During photosynthetic water oxidation, the Mn4Ca cluster in Photosystem II progresses through five intermediate Si (i = 0-4) states. X-ray crystallography studies have reported the insertion of one new O ligand during the formation of the S3 state, but recent studies question the presence of this additional ligand based on cryo-EM and earlier room-temperature crystallography data. There is also controversy about whether the O-O bond interaction already occurs in the S3 state or in the subsequent S3 to S0 transition. Here we report conventional high-resolution data for the S1, S2, and S3 states to a resolution of ~1.9 Å, and anomalous diffraction data at two energies (9.5 keV and 7 keV), that was used to model the Mn positions, followed by determination of oxygen positions using the high-resolution maps. We show that the new oxygen atom, OX (or O6), in the S3 state is observable as a distinct peak without any restraints, confirming its ligation to Mn1 and Ca. The OX-O5 distance is ~2.1 Å, supporting no strong interaction between them in the S3 state, suggesting that if this is the O-O bond formation site, it is formed during the S3 to S0 transition initiated by the final oxidation of the cluster.

The influence of poly(ethylene glycol) (PEG with molecular weights between 400 and 4000) on the critical micelle concentration (CMC) of nonionic detergents with maltose as well as oligo(ethylene glycol) head groups is determined by using 8-anilinonaphthalene-1-sulfonate (ANS) as fluorescence probe. The CMC is found to increase with the concentration of PEG (0%-30% (w/v)) in a way that is determined by the molar concentration of oxyethylene (OE) units and independent of the molecular weight of the added polymer. The effect is explained with the extended conformation of PEG in aqueous solution allowing for an interaction of detergent monomers with individual OE units via their alkyl tails. The fluorescence spectra of ANS are found to exhibit two major emission peaks that are affected in position and intensity by binding to micelles as well as PEG. A model with two conformations of ANS combined with two binding sites in the micelles is used to explain the spectra and their correlation with detergent properties. The shapes of the titration curves are shown to depend on the aggregation number and the equilibrium constant describing binding of ANS to micelles and are analyzed to find that PEG competes with micelles for binding of ANS.

Photosystem II starts the photosynthetic electron transport chain that converts solar energy into chemical energy and thereby sustains life on Earth. It catalyzes two chemical reactions, plastoquinone reduction and water oxidation to molecular oxygen, which both are performed at sequestered sites. While it is known that proton-coupled electron transfer is crucial for these processes, the molecular details have remained speculative due to incomplete structural data. Thus, we collected high-resolution cryo-EM data of photosystem II from Thermosynechococcus vestitus . The advanced structure (1.71 Å) reveals several previously unditected occupied water binding sites and more than half of the hydrogen and proton positions of the protein. This unprecedented insight into the structure of photosystem II significantly enhances our understanding of its intricate protein-water-cofactor interactions enabling solar-driven catalysis. One sentence summary Cryo-EM structure of PSII at 1.71 Å resolution reveals over 50% of hydrogen and proton sites and additional water binding sites, aiding catalytic insight. ### Competing Interest Statement The authors have declared no competing interest.

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.

The influence of poly(ethylene glycol) (PEG) polymers H–(O–CH2–CH2)p–OH with different average molecular sizes p on the micelle formation of n-alkyl-β-D-maltoside detergents with the number of carbon atoms in the alkyl chain ranging from 10 to 12 is investigated with the aim to learn more about the detergent behavior under conditions suitable for the crystallization of the photosynthetic pigment–protein complex photosystem II. PEG is shown to increase the critical micelle concentration (CMC) of all three detergents in the crystallization buffer in a way that the free energy of micelle formation increases linearly with the concentration of oxyethylene units (O–CH2–CH2) irrespective of the actual molecular weight of the polymer. The CMC shift is modeled by assuming for simplicity that it is dominated by the interaction between PEG and detergent monomers and is interpreted in terms of an increase of the transfer free energy of a methylene group of the alkyl chain by 0.2 kJ mol−1 per 1 mol L−1 increase of the concentration of oxyethylene units at 298 K. Implications of this effect for the solubilization and crystallization of protein–detergent complexes as well as detergent extraction from crystals are discussed.

