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• Photosystem II (PS II) is a large membrane-bound protein complex that catalyses light-driven water oxidation in plants and cyanobacteria. The structure of PS II is well studied in cyanobacteria; however, there are very few PS II structures from plants. The currently available plant PS II structures are comparatively low resolution and are frequently incomplete, that is, missing subunits or cofactors.
• We optimized the procedure for isolating PS II from Arabidopsis thaliana and employed cryo-electron microscopy to generate a high-resolution structure of an intact and oxygen-evolving PS II from Arabidopsis thaliana at 2.44 Å resolution, which to date represents the highest resolution structure of PS II from higher plants.
• At this resolution, many water molecules within the PS II structure can be detected, including waters around the water-splitting manganese cluster, the nonheme iron, and within the water/proton channels connecting these active sites to the protein exterior, allowing for the first detailed description of the water networks in Arabidopsis thaliana and comparison with the highly resolved cyanobacterial PS II.
• Our findings further the understanding of design principles of protein–water–cofactor interactions in photosynthetic water splitting, quinone reduction/exchange, and about the role of lipids at the interface between PS II and the light-harvesting proteins.

Oligomers of the SARS-CoV-2 nucleocapsid (N) protein are characterized by pronounced instability resulting in fast degradation. This property likely relates to two contrasting behaviors of the N protein: genome stabilization through a compact nucleocapsid during cell evasion and genome release by nucleocapsid disassembling during infection. In vivo, the N protein forms rounded complexes of high molecular mass from its interaction with the viral genome. To study the N protein and understand its instability, we analyzed degradation profiles under different conditions by size-exclusion chromatography and characterized samples by mass spectrometry and cryo-electron microscopy. We identified self-cleavage properties of the N protein based on specific Proprotein convertases activities, with Cl^- playing a key role in modulating stability and degradation. These findings allowed isolation of a stable oligomeric complex of N, for which we report the 3D structure at ∼6.8 Å resolution. Findings are discussed considering available knowledge about the coronaviruses’ infection cycle.

Light-driven water oxidation by photosystem II sustains life on Earth by providing the electrons and protons for the reduction of CO2 to carbohydrates and the molecular oxygen we breathe. The inorganic core of the oxygen evolving complex is made of the earth-abundant elements manganese, calcium and oxygen (Mn4CaO5 cluster), and is situated in a binding pocket that is connected to the aqueous surrounding via water-filled channels that allow water intake and proton egress. Recent serial crystallography and infrared spectroscopy studies performed with PSII isolated from Thermosynechococcus vestitus (T. vestitus) support that one of these channels, the O1 channel, facilitates water access to the Mn4CaO5 cluster during its S2→S3 and S3→S4→S0 state transitions, while a subsequent CryoEM study concluded that this channel is blocked in the cyanobacterium Synechocystis sp. PCC 6803, questioning the role of the O1 channel in water delivery. Employing site-directed mutagenesis we modified the two O1 channel bottleneck residues D1-E329 and CP43-V410 (T. vestitus numbering) and probed water access and substrate exchange via time resolved membrane inlet mass spectrometry. Our data demonstrates that water reaches the Mn4CaO5 cluster via the O1 channel in both wildtype and mutant PSII. In addition, the detailed analysis provides functional insight into the intricate protein-water-cofactor network near the Mn4CaO5 cluster that includes the pentameric, near planar ‘water wheel’ of the O1 channel.

Photosystem II starts the photosynthetic electron transport chain that converts solar energy into chemical energy and thus sustains life on Earth. It catalyzes two chemical reactions: water oxidation to molecular oxygen and plastoquinone reduction. Coupling of electron and proton transfer is crucial for efficiency; however, the molecular basis of these processes remains speculative owing to uncertain water binding sites and the lack of experimentally determined hydrogen positions. We thus collected high-resolution cryo-electron microscopy data of fully hydrated photosystem II from the thermophilic cyanobacterium Thermosynechococcus vestitus to a final resolution of 1.71 angstroms. The structure reveals several previously undetected partially occupied water binding sites and more than half of the hydrogen and proton positions. This clarifies the pathways of substrate water binding and plastoquinone B protonation.

