Matthias Broser

Cyanobacterial photosystem II at 2.9-A resolution and the role of quinones, lipids, channels and chloride.

Photosystem II (PSII) is a large homodimeric protein–cofactor complex located in the photosynthetic thylakoid membrane that acts as light-driven water:plastoquinone oxidoreductase. The crystal structure of PSII from Thermosynechococcus elongatus at 2.9-Å resolution allowed the unambiguous assignment of all 20 protein subunits and complete modeling of all 35 chlorophyll a molecules and 12 carotenoid molecules, 25 integral lipids and 1 chloride ion per monomer. The presence of a third plastoquinone QC and a second plastoquinone-transfer channel, which were not observed before, suggests mechanisms for plastoquinol-plastoquinone exchange, and we calculated other possible water or dioxygen and proton channels. Putative oxygen positions obtained from a Xenon derivative indicate a role for lipids in oxygen diffusion to the cytoplasmic side of PSII. The chloride position suggests a role in proton-transfer reactions because it is bound through a putative water molecule to the Mn4Ca cluster at a distance of 6.5 Å and is close to two possible proton channels.

Structural Changes of the Oxygen-evolving Complex in Photosystem II during the Catalytic Cycle

Background: Mn4CaO5 cluster catalyzes water oxidation in photosystem II. Results: Mn-Mn/Ca/ligand distances and changes in the structure of the Mn4CaO5 cluster are determined for the intermediate states in the reaction using x-ray spectroscopy. Conclusion: Position of one bridging oxygen and related geometric changes may be critical during catalysis. Significance: Knowledge about structural changes during catalysis is crucial for understanding the O–O bond formation mechanism in PSII. The oxygen-evolving complex (OEC) in the membrane-bound protein complex photosystem II (PSII) catalyzes the water oxidation reaction that takes place in oxygenic photosynthetic organisms. We investigated the structural changes of the Mn4CaO5 cluster in the OEC during the S state transitions using x-ray absorption spectroscopy (XAS). Overall structural changes of the Mn4CaO5 cluster, based on the manganese ligand and Mn-Mn distances obtained from this study, were incorporated into the geometry of the Mn4CaO5 cluster in the OEC obtained from a polarized XAS model and the 1.9-Å high resolution crystal structure. Additionally, we compared the S1 state XAS of the dimeric and monomeric form of PSII from Thermosynechococcus elongatus and spinach PSII. Although the basic structures of the OEC are the same for T. elongatus PSII and spinach PSII, minor electronic structural differences that affect the manganese K-edge XAS between T. elongatus PSII and spinach PSII are found and may originate from differences in the second sphere ligand atom geometry.

Recent progress in the crystallographic studies of photosystem II.

The photosynthetic oxygen-evolving photosystem II (PSII) is the only known biochemical system that is able to oxidize water molecules and thereby generates almost all oxygen in the Earths atmosphere. The elucidation of the structural and mechanistic aspects of PSII keeps scientists all over the world engaged since several decades. In this Minireview, we outline the progress in understanding PSII based on the most recent crystal structure at 2.9 A resolution. A likely position of the chloride ion, which is known to be required for the fast turnover of water oxidation, could be determined in native PSII and is compared with work on bromide and iodide substituted PSII. Moreover, eleven new integral lipids could be assigned, emphasizing the importance of lipids for the perfect function of PSII. A third plastoquinone molecule (Q(C)) and a second quinone transfer channel are revealed, making it possible to consider different mechanisms for the exchange of plastoquinone/plastoquinol molecules. In addition, possible transport channels for water, dioxygen and protons are identified.

Probing the accessibility of the Mn(4)Ca cluster in photosystem II: channels calculation, noble gas derivatization, and cocrystallization with DMSO.

Using the 2.9 A resolution structure of the membrane-intrinsic protein-cofactor complex photosystem II (PSII) from the cyanobacterium Thermosynechococcus elongatus, we calculated and characterized nine possible substrate/product channels leading to/away from the Mn(4)Ca cluster, where water is oxidized to dioxygen, protons, and electrons. Five narrow channels could function in proton transport, assuming that no large structural changes are associated with water oxidation. Four wider channels could serve to supply water to or remove oxygen from the Mn(4)Ca cluster. One of them might be regulated by conformational changes of Lys134 in subunit PsbU. Data analyses of Kr derivatized crystals and complexes with dimethyl sulfoxide (DMSO) confirm the accessibility of the proposed dioxygen channels to other molecules. Results from Xe derivatization suggest that the lipid clusters within PSII could serve as a drain for oxygen because of their predominant hydrophobic character and mediate dioxygen release from the lumen.