In natural photosynthesis, the light-driven splitting of water into electrons, protons and molecular oxygen forms the first step of the solar-to-chemical energy conversion process. The reaction takes place in photosystem II, where the Mn 4 CaO 5 cluster first stores four oxidizing equivalents, the S 0 to S 4 intermediate states in the Kok cycle, sequentially generated by photochemical charge separations in the reaction center and then catalyzes the O–O bond formation chemistry 1 – 3 . Here, we report room temperature snapshots by serial femtosecond X-ray crystallography to provide structural insights into the final reaction step of Kok’s photosynthetic water oxidation cycle, the S 3 →[S 4 ]→S 0 transition where O 2 is formed and Kok’s water oxidation clock is reset. Our data reveal a complex sequence of events, which occur over micro- to milliseconds, comprising changes at the Mn 4 CaO 5 cluster, its ligands and water pathways as well as controlled proton release through the hydrogen-bonding network of the Cl1 channel. Importantly, the extra O atom O x , which was introduced as a bridging ligand between Ca and Mn1 during the S 2 →S 3 transition 4 – 6 , disappears or relocates in parallel with Y z reduction starting at approximately 700 μs after the third flash. The onset of O 2 evolution, as indicated by the shortening of the Mn1–Mn4 distance, occurs at around 1,200 μs, signifying the presence of a reduced intermediate, possibly a bound peroxide.

Photosynthesis as one of the most fascinating natural processes uses sunlight and CO2 for the preparation of various organic substances. Taking nature as inspiration the present work applies two proteins in an artificial matrix for constructing a biohybrid system allowing light-to-chemical photocataysis. Thus, the protein complexes photosystem I (PSI) and formate dehydrogenase (FDH) are combined with a porous three-dimensional indium tin oxide structure. Direct electron transfer takes place between the electrode material and PSI resulting in cathodic photocurrents. Instead of maximizing the current output, the study focuses on the usage of the excited electrons to drive FDH catalysis. At the enzyme carbon dioxide is reduced to formate with an efficiency of about 15% relative to the number of enhanced photoelectrons. The arrangement is favorable since it enables the conversion of CO2 at rather small applied potential of −0.2 V vs Ag/AgCl and over a longer period of illumination. Conversion efficiency and stability provide a good starting point for further investigations. The reported ITO/PSI/FDH electrode represents a system that combines photocatalysis with enzymatic catalysis allowing the storage of light energy in chemical energy.

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.

The critical micelle concentration (CMC) of nonionic detergents is defined as the breaking point in the monomer concentration as a function of the total detergent concentration, identified by setting the third derivate of this function to zero. Combined with a mass action model for micelle formation, this definition yields analytic formulae for the concentration ratio of monomers to total detergent at the CMC and the relationship between the CMC and the free energy of micellization g mic. The theoretical breaking point is shown to coincide with the breaking point of the experimental titration curve, if the fluorescence enhancement of 8-anilino-1-naphthalene-sulfonic acid (ANS) or a similar probe dye is used to monitor micelle formation. Application to a series of n-alkyl-β-d-maltosides with the number of carbon atoms in the alkyl chain ranging from 8 to 12 demonstrates the good performance of a molecular thermodynamic model, in which the free energy of micellization is given by g mic = σΦ + g pack + g st. In this model, σ is a fit parameter with the dimension of surface tension, Φ represents the change in area of hydrophobic molecular surfaces in contact with the aqueous phase, and g pack and g st are contributions, respectively, from alkyl chain packing in the micelle interior and steric repulsion of detergent head groups. The analysis of experimental data from different sources shows that varying experimental conditions such as co-solutes in the aqueous phase can be accounted for by adapting only σ, if the co-solutes do not bind to the detergent to an appreciable extent. The model is considered a good compromise between theory and practicability to be applied in the context of in vitro investigations of membrane proteins.

