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Humboldt-Universität zu Berlin - Institut für Biologie

Structural elucidation of the light-driven self-assembly process of the water-oxidizing Mn4CaO5 complex

 

In plants, algae and cyanobacteria, Photosystem II (PSII) catalyzes the light-driven oxidation of water by a protein-bound Mn4CaO5 cluster, the water-oxidizing complex (WOC). The first 3D crystal structure of PSII was obtained by Zouni and coworkers at a resolution of 3.8 Å, thus initiating an era of structure-based investigation of water oxidation in PSII at the atomic level. The recent progress in improvement of crystal qualities allowed collecting high-resolution structures of PSII for both static (at 1.9 Å resolution under cryogenic conditions; Umena et al., Nature 2011) and dynamic measurements (at 2.0 Å resolution for all S states in the Kok cycle measured at room-temperature by using an fs-X-ray free electron laser (XFEL) as radiation source; Kern at al., Nature 2018). A resolution that is better than 2.0 Å enabled detailed structural insights into the mechanism of light-driven water-splitting. However, the self-assembly process of the Mn4CaO5 complex is not fully understood until now.

In the intact organism, the assembly of the Mn4CaO5 cluster is a light-driven oxidative process that is interfaced with an exchange of the D1 protein binding the WOC. During catalytic water oxidation, partial degradation of the Mn4CaO5 cluster can occur, but is efficiently counteracted by self-repair. During this repair, a situation may occur, where the WOC is absent or partially assembled, but the photosynthetic reaction center (RC), to which the WOC is normally attached, is fully active. Then, harmful photochemical side reaction may happen. A way to reduce these side reactions is the modulation of the redox potential of the electron acceptor QA in the RC, which is situated at the acceptor side of the RC opposite to the WOC (donor side). To understand the self-repair process of the Mn4CaO5 cluster, a crystal structure is needed in which the metal cluster is completely depleted from the photosystem. The structure of such an apo-PSII thus serves as a starting point for the further elucidation of the self-assembly process of the Mn4CaO5 cluster.

 

Cartoon on water-oxidizing Mn oxides related to the biological Mn4CaO5-cluster of PSII. Figure taken from A. Indra et al., Uncovering Structure–Activity Relationships in Manganese-Oxide-Based Heterogeneous Catalysts for Efficient Water Oxidation, ChemSusChem, 2015, DOI: 10.1002/cssc.201402812.

 

Recent results

Recently, we have obtained a crystal structure of apo-PSII. To our surprise, we found that all cluster-coordinating amino acid residues and nearby water molecules remained essentially in the same position as found in the fully assembled Mn4CaO5 cluster without any major deformation of the binding pocket. Inside this cavity, we identified two putative molecules that fill the void and maintain the geometry of the ligand sphere of the Mn4CaO5 cluster. Based on these structural data, we concluded that there exists a pre-organized protein matrix facilitating kinetically competent and error-free light-driven formation of the Mn4CaO5 cluster. Removal of the Mn4CaO5 cluster did not result in any discernible movement of subunits or domains, neither at the PSII donor side nor in the region of the membrane spanning helices or at the acceptor side.

In addition to the structure of apo-PSII, we also obtained a putative assembly intermediate, a binuclear Mn complex, [Mn1-(µ-O)2-Mn2], possibly involving two Mn (III) ions interconnected by di-µ-oxo bridges. Such an intermediate may be formed during both the assembly and disassembly process of the Mn4CaO5 cluster. According to the so-called two-quantum model, there is a stable intermediate state with two Mn sites occupied by Mn(III) ions that is consistent with our structural data. However, the incorporation of the two remaining Mn ions and the binding of a Ca ion to fully assemble the Mn4CaO5 cluster have not been kinetically resolved.

Objectives and preliminary results

The main objective of the UniSysCat project is to fully understand the self-assembly process of the Mn4CaO5 cluster. The preliminary experiments of photoactivation performed on apo-PSII microcrystal suspensions using optimized concentration ratios of Mn:Ca and illumination conditions were conducted by membrane inlet mass spectrometry (MIMS) in collaboration with Prof. Johannes Messinger´s group.

With the optimized reassembly conditions, almost 50 % of the original oxygen activity could be restored inside the apo-PSII crystals. This result will allow determining the conditions to capture the PSII intermediate states formed during the photoactivation.

Future plans and cooperation

The structure of apo-PSII represents the likely starting state for metal-cluster reassembly that serves as a basis to understand the mechanism of WOC assembly/disassembly. Our approach paves the way for capturing photo-assembly intermediates at a high resolution.

  1. Based on the optimized MIMS data, the protocol for the light-induced reconstitution experiments on apo-PSII microcrystals will be further studied by using MIMS (Zouni/Messinger; Uppsala, Sweden).
  2. Based on our recent structural studies on apo-PSII, we plan to study the photo-activation reaction that leads to the incorporation of the Mn ions to form the Mn4CaO5 cluster. For that reason, we intend to use the optimized reconstitution protocol for SFX at LCLS to obtain a high-resolution room temperature structure without radiation damage of these two intermediates (Zouni/Dobbek/Kern/Yachandra/Yano; SLAC Stanford USA).

 

Pre-organized ligand shell under conditions of cluster removal with minimal restructuring. https://doi.org/10.7554/eLife.26933.014

 

Further cooperation within UniSysCat

Microseeding protocol and post-crystallization experiments on single crystals (Zouni/Dobbek)

DLS, EPR (Teutloff, FU), Fluorescence spectroscopy and SAXS (Pieper, Estonia), XRD (BESSY, DESY)

Measurements of polarized X-ray absorption spectroscopy at the Mn-edge on different Mn intermediate PSII variants (Dau/Zouni)

Spectroscopic characterization of intermediate clusters formed in Mn-depleted PSII by various techniques and theory

EPR/ENDOR (Teutloff), XAS/EXAFS (Dau/Haumann), IR (Hildebrandt)

DFT calculations (Luber/Kaupp)

Insertion of artificial Mn or other metal clusters into apo-PSII (Driess/Dobbek/Dau)

External Cooperation

Prof. V. Yachandra and J. Yano: polarized XAS at the Mn K-edge on our PSIIcc crystals (Berkeley, USA).

 

Recently published research article

Zhang M, Bommer M, Chatterjee R, Hussein R, Yano J, Dau H, Kern J, Dobbek H, Zouni A. Structural insights into the light-driven auto-assembly process of the water-oxidizing Mn4CaO5-cluster in photosystem II. Elife. 2017 Jul 18;6. pii: e26933. doi: 10.7554/eLife.26933. https://elifesciences.org/articles/26933

 

For further questions to this project, please contact Miao Zhang.

 

         Email: zhangmia@hu-berlin.de

         Phone: +49(0)30209347931/47933