Crystal structure of monomeric photosystem II from Thermosynechococcus elongatus at 3.6 Å resolution

The membrane-embedded photosystem II core complex (PSIIcc) uses light energy to oxidize water in photosynthesis. Information about the spatial structure of PSIIcc obtained from x-ray crystallography was so far derived from homodimeric PSIIcc of thermophilic cyanobacteria. Here, we report the first crystallization and structural analysis of the monomeric form of PSIIcc with high oxygen evolution capacity, isolated from Thermosynechococcus elongatus. The crystals belong to the space group C2221, contain one monomer per asymmetric unit, and diffract to a resolution of 3.6 Å. The x-ray diffraction pattern of the PSIIcc-monomer crystals exhibit less anisotropy (dependence of resolution on crystal orientation) compared with crystals of dimeric PSIIcc, and the packing of the molecules within the unit cell is different. In the monomer, 19 protein subunits, 35 chlorophylls, two pheophytins, the non-heme iron, the primary plastoquinone QA, two heme groups, 11 β-carotenes, 22 lipids, seven detergent molecules, and the Mn4Ca cluster of the water oxidizing complex could be assigned analogous to the dimer. Based on the new structural information, the roles of lipids and protein subunits in dimer formation of PSIIcc are discussed. Due to the lack of non-crystallographic symmetry and the orientation of the membrane normal of PSIIcc perpendicular (∼87°) to the crystallographic b-axis, further information about the structure of the Mn4Ca cluster is expected to become available from orientation-dependent spectroscopy on this new crystal form.

Structural Basis of Cyanobacterial Photosystem II Inhibition by the Herbicide Terbutryn

Herbicides that target photosystem II (PSII) compete with the native electron acceptor plastoquinone for binding at the QB site in the D1 subunit and thus block the electron transfer from QA to QB. Here, we present the first crystal structure of PSII with a bound herbicide at a resolution of 3.2 Å. The crystallized PSII core complexes were isolated from the thermophilic cyanobacterium Thermosynechococcus elongatus. The used herbicide terbutryn is found to bind via at least two hydrogen bonds to the QB site similar to photosynthetic reaction centers in anoxygenic purple bacteria. Herbicide binding to PSII is also discussed regarding the influence on the redox potential of QA, which is known to affect photoinhibition. We further identified a second and novel chloride position close to the water-oxidizing complex and in the vicinity of the chloride ion reported earlier (Guskov, A., Kern, J., Gabdulkhakov, A., Broser, M., Zouni, A., and Saenger, W. (2009) Nat. Struct. Mol. Biol. 16, 334–342). This discovery is discussed in the context of proton transfer to the lumen.

Electronic structure of the tyrosine D radical and the water-splitting complex from pulsed ENDOR spectroscopy on photosystem II single crystals

Pulsed electron nuclear double resonance (ENDOR) spectroscopy at Q- and W-band frequencies was applied to single crystals of photosystem II from Th. elongatus. W-Band (1)H-ENDOR on the dark-stable radical state Y of the redox-active tyrosine residue Y(D) yields a complete mapping of the electronic structure of this amino acid radical in terms of an assignment of all hyperfine coupling tensors of the protons covalently bound to the side chain. This study can serve as a model case for the potential of high-field/high-frequency ENDOR on protein single crystals for obtaining highly resolved electronic structure information. Q-band (55)Mn-ENDOR was applied to the S(2) oxidation state of the water-splitting complex in photosystem II single crystals. Irrespective of the difficulties arising from the extremely broad electron paramagnetic resonance (EPR) spectroscopy ( approximately 200 mT) and ENDOR ( approximately 100 MHz) spectra a tentative assignment of the Mn ion in the formal oxidation state III to a Mn position in the structural model of PSII is possible on the basis of the ENDOR data.

Modeling of variant copies of subunit D1 in the structure of photosystem II from Thermosynechococcus elongatus

Abstract In the cyanobacterium Thermosynechococcus elongatus BP-1, living in hot springs, the light environment directly regulates expression of genes that encode key components of the photosynthetic multi-subunit protein-pigment complex photosystem II (PSII). Light is not only essential as an energy source to power photosynthesis, but leads to formation of aggressive radicals which induce severe damage of protein subunits and organic cofactors. Photosynthetic organisms develop several protection mechanisms against this photo-damage, such as the differential expression of genes coding for the reaction center subunit D1 in PSII. Testing the expression of the three different genes (psbAI, psbAII, psbAIII) coding for D1 in T. elongatus under culture conditions used for preparing the material used in crystallization of PSII showed that under these conditions only subunit PsbA1 is present. However, exposure to high-light intensity induced partial replacement of PsbA1 with PsbA3. Modeling of the variant amino acids of the three different D1 copies in the 3.0 Å resolution crystal structure of PSII revealed that most of them are in the direct vicinity to redox-active cofactors of the electron transfer chain. Possible structural and mechanistic consequences for electron transfer are discussed.

Photosystem II — Details of Cofactor-Protein Interactions in the Light of the 3 Å Resolution Crystal Structure

The large membrane intrinsic protein complex Photosytem II (PSII) catalyses light-driven charge separation accompanied by the oxidation of water during oxygenic photosynthesis. In this contribution we will discuss the recent X-ray crystallographic structural model at 3 A resolution (Loll et al. 2005) in relation to various spectroscopic and biochemical data. Special emphasis will be given on: (A) the quinone binding pockets, the proposed diffusion pathway of quinones into the QB binding site and the possible binding of additional quinones within the complex; (B) the catalytic center for light-induced water oxidation (the Mn4-Ca cluster). The arrangement of metal cations in the cluster, their coordination and protein surroundings are discussed with regard to spectroscopic and mutagenesis studies. Limitations of the presently available structural data are pointed out and the very recent results of X-ray spectroscopy (XANES and EXAFS) on PSII single crystals (Yano et al. 2006) are described in comparison with the X-ray crystallographic model of the water oxidizing complex.