One restriction for biohybrid photovoltaics is the limited conversion of green light by most natural photoactive components. The present study aims to fill the green gap of photosystem I (PSI) with covalently linked fluorophores, ATTO 590 and ATTO 532. Photobiocathodes are prepared by combining a 20 μm thick 3D indium tin oxide (ITO) structure with these constructs to enhance the photocurrent density compared to setups based on native PSI. To this end, two electron transfer mechanisms, with and without a mediator, are studied to evaluate differences in the behavior of the constructs. Wavelength-dependent measurements confirm the influence of the additional fluorophores on the photocurrent. The performance is significantly increased for all modifications compared to native PSI when cytochrome c is present as a redox-mediator. The photocurrent almost doubles from -32.5 to up to -60.9 μA cm-2. For mediator-less photobiocathodes, interestingly, drastic differences appear between the constructs made with various dyes. While the turnover frequency (TOF) is doubled to 10 e-/PSI/s for PSI-ATTO590 on the 3D ITO compared to the reference specimen, the photocurrents are slightly smaller since the PSI-ATTO590 coverage is low. In contrast, the PSI-ATTO532 construct performs exceptionally well. The TOF increases to 31 e-/PSI/s, and a photocurrent of -47.0 μA cm-2 is obtained. This current is a factor of 6 better than the reference made with native PSI in direct electron transfer mode and sets a new record for mediator-free photobioelectrodes combining 3D electrode structures and light-converting biocomponents.

One of the reasons for the high efficiency and selectivity of biological catalysts arise from their ability to control the pathways of substrates and products using protein channels, and by modulating the transport in the channels using the interaction with the protein residues and the water/hydrogen-bonding network. This process is clearly demonstrated in Photosystem II (PS II), where its light-driven water oxidation reaction catalyzed by the Mn 4 CaO 5 cluster occurs deep inside the protein complex and thus requires the transport of two water molecules to and four protons from the metal center to the bulk water. Based on the recent advances in structural studies of PS II from X-ray crystallography and cryo-electron microscopy, in this review we compare the channels that have been proposed to facilitate this mass transport in cyanobacteria, red and green algae, diatoms, and higher plants. The three major channels (O1, O4, and Cl1 channels) are present in all species investigated; however, some differences exist in the reported structures that arise from the different composition and arrangement of membrane extrinsic subunits between the species. Among the three channels, the Cl1 channel, including the proton gate, is the most conserved among all photosynthetic species. We also found at least one branch for the O1 channel in all organisms, extending all the way from Ca/O1 via the ‘water wheel’ to the lumen. However, the extending path after the water wheel varies between most species. The O4 channel is, like the Cl1 channel, highly conserved among all species while having different orientations at the end of the path near the bulk. The comparison suggests that the previously proposed functionality of the channels in T. vestitus (Ibrahim et al., Proc Natl Acad Sci USA 117:12624–12635, 2020; Hussein et al., Nat Commun 12:6531, 2021) is conserved through the species, i.e. the O1-like channel is used for substrate water intake, and the tighter Cl1 and O4 channels for proton release. The comparison does not eliminate the potential role of O4 channel as a water intake channel. However, the highly ordered hydrogen-bonded water wire connected to the Mn 4 CaO 5 cluster via the O4 may strongly suggest that it functions in proton release, especially during the S 0 → S 1 transition (Saito et al., Nat Commun 6:8488, 2015; Kern et al., Nature 563:421–425, 2018; Ibrahim et al., Proc Natl Acad Sci USA 117:12624–12635, 2020; Sakashita et al., Phys Chem Chem Phys 22:15831–15841, 2020; Hussein et al., Nat Commun 12:6531, 2021).

Synthetic materials can be combined with biological components in many ways. One example that provides scientists with multiple challenges is a photobioelectrode that converts sunlight into electrons in a biohybrid approach. In the present study several key parameters are evaluated concerning their influence on the direct electron transfer from a 3D indium tin oxide (ITO) electrode material to photosystem I (PSI) as a light-harvesting biomolecule. In contrast to previous investigations, no mediating molecule is added to shuttle the electrons to the luminal side of PSI. Thus, this setup is less complex than foregoing ones. The solution composition drastically influences the interaction of PSI with the ITO surface. Here, the application of higher buffer concentrations and the addition of salts are advantageous, whereas the nature of the buffer ions plays a minor role. The artificial electrode material's thickness is adjustable since a spin-coating procedure is used for preparation. With a 30 mu m thick structure and immobilized PSI cathodic photocurrents up to 10.1 mu A cm-2 are obtained at 100 mW cm-2 illumination intensity and an applied potential of-0.1V vs. Ag/AgCl. Over a period of three days the photo-bioelectrodes are illuminated for a total of 90 min and stored between the measurements at ambient tempera-ture. The stability of the setup is noteworthy as still about 90% of the photocurrent is retained. The photocathode described here offers many positive features, including a high onset potential for the photocurrent starting sligthly above the redox potentail of P700, and applicability in a wide pH range from pH 5 to 8.

