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 ?. Very recently, in a striking breakthrough, the crystal structure of dimeric PSII from a related thermophilic cyanobacterium (T. vulcanus) was solved at 1.9 ? 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 ? 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 ?. 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.