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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 Mn4CaO5 cluster first stores four oxidizing equivalents, the S0 to S4 intermediate states in the Kok cycle, sequentially generated by photochemical charge separations in the reaction center and then catalyzes the O–O bond formation chemistry1–3. 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 S3→[S4]→S0 transition where O2 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 Mn4CaO5 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 Ox, which was introduced as a bridging ligand between Ca and Mn1 during the S2→S3 transition4–6, disappears or relocates in parallel with Yz reduction starting at approximately 700 μs after the third flash. The onset of O2 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.

Photosynthetic water oxidation by Photosystem II (PSII) is a fascinating process because it sustains life on Earth and serves as a blue print for scalable synthetic catalysts required for renewable energy applications. The biophysical, computational, and structural description of this process, which started more than 50 years ago, has made tremendous progress over the past two decades, with its high-resolution crystal structures being available not only of the dark-stable state of PSII, but of all the semi-stable reaction intermediates and even some transient states. Here, we summarize the current knowledge on PSII with emphasis on the basic principles that govern the conversion of light energy to chemical energy in PSII, as well as on the illustration of the molecular structures that enable these reactions. The important remaining questions regarding the mechanism of biological water oxidation are highlighted, and one possible pathway for this fundamental reaction is described at a molecular level.

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.

We present the synthesis and characterization of an efficient and low cost solar-driven electrolyzer consisting of Earth-abundant materials. The trimetallic NiFeMo electrocatalyst takes the shape of nanometer-sized flakes anchored to a fully carbon-based current collector comprising a nitrogen-doped carbon nanotube network, which in turn is grown on a carbon fiber paper support. This catalyst electrode contains solely Earth-abundant materials, and the carbon fiber support renders it effective despite a low metal content. Notably, a bifunctional catalyst–electrode pair exhibits a low total overpotential of 450 mV to drive a full water-splitting reaction at a current density of 10 mA cm–2 and a measured hydrogen Faradaic efficiency of ∼100%. We combine the catalyst–electrode pair with solution-processed perovskite solar cells to form a lightweight solar-driven water-splitting device with a high peak solar-to-fuel conversion efficiency of 13.8%. Originally included in thesis in manuscript form.

Green organisms evolve O2via photosynthesis and consume by respiration. Net O2consumption only becomes dominant when photosynthesis is suppressed at night. Here, we show that green thylakoid membranes of Scots pine (Pinus sylvestris L) and Norway spruce (Picea abies) needles demonstrate strong O2consumption even in the presence of light when extremely low temperatures coincide with high solar irradiation during early spring. This phenomenon deviates from the general finding that photosynthetic organisms evolve O2upon illumination. By using different electron transport chain inhibitors, we showed that O2consumption occurred around photosystem (PS) I and correlated with higher abundance of flavodiiron (Flv) A protein in ES thylakoid membranes. Furthermore, by measuring P700 absorption changes, we separated different alternative electron flow pathways and demonstrated that electron scavenging from the acceptor-side of PSI via O2photoreduction is a major alternative pathway in ES. This photoprotection mechanism in vascular plants indicates that conifers have developed an adaptative evolution trajectory for growing in harsh environments.

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 Mn4 CaO5 complex, in the intermediate Si (i= 0-4)-states of the Kok cycle, obtained using XFELs.

Ammonia production via the electrochemical N2 reduction reaction (NRR) at ambient conditions is highly desired as an alternative to the Haber-Bosch process, but remains a great challenge due to the low efficiency and selectivity caused by the competing hydrogen evolution reaction (HER). Herein we investigate the effect of availabilities of reactants (protons, electrons and N2) on NRR using a FeOx-coated carbon fiber paper cathode in various electrochemical configurations. NRR is found viable only under the conditions of low proton- and high N2 availabilities, which are achieved using 0.12 vol% water in LiClO4-ethyl acetate electrolyte and gaseous N2 supplied to the membrane-electrode assembly cathode. This results in an NRR rate of 29 ± 19 pmolNH3 s−1 cm−2 at a Faradaic efficiency of 70 ± 24% at the applied potential of −0.1 V vs. NHE. Other conditions (high proton-, or low N2-availability, or both) yield a lower or negligible amount of ammonia due to the competing HER. Our work shows that promoting NRR by suppressing the HER requires optimization of the operational variables, which serves as a complementary strategy to the development of NRR catalysts.

