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The water oxidation reaction in photosystem II (PS II) produces most of the molecular oxygen in the atmosphere, which sustains life on Earth, and in this process releases four electrons and four protons that drive the downstream process of CO2 fixation in the photosynthetic apparatus. The catalytic center of PS II is an oxygen-bridged Mn4Ca complex (Mn4CaO5) which is progressively oxidized upon the absorption of light by the chlorophyll of the PS II reaction center, and the accumulation of four oxidative equivalents in the catalytic center results in the oxidation of two waters to dioxygen in the last step. The recent emergence of X-ray free-electron lasers (XFELs) with intense femtosecond X-ray pulses has opened up opportunities to visualize this reaction in PS II as it proceeds through the catalytic cycle. In this review, we summarize our recent studies of the catalytic reaction in PS II by following the structural changes along the reaction pathway via room-temperature X-ray crystallography using XFELs. The evolution of the electron density changes at the Mn complex reveals notable structural changes, including the insertion of OX from a new water molecule, which disappears on completion of the reaction, implicating it in the O—O bond formation reaction. We were also able to follow the structural dynamics of the protein coordinating with the catalytic complex and of channels within the protein that are important for substrate and product transport, revealing well orchestrated conformational changes in response to the electronic changes at the Mn4Ca cluster.

Abstract Photosystem I (PS I) has a symmetric structure with two highly similar branches of pigments at the center that are involved in electron transfer, but shows very different efficiency along the two branches. We have determined the structure of cyanobacterial PS I at room temperature (RT) using femtosecond X-ray pulses from an X-ray free electron laser (XFEL) that shows a clear expansion of the entire protein complex in the direction of the membrane plane, when compared to previous cryogenic structures. This trend was observed by complementary datasets taken at multiple XFEL beamlines. In the RT structure of PS I, we also observe conformational differences between the two branches in the reaction center around the secondary electron acceptors A1A and A1B. The π-stacked Phe residues are rotated with a more parallel orientation in the A-branch and an almost perpendicular confirmation in the B-branch, and the symmetry breaking PsaB-Trp673 is tilted and further away from A1A. These changes increase the asymmetry between the branches and may provide insights into the preferential directionality of electron transfer.

The oxygen-evolving complex in Photosystem II (PSII) catalyzes the light-driven oxidation of water to oxygen and it is still under debate how the water reaches the active site. Here, the authors analyse time-resolved XFEL-based crystal structures of PSII that were determined at room temperature and report the structures of the waters in the putative channels surrounding the active site at various time-points during the reaction cycle and conclude that the O1 channel is the likely water intake pathway and the Cl1 channel the likely proton release pathway.

In the last ten years, x-ray free-electron lasers (XFELs) have been successfully employed to characterize metalloproteins at room temperature using various techniques including x-ray diffraction, scattering, and spectroscopy. The approach has been to outrun the radiation damage by using femtosecond (fs) x-ray pulses. An example of an important and damage sensitive active metal center is the Mn4CaO5 cluster in photosystem II (PS II), the catalytic site of photosynthetic water oxidation. The combination of serial femtosecond x-ray crystallography and Kβ x-ray emission spectroscopy (XES) has proven to be a powerful multimodal approach for simultaneously probing the overall protein structure and the electronic state of the Mn4CaO5 cluster throughout the catalytic (Kok) cycle. As the observed spectral changes in the Mn4CaO5 cluster are very subtle, it is critical to consider the potential effects of the intense XFEL pulses on the Kβ XES signal. We report here a systematic study of the effects of XFEL peak power, beam focus, and dose on the Mn Kβ1,3 XES spectra in PS II over a wide range of pulse parameters collected over seven different experimental runs using both microcrystal and solution PS II samples. Our findings show that for beam intensities ranging from ∼5 × 1015 to 5 × 1017 W/cm2 at a pulse length of ∼35 fs, the spectral effects are small compared to those observed between S-states in the Kok cycle. Our results provide a benchmark for other XFEL-based XES studies on metalloproteins, confirming the viability of this approach.

X-ray absorption spectroscopy at the L-edge of 3d transition metals provides unique information on the local metal charge and spin states by directly probing 3d-derived molecular orbitals through 2p-3d transitions. However, this soft x-ray technique has been rarely used at synchrotron facilities for mechanistic studies of metalloenzymes due to the difficulties of x-ray-induced sample damage and strong background signals from light elements that can dominate the low metal signal. Here, we combine femtosecond soft x-ray pulses from a free-electron laser with a novel x-ray fluorescence-yield spectrometer to overcome these difficulties. We present L-edge absorption spectra of inorganic high-valent Mn complexes (Mn ∼ 6–15 mmol/l) with no visible effects of radiation damage. We also present the first L-edge absorption spectra of the oxygen evolving complex (Mn4CaO5) in Photosystem II (Mn

In plants, algae and cyanobacteria, Photosystem II (PSII) catalyzes the light-driven splitting of water at a protein-bound Mn4CaO5-cluster, the water-oxidizing complex (WOC). In the photosynthetic organisms, the light-driven formation of the WOC from dissolved metal ions is a key process because it is essential in both initial activation and continuous repair of PSII. Structural information is required for understanding of this chaperone-free metal-cluster assembly. For the first time, we obtained a structure of PSII from Thermosynechococcus elongatus without the Mn4CaO5-cluster. Surprisingly, cluster-removal leaves the positions of all coordinating amino acid residues and most nearby water molecules largely unaffected, resulting in a pre-organized ligand shell for kinetically competent and error-free photo-assembly of the Mn4CaO5-cluster. First experiments initiating (i) partial disassembly and (ii) partial re-assembly after complete depletion of the Mn4CaO5-cluster agree with a specific bi-manganese cluster, likely a di-µ-oxo bridged pair of Mn(III) ions, as an assembly intermediate.

In photosynthesis, photosystem II (PSII) is the multi-subunit membrane protein complex that catalyzes photo-oxidation of water into dioxygen through the oxygen evolving complex (OEC). To understand the water oxidation reaction, it is important to get structural information about the transient and intermediate states of the OEC in the dimeric PSII core complex (dPSIIcc). In recent times, femtosecond X-ray pulses from the free electron laser (XFEL) are being used to obtain X-ray diffraction (XRD) data of dPSIIcc microcrystals at room temperature that are free of radiation damage. In our experiments at the XFEL, we used an electrospun liquid microjet setup that requires microcrystals less than 40 μm in size. In this study, we explored various microseeding techniques to get a high yield of monodisperse uniform-sized microcrystals. Monodisperse microcrystals of dPSIIcc of uniform size were a key to improve the stability of the jet and the quality of XRD data obtained at the XFEL. This was evident by an improvement of the quality of the datasets obtained, from 6.5 Å, using crystals grown without the micro seeding approach, to 4.5 Å using crystals generated with the new method.