowing us to detect, inter alia, small conformational changes caused by proton uptake. In the second funding period we focus on structure-function relations, on protonation-state changes of complete PSIIcc, as well as isolated PsbO-beta, and the role of individual residues in proton translocation. To reach our goals we plan to optimize the new detergent-depleted crystals to improve the resolution of PSIIcc variant crystals. The microseeding protocol for X-ray free electron laser (XFEL) measurements will be further improved by using dynamic light scattering to probe aggregation of PSIIcc under crystallization conditions. Furthermore, a macroseeding protocol for the growth of large crystals of PSIIcc for neutron diffraction will be improved. Further, we will measure residue-specific pKa values of PsbO by NMR. Additionally, the PSIIcc variants and PsbO will be studied in collaboration with other groups.

Principal Investigators
Zouni, Athina (Details) (Experimental Biophysics)
Dobbek, Holger Prof. Dr. rer. nat. (Details) (Structural Biology / Biochemistry)
Ernsting, Nikolaus Prof. Ph.D. (Details) (Senior Professor and Professors retired)
Hegemann, Peter Prof. Dr. (Details) (Experimental Biophysics)

Duration of Project
Start date: 01/2013
End date: 03/2021

Research Areas
Basic Research in Biology and Medicine, Biophysics, Life Sciences, Molecular Chemistry, Physical and Theoretical Chemistry, Physical Chemistry of Molecules, Interfaces and Liquids - Spectroscopy, Kinetics, Plant Biochemistry and Biophysics, Structural Biology

Research Areas
Erneuerbare Energien, Membran

Kern J, Alonso-Mori R, Tran R, Hattne J, Gildea R.G., Echols N, Glöckner C, Hellmich J, Laksmono H, Sierra R G, Lassalle-Kaiser B, Koroidov S, Lampe A, Han G, Gul S, DiFiore D, Milathianaki D, Fry A R, Miahnari A, Schafer D W, Messerschmidt M, Seibert M M, Jason Koglin E, Sokaras D, Weng T C, Sellberg J, Latimer M J, Grosse-Kunstleve R W, Zwart P H, White W H, Glatzel P, Adams P D, Bogan M J, Williams G j, Boutet S, Messinger J, Zouni A, Sauter N K, Yachandra V K, Bergmann U, Yano J, Simultaneous Femtosecond X-Ray Spectroscopy and Diffraction of Photosystem II at Room Temperature. Science 2013, 340, 491-495

Glöckner C, Kern J, Broser M, Zouni A, Yachandra V, Junko Y, Structural Changes of the Oygen-evolving Complex in Photosystem II during the Catalytic Cycle, J. Biol. Chem. 2013, 288, 22607-22620

Müh F., Zouni A., The nonheme iron in photosystem II, Photosynth Res (2013) 116: 295-414, DOI 10.1007/s11120-013-9926-y.

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. 2014, 5, DOI: 10.1038/ncomms5371

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 2014, 22, 1607-1615

Tran R., Kern J., Hattne J., Kroidov S., Hellmich J., Alonso-Mori R., Sauter N.K., Bergmann U., Messinger J., Zouni A., Yano J., Yachandra V.K., The Mn4Ca photosynthetic water-oxidation catalyst studied by simultaneous X-ray spectroscopy and crystallography using an X-ray free-electron laser, Phil. Trans. R. Soc. B (2014): 2014369 20130324, DOI: 10.1098/rstb.2013.0324

Brecht M, Skandary S., Hellmich J., Glöckner C., Konrad A., Hussels M., Meixner A. J., Zouni A., Schlodder E., Spectroscopic properties of photosystem II core complexes from Thermosynechococcus elongatus revealed by single-molecule experiments, Biochim Biophys Acta 2014, 1837, 773-781

Skandary S, Hussels M, Konrad A, Renger T, Müh F, Bommer M, Zouni A, Meixner A.J, and Brecht M, Variation of Exciton-Vibrational Coupling in Photosystem II Core Complexes from Thermosynechococcus elongatus As Revealed by Single-Molecule Spectroscopy, The Journal of Physical Chemistry B 02/2015; 119(11). DOI:10.1021/jp510631x

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

Müh F, DiFiore D, and Zouni A, Influence of poly(ethylene glycol) on the micelle formation of alkyl maltosides used in membrane protein crystallization, Physical Chemistry Chemical Physics 03/2015; 17(17). DOI: 10.1039/C5CP00431D

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

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

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 Crystallography. Structure. 2016 Apr 5;24(4):631-40. doi: 10.1016/j.str.2016.02.007. Epub 2016 Mar 17.

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. Biochemistry, 2016, 55 (33), pp 4626–4635 DOI: 10.1021/acs.biochem.6b00441

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.

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

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

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.

Hussein R, Mohamed Ibrahim M, Chatterjee R, Coates L, Müh F, Vittal K. Yachandra V.K., 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.

Skandary S, Müh F, Ashraf I, Ibrahim M, Metzger M, Zouni A, Meixner AJ, and Brecht M. Role of missing carotenoid in reducing the fluorescence of single monomeric photosystem II core complexes. Phys Chem Chem Phys. 2017 May 24;19(20):13189-13194. doi: 10.1039/c6cp07748j.!divAbstract

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).

Riedel M, Wersig J, Ruff A, Schuhmann W, Zouni A, Lisdat F. A Z-Scheme-Inspired Photobioelectrochemical H2 O/O2 Cell with a 1 V Open-Circuit Voltage Combining Photosystem II and PbS Quantum Dots. Angew Chem Int Ed Engl. 2019 Jan 14;58(3):801-805. doi: 10.1002/anie.201811172. Epub 2018 Dec 13.

Kemmler, L.; Ibrahim, M.; Dobbek, H.; Zouni, A.; Bondar, A.-N., Dynamic water bridging and proton transfer at a surface carboxylate cluster of photosystem II. Physical Chemistry Chemical Physics 2019, 21 (45), 25449-25466.

Sidabras, J. W.; Duan, J.; Winkler, M.; Happe, T.; Hussein, R.; Zouni, A.; Suter, D.; Schnegg, A.; Lubitz, W.; Reijerse, E. J., Extending electron paramagnetic resonance to nanoliter volume protein single crystals using a self-resonant microhelix. Science advances 2019, 5 (10).

Kern, J.; Müh, F.; Zouni, A., Structural studies on tetrapyrrole containing proteins enabled by femtosecond X-ray pulses. Metabolism, Structure and Function of Plant Tetrapyrroles: Control Mechanisms of Chlorophyll Biosynthesis and Analysis of Chlorophyll-Binding Proteins 2019, 33.

Ruocheng Han, Katharina Rempfer, Miao Zhang, Prof. Dr. Holger Dobbek, Prof. Dr. Athina Zouni, Prof. Dr. Holger Dau, Prof. Dr. Sandra Luber Investigating the Structure and Dynamics of Apo‑Photosystem II ChemCatChem, 21 June 2019,

Chatterjee R, Lasalle L, Gul S, Fuller FD, Young ID, Ibrahim M, de Lichtenberg C, Cheah MH, Zouni A, Messinger J, Yachandra VK, Kern J, Yano J Structural isomers of the S2 state in photosystem II: do they exist at room temperature and are they important for function? Physiol Plant. 2019 May; 166(1):60-72. Epub 2019 Mar 15

Last updated on 2021-04-01 at 17:46