Photosynthetic water oxidation is catalyzed by a manganese–calcium oxide cluster, which experiences five “S-states” during a light-driven reaction cycle. The unique “distorted chair”-like geometry of the Mn4CaO5(6) cluster shows structural flexibility that has been frequently proposed to involve “open” and “closed”-cubane forms from the S1 to S3 states. The isomers are interconvertible in the S1 and S2 states, while in the S3 state, the open-cubane structure is observed to dominate inThermosynechococcus elongatus (cyanobacteria) samples. In this work, using density functional theory calculations, we go beyond the S3+Yz state to the S3nYz• → S4+Yz step, and report for the first time that the reversible isomerism, which is suppressed in the S3+Yz state, is fully recovered in the ensuing S3nYz• state due to the proton release from a manganese-bound water ligand. The altered coordination strength of the manganese–ligand facilitates formation of the closed-cubane form, in a dynamic equilibrium with the open-cubane form. This tautomerism immediately preceding dioxygen formation may constitute the rate limiting step for O2 formation, and exert a significant influence on the water oxidation mechanism in photosystem II. Swedish Research Council 2020-03809

Significance A new bridging oxygen ligand is incorporated between one of the Mn atoms and Ca in the Mn4Ca cluster during the transition from the one-photon induced S2 intermediate state to the two-photon induced S3 state in the catalytic water oxidation reaction in photosystem II. However, the sequence of events leading to this change is not known. Here we report an X-ray crystallography and spectroscopy study at room temperature using an X-ray free electron laser to collect a “molecular movie” of the structural and oxidation state change steps leading to the insertion of this new oxygen bridge, in the 50 µs to 200 ms time scales after photon absorption, which triggers the S2 → S3 state transition., In oxygenic photosynthesis, light-driven oxidation of water to molecular oxygen is carried out by the oxygen-evolving complex (OEC) in photosystem II (PS II). Recently, we reported the room-temperature structures of PS II in the four (semi)stable S-states, S1, S2, S3, and S0, showing that a water molecule is inserted during the S2 → S3 transition, as a new bridging O(H)-ligand between Mn1 and Ca. To understand the sequence of events leading to the formation of this last stable intermediate state before O2 formation, we recorded diffraction and Mn X-ray emission spectroscopy (XES) data at several time points during the S2 → S3 transition. At the electron acceptor site, changes due to the two-electron redox chemistry at the quinones, QA and QB, are observed. At the donor site, tyrosine YZ and His190 H-bonded to it move by 50 µs after the second flash, and Glu189 moves away from Ca. This is followed by Mn1 and Mn4 moving apart, and the insertion of OX(H) at the open coordination site of Mn1. This water, possibly a ligand of Ca, could be supplied via a “water wheel”-like arrangement of five waters next to the OEC that is connected by a large channel to the bulk solvent. XES spectra show that Mn oxidation (τ of ∼350 µs) during the S2 → S3 transition mirrors the appearance of OX electron density. This indicates that the oxidation state change and the insertion of water as a bridging atom between Mn1 and Ca are highly correlated.

Worldwide significant efforts are ongoing to develop devices that store solar energy as fuels. In one such approach, solar energy is absorbed by semiconductors and utilized directly by catalysts at their surfaces to split water into H-2 and O-2. To protect the semiconductors in these photo-electrochemical cells (PEC) from corrosion, frequently thin TiO2 interlayers are applied. Employing a well-performing photoanode comprised of 1-D n-Si microwires (MWs) covered with a mesoporous (mp) TiO2 interlayer fabricated by solution processing and functionalized with alpha-Fe2O3 nanorods, we studied here the function of this TiO2 interlayer by high-energy resolution fluorescence detected X-ray absorption near edge structure (HERFD-XANES) spectroscopy, along with X-ray emission spectroscopy (XES) and standard characterization techniques. Our data reveal that the TiO2 interlayer not only protects the n-Si MW surface from corrosion, but that it also acts as a template for the hydrothermal growth of alpha-Fe2O3 nanorods and improves the photocatalytic efficiency. We show that the latter effect correlates with the presence of stable oxygen vacancies at the interface between mp-TiO2 and alpha-Fe2O3, which act as electron traps and thereby substantially reduce the charge recombination rate at the hematite surface.

Photosynthesis stores solar light as chemical energy and efficiency of this process isv highly important. The electrons required for CO2 reduction are extracted from water in a reaction driven by light-induced charge separations in the Photosystem II reaction center and catalyzed by the CaMn4O5-cluster. This cyclic process involves five redox intermediates known as the S-0-S-4 states. In this study, we quantify the flash-induced turnover efficiency of each S state by electron paramagnetic resonance spectroscopy. Measurements were performed in photosystem II membrane preparations from spinach in the presence of an exogenous electron acceptor at selected temperatures between -10 degrees C and +20 degrees C and at flash frequencies of 1.25, 5 and 10 Hz. The results show that at optimal conditions the turnover efficiencies are limited by reactions occurring in the water oxidizing complex, allowing the extraction of their S state dependence and correlating low efficiencies to structural changes and chemical events during the reaction cycle. At temperatures 10 degrees C and below, the highest efficiency (i.e. lowest miss parameter) was found for the S-1 -> S-2 transition, while the S-2 -> S-3 transition was least efficient (highest miss parameter) over the whole temperature range. These electron paramagnetic resonance results were confirmed by measurements of flash-induced oxygen release patterns in thylakoid membranes and are explained on the basis of S state dependent structural changes at the CaMn4O5-cluster that were determined recently by femtosecond X-ray crystallography. Thereby, possible "molecular errors" connected to the e(-) transfer, H+ transfer, H2O binding and O-2 release are identified.

The design of molecular water oxidation catalysts (WOCs) requires a rational approach that considers the intermediate steps of the catalytic cycle, including water binding, deprotonation, storage of oxidizing equivalents, O-O bond formation, and O2 release. We investigated several of these properties for a series of base metal complexes (M = Mn, Fe, Co, Ni) bearing two variants of a pentapyridyl ligand framework, of which some were reported previously to be active WOCs. We found that only [Fe(Py5OMe)Cl]+ (Py5OMe = pyridine-2,6-diylbis[di-(pyridin-2-yl)methoxymethane]) showed an appreciable catalytic activity with a turnover number (TON) = 130 in light-driven experiments using the [Ru(bpy)3]2+/S2O82- system at pH 8.0, but that activity is demonstrated to arise from the rapid degradation in the buffered solution leading to the formation of catalytically active amorphous iron oxide/hydroxide (FeOOH), which subsequently lost the catalytic activity by forming more extensive and structured FeOOH species. The detailed analysis of the redox and water-binding properties employing electrochemistry, X-ray absorption spectroscopy (XAS), UV-vis spectroscopy, and density-functional theory (DFT) showed that all complexes were able to undergo the MIII/MII oxidation, but none was able to yield a detectable amount of a MIV state in our potential window (up to +2 V vs SHE). This inability was traced to (i) the preference for binding Cl- or acetonitrile instead of water-derived species in the apical position, which excludes redox leveling via proton coupled electron transfer, and (ii) the lack of sigma donor ligands that would stabilize oxidation states beyond MIII. On that basis, design features for next-generation molecular WOCs are suggested.

Structural characterization of transient electrochemical species in the sub-millisecond time scale is the holy grail of electrochemistry. Presently, common time resolution of structural spectro-electrochemical methods is about 0.1 seconds. Herein, a transient spectro-electrochemical Raman setup of easy implementation is described which allows sub-ms time resolution. The technique studies electrochemical processes by initiating the reaction with an electric potential (or current) pulse and analyses the product with a synchronized laser pulse of the modified Raman spectrometer. The approach was validated by studying a known redox driven isomerization of a Ru-based molecular switch grafted, as monolayer, on a SERS active Au microelectrode. Density-functional-theory calculations confirmed the spectral assignments to sub-ms transient species. This study paves the way to a new generation of time-resolved spectro-electrochemical techniques which will be of fundamental help in the development of next generation electrolizers, fuel cells and batteries.