This chapter compares two different techniques for monitoring photosynthetic O₂ production; the wide-spread Clark-type O₂ electrode and the more sophisticated membrane inlet mass spectrometry (MIMS) technique. We describe how a simple membrane inlet for MIMS can be made out of a commercial Clark-type cell and outline the advantages and drawbacks of the two techniques to guide researchers in deciding which method to use. Protocols and examples are given for measuring O₂ evolution rates and for determining the number of chlorophyll molecules per active photosystem II reaction center.

Chlorophyll is essential in photosynthesis, converting sunlight into chemical energy in plants, algae, and certain bacteria. Its structure, featuring a porphyrin ring enclosing a central magnesium ion, varies in forms like chlorophyll a, b, c, d, and f, allowing light absorption at a broader spectrum. With a 20-carbon phytyl tail (except for chlorophyll c), chlorophyll is anchored to proteins. Previous findings suggested the presence of chlorophyll with a modified farnesyl tail in thermophilic cyanobacteria Thermosynechoccocus vestitus. In our Arabidopsis thaliana PSII cryo-EM map, specific chlorophylls showed incomplete phytyl tails, suggesting potential farnesyl modifications. However, further high-resolution mass spectrometry (HRMS) analysis in A. thaliana and T. vestitus did not confirm the presence of any farnesyl tails. Instead, we propose the truncated tails in PSII models may result from binding pocket flexibility rather than actual modifications.

Salinibacter ruber is an extremophilic bacterium able to grow in high-salts environments, such as saltern crystallizer ponds. This halophilic bacterium is red-pigmented due to the production of several carotenoids and their derivatives. Two of these pigment molecules, salinixanthin and retinal, are reported to be essential cofactors of the xanthorhodopsin, a light-driven proton pump unique to this bacterium. Here, we isolate and characterize an outer membrane porin-like protein that retains salinixanthin. The characterization by mass spectrometry identified an unknown protein whose structure, predicted by AlphaFold, consists of a 8 strands beta-barrel transmembrane organization typical of porins. The protein is found to be part of a functional network clearly involved in the outer membrane trafficking. Cryo-EM micrographs showed the shape and dimensions of a particle comparable with the ones of the predicted structure. Functional implications, with respect to the high representativity of this protein in the outer membrane fraction, are discussed considering its possible role in primary functions such as the nutrients uptake and the homeostatic balance. Finally, also a possible involvement in balancing the charge perturbation associated with the xanthorhodopsin and ATP synthase activities is considered.

The correct development of seeds is a pivotal requirement for species preservation. This process depends on the balance between sensing the environmental stimuli/stressors and hormone-mediated transduction, which results in physiological responses. The red and blue regions of the electromagnetic spectrum are known to influence seed dormancy and germination. Here, we report on the effects induced by the blue (peak at 430 nm) and red (peak at 650 nm) regions of the electromagnetic spectrum on seeds from photo- and skotomorphogenetic capsules developed under white, blue, or red light. Regardless of exposure, seeds from skotomorphogenetic capsules showed an almost absent dormancy in association with altered germination kinetics. Conversely, in seeds from photomorphogenetic capsules, the exposure to the blue region induced skotomorphogenetic-like effects, while the exposure to the whole visible range (350–750 nm), as well as to only the red region, showed a dose-related trend. The observed differences appeared to be dependent on the wavelengths in the red and to be based on transduction mechanisms taking place in fruits. While the molecular bases of this differential effect need to be clarified, the results hint at the role played by different light wavelengths and intensities in seed development and germination. These findings may be relevant for applications in crop production and species safeguarding.

In higher plants, the photosynthetic process is performed and regulated by Photosystem II (PSII). Arabidopsis thaliana was the first higher plant with a fully sequenced genome, conferring it the status of a model organism; nonetheless, a high-resolution structure of its Photosystem II is missing. We present the first Cryo-EM high-resolution structure of Arabidopsis PSII supercomplex with average resolution of 2.79 Å, an important model for future PSII studies. The digitonin extracted PSII complexes demonstrate the importance of: the LHG2630-lipid-headgroup in the trimerization of the light-harvesting complex II; the stabilization of the PsbJ subunit and the CP43-loop E by DGD520-lipid; the choice of detergent for the integrity of membrane protein complexes. Furthermore, our data shows at the anticipated Mn4CaO5-site a single metal ion density as a reminiscent early stage of Photosystem II photoactivation.