Ever since the discovery that Mn was required for oxygen evolution in plants by Pirson in 1937 and the period-four oscillation in flash-induced oxygen evolution by Joliot and Kok in the 1970s, understanding of this process has advanced enormously using state-of-the-art methods. The most recent in this series of innovative techniques was the introduction of X-ray free-electron lasers (XFELs) a decade ago, which led to another quantum leap in the understanding in this field, by enabling operando X-ray structural and X-ray spectroscopy studies at room temperature. This review summarizes the current understanding of the structure of Photosystem II (PS II) and its catalytic centre, the Mn4CaO5 complex, in the intermediate S-i (i = 0-4)-states of the Kok cycle, obtained using XFELs.

Wang their own analysis of our crystallographic data (2) and our, and similar previous (3 – 5), results that a newly inserted water (Ox) in the catalytic center of photosystem II (PSII) can be uniquely identified in the room temperature crystal structures taken at various time points during the S 2 -to-S 3 transition of the water oxidation reaction in PSII. They suggest that the results can be explained by the movement of the existing ligand, O5, without incorporating a new water (Ox). The following four points describe why their claim is not valid.

High-resolution structures of photosynthetic pigment–protein complexes are often determined using crystallography or cryo-electron microscopy (cryo-EM), which are restricted to the use of protein crystals or to low temperatures, respectively. However, functional studies and biotechnological applications of photosystems necessitate the use of proteins isolated in aqueous solution, so that the relevance of high-resolution structures has to be independently verified. In this regard, small-angle neutron and X-ray scattering (SANS and SAXS, respectively) can serve as the missing link because of their capability to provide structural information for proteins in aqueous solution at physiological temperatures. In the present review, we discuss the principles and prototypical applications of SANS and SAXS using the photosynthetic pigment–protein complexes phycocyanin (PC) and Photosystem I (PSI) as model systems for a water-soluble and for a membrane protein, respectively. For example, the solution structure of PSI was studied using SAXS and SANS with contrast matching. A Guinier analysis reveals that PSI in solution is virtually free of aggregation and characterized by a radius of gyration of about 75 Å. The latter value is about 10% larger than expected from the crystal structure. This is corroborated by an ab initio structure reconstitution, which also shows a slight expansion of Photosystem I in buffer solution at room temperature. In part, this may be due to conformational states accessible by thermally activated protein dynamics in solution at physiological temperatures. The size of the detergent belt is derived by comparison with SANS measurements without detergent match, revealing a monolayer of detergent molecules under proper solubilization conditions.

We describe the development of biohybrid electrodes constructed via combination of electrospun (e spun) 3D indium tin oxide (ITO) with the trimeric supercomplex photosystem I and the small electrochemically active protein cytochrome c (cyt c). The developed 3D surface of ITO has been created by electrospinning of a mixture of polyelthylene oxide (PEO) and ITO nanoparticles onto ITO glass slides followed by a subsequent elimination of PEO by sintering the composite. Whereas the photosystem I alone shows only small photocurrents at these 3D electrodes, the co-immobilization of cyt c to the e spun 3D ITO results in well-defined photoelectrochemical signals. The scaling of thickness of the 3D ITO layers by controlling the time (10 min and 60 min) of electrospinning results in enhancement of the photocurrent. Several performance parameters of the electrode have been analyzed for different illumination intensities. (c) 2021 Elsevier B.V. All rights reserved.

The attraction of bioelectrochemistry has risen constantly in recent times. Here, nature is imitated in biohybrid systems. One interesting example are photobioelectrodes where artificial materials are linked to biological components which are responsible for photosynthesis in plants or cyanobacteria. To date, biophotovoltaics using bacterial reaction centers, photosystem I or II (PSI or PSII), thylakoid membranes or whole cells have been set up successfully. [1] In this way, not only the generation of photocurrent is possible. Additionally, photosystem-based biosensors as well as devices that convert light to valuable chemicals have been constructed. [2, 3]

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.