Wang et al. (1) performed their own analysis of our crystallographic data (2) and questioned 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 S2-to-S3 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. [↵][1]1To whom correspondence may be addressed. Email: jyano{at}lbl.gov. [1]: #xref-corresp-1-1

Molecular hydrogen, H2, is an energy carrier that is increasing in popularity as an alternative fuel. Presently, the major part of commercially available H2 is produced from fossil resources. To make H2 a true contender to fossil fuels, a sustainable production via water splitting by renewable electricity in combination with earth-abundant catalysts or by photosynthetic microorganisms is required.

Significance Recent results have shown that nature’s water splitting catalyst inserts an additional water molecule into what appears to be a solvent inaccessible site late in its reaction cycle. The emerging consensus of the field is that this water molecule is one of the substrates of the reaction. Here, we show that this water molecule does not come directly from solvent. It instead represents an earlier bound water, which is inserted into this site via facile structural tautomerism. The trigger for this process is cofactor oxidation. This then allows an additional water to bind from solvent to a more open site of the cofactor. In this way the cofactor carefully regulates water uptake, preventing water insertion earlier in the reaction cycle.

In nature, an oxo‐bridged Mn4CaO5 cluster embedded in photosystem II (PSII), a membrane‐bound multi‐subunit pigment protein complex, catalyzes the water oxidation reaction that is driven by light‐i ...

High solar flux is known to diminish photosynthetic growth rates, reducing biomass productivity and lowering disease tolerance. Photosystem II (PSII) of plants is susceptible to photodamage (also known as photoinactivation) in strong light, resulting in severe loss of water oxidation capacity and destruction of the water-oxidizing complex (WOC). The repair of damaged PSIIs comes at a high energy cost and requires de novo biosynthesis of damaged PSII subunits, reassembly of the WOC inorganic cofactors and membrane remodeling. Employing membrane-inlet mass spectrometry and O

Light-driven oxidation of water to molecular oxygen is catalyzed by the oxygen-evolving complex (OEC) in Photosystem II (PS II). This multi-electron, multi-proton catalysis requires the transport of two water molecules to and four protons from the OEC. A high-resolution 1.89 Å structure obtained by averaging all the S states and refining the data of various time points during the S2 to S3 transition has provided better visualization of the potential pathways for substrate water insertion and proton release. Our results indicate that the O1 channel is the likely water intake pathway, and the Cl1 channel is the likely proton release pathway based on the structural rearrangements of water molecules and amino acid side chains along these channels. In particular in the Cl1 channel, we suggest that residue D1-E65 serves as a gate for proton transport by minimizing the back reaction. The results show that the water oxidation reaction at the OEC is well coordinated with the amino acid side chains and the H-bonding network over the entire length of the channels, which is essential in shuttling substrate waters and protons., 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.

The molecular oxygen we breathe is produced from water-derived oxygen species bound to the Mn4CaO5 cluster in photosystem II (PSII). Present research points to the central oxo-bridge O5 as the ‘slow exchanging substrate water (Ws)’, while, in the S2 state, the terminal water ligands W2 and W3 are both discussed as the ‘fast exchanging substrate water (Wf)’. A critical point for the assignment of Wf is whether or not its exchange with bulk water is limited by barriers in the channels leading to the Mn4CaO5 cluster. In this study, we measured the rates of H216O/H218O substrate water exchange in the S2 and S3 states of PSII core complexes from wild-type (WT) Synechocystis sp. PCC 6803, and from two mutants, D1-D61A and D1-E189Q, that are expected to alter water access via the Cl1/O4 channels and the O1 channel, respectively. We found that the exchange rates of Wf and Ws were unaffected by the E189Q mutation (O1 channel), but strongly perturbed by the D61A mutation (Cl1/O4 channel). It is concluded that all channels have restrictions limiting the isotopic equilibration of the inner water pool near the Mn4CaO5 cluster, and that D61 participates in one such barrier. In the D61A mutant this barrier is lowered so that Wf exchange occurs more rapidly. This finding removes the main argument against Ca-bound W3 as fast substrate water in the S2 state, namely the indifference of the rate of Wf exchange towards Ca/Sr substitution., Access to the oxygen-evolving complex in photosynthesis is restricted by specific barriers in the channels connecting the Mn4CaO5 catalyst with bulk water. Together with other recent data, this finding allows assigning the two substrate waters.

Developing new transition metal catalysts requires understanding of how both metal and ligand properties determine reactivity. Since metal complexes bearing ligands of the Py5 family (2,6-bis-[(2-pyridyl)methyl] pyridine) have been employed in many fields in the past 20 years, we set out here to understand their redox properties by studying a series of base metal ions (M = Mn, Fe, Co, and Ni) within the Py5OH (pyridine-2,6-diylbis[di-(pyridin-2-yl)methanol]) variant. Both reduced (M-II) and the one-electron oxidized (M-III) species were carefully characterized using a combination of X-ray crystallography, X-ray absorption spectroscopy, cyclic voltammetry, and density-functional theory calculations. The observed metal-ligand interactions and electrochemical properties do not always follow consistent trends along the periodic table. We demonstrate that this observation cannot be explained by only considering orbital and geometric relaxation, and that spin multiplicity changes needed to be included into the DFT calculations to reproduce and understand these trends. In addition, exchange reactions of the sixth ligand coordinated to the metal, were analysed. Finally, by including published data of the extensively characterised Py5OMe (pyridine-2,6-diylbis[di-(pyridin-2-yl)methoxymethane])complexes, the special characteristics of the less common Py5OH ligand were extracted. This comparison highlights the non-innocent effect of the distal OH functionalization on the geometry, and consequently on the electronic structure of the metal complexes. Together, this gives a complete analysis of metal and ligand degrees of freedom for these base metal complexes, while also providing general insights into how to control electrochemical processes of transition metal complexes.

Worldwide significant efforts are ongoing to develop devices that store solar energy as fuels. In one such approach, solar energy is absorbed by semiconductors and utilized directly by catalysts at their surfaces to split water into H2 and O2. To protect the semiconductors in these photo-electrochemical cells (PEC) from corrosion, frequently thin TiO2 interlayers are applied. Employing a well-performing photoanode comprised of 1-D n-Si microwires (MWs) covered with a mesoporous (mp) TiO2 interlayer fabricated by solution processing and functionalized with α-Fe2O3 nanorods, we studied here the function of this TiO2 interlayer by high-energy resolution fluorescence detected X-ray absorption near edge structure (HERFD-XANES) spectroscopy, along with X-ray emission spectroscopy (XES) and standard characterization techniques. Our data reveal that the TiO2 interlayer not only protects the n-Si MW surface from corrosion, but that it also acts as a template for the hydrothermal growth of α-Fe2O3 nanorods and improves the photocatalytic efficiency. We show that the latter effect correlates with the presence of stable oxygen vacancies at the interface between mp-TiO2 and α-Fe2O3, which act as electron traps and thereby substantially reduce the charge recombination rate at the hematite surface.

Significance Photosynthetic H2 production in the green alga Chlamydomonas reinhardtii is catalyzed by O2-sensitive [FeFe]-hydrogenases, which accept electrons from photosynthetically reduced ferredoxin and reduce protons to H2. Since the process occurs downstream of photosystem I, the contribution of photosystem II (PSII) in H2 photoproduction has long been a subject of debate. Indeed, water oxidation by PSII results in O2 accumulation in chloroplasts, which inhibits H2 evolution. Therefore, clear evidence for direct water biophotolysis resulting in simultaneous H2 and O2 releases in algae has never been presented. This paper demonstrates that sustained H2 photoproduction in C. reinhardtii is directly linked to PSII-dependent water oxidation and brings insights into regulation of PSII activity and H2 production by CO2/HCO3– under microoxic conditions., The unicellular green alga Chlamydomonas reinhardtii is capable of photosynthetic H2 production. H2 evolution occurs under anaerobic conditions and is difficult to sustain due to 1) competition between [FeFe]-hydrogenase (H2ase), the key enzyme responsible for H2 metabolism in algae, and the Calvin–Benson–Bassham (CBB) cycle for photosynthetic reductants and 2) inactivation of H2ase by O2 coevolved in photosynthesis. Recently, we achieved sustainable H2 photoproduction by shifting algae from continuous illumination to a train of short (1 s) light pulses, interrupted by longer (9 s) dark periods. This illumination regime prevents activation of the CBB cycle and redirects photosynthetic electrons to H2ase. Employing membrane-inlet mass spectrometry and H218O, we now present clear evidence that efficient H2 photoproduction in pulse-illuminated algae depends primarily on direct water biophotolysis, where water oxidation at the donor side of photosystem II (PSII) provides electrons for the reduction of protons by H2ase downstream of photosystem I. This occurs exclusively in the absence of CO2 fixation, while with the activation of the CBB cycle by longer (8 s) light pulses the H2 photoproduction ceases and instead a slow overall H2 uptake is observed. We also demonstrate that the loss of PSII activity in DCMU-treated algae or in PSII-deficient mutant cells can be partly compensated for by the indirect (PSII-independent) H2 photoproduction pathway, but only for a short (

In photosynthesis, the oxygen-evolving complex (OEC) of the pigment-protein complex photosystem II (PSII) orchestrates the oxidation of water. Introduction of the V185N mutation into the D1 protein was previously reported to drastically slow O

In photosynthesis, dioxygen formation from water is catalyzed by the oxygen evolving complex (OEC) in Photosystem II (PSII) that harbours the Mn

In photosynthesis, dioxygen formation from water is catalyzed by the oxygen evolving complex (OEC) in Photosystem II (PSII) that harbours the Mn4Ca cluster. During catalysis, the OEC cycles through five redox states, S0 to S4. In the S2 state, the Mn4Ca cluster can exist in two conformations, which are signified by the low-spin (LS) g = 2 EPR multiline signal and the high-spin (HS) g = 4.1 EPR signal. Here, we employed time-resolved membrane inlet mass spectrometry to measure the kinetics of H218O/H216O exchange between bulk water and the two substrate waters bound at the Mn4Ca cluster in the SLS2, SHS2, and the S3 states in both Ca-PSII and Sr-PSII core complexes from T. elongatus. We found that the slowly exchanging substrate water exchanges 10 times faster in the SHS2 than in the SLS2 state, and that the SLS2 → SHS2 conversion has at physiological temperature an activation barrier of 17 ± 1 kcal mol−1. Of the presently suggested SHS2 models, our findings are best in agreement with a water exchange pathway involving a SHS2state that has an open cubane structure with a hydroxide bound between Ca and Mn1. We also show that water exchange in the S3 state is governed by a different equilibrium than in S2, and that the exchange of the fast substrate water in the S2 state is unaffected by Ca/Sr substitution. These findings support that (i) O5 is the slowly exchanging substrate water, with W2 being the only other option, and (ii) either W2 or W3 is the fast exchanging substrate. The three remaining possibilities for O–O bond formation in PSII are discussed.

The effect of bicarbonate (HCO3-) on photosystem II (PSII) activity was discovered in the 1950s, but only recently have its molecular mechanisms begun to be clarified. Two chemical mechanisms have been proposed. One is for the electron-donor side, in which mobile HCO3- enhances and possibly regulates water oxidation by acting as proton acceptor, after which it dissociates into CO2 and H2O. The other is for the electron-acceptor side, in which (i) reduction of the Q(A) quinone leads to the release of HCO(3)(- )from its binding site on the non-heme iron and (ii) the E-m potential of the Q(A)/Q(.-) couple increases when HCO3- dissociates. This suggested a protective/regulatory role of HCO3- as it is known that increasing the E-m of Q(A) decreases the extent of back-reaction-associated photodamage. Here we demonstrate, using plant thylakoids, that time-resolved membrane-inlet mass spectrometry together with C-13 isotope labeling of HCO3- allows donor- and acceptor-side formation of CO2 by PSII to be demonstrated and distinguished, which opens the door for future studies of the importance of both mechanisms under in vivo conditions.

With this Tribute, we remember and honor Thomas John (Tom) Wydrzynski. Tom was a highly innovative, independent and committed researcher, who had, early in his career, defined his life-long research goal. He was committed to understand how Photosystem II produces molecular oxygen from water, using the energy of sunlight, and to apply this knowledge towards making artificial systems. In this tribute, we summarize his research journey, which involved working on 'soft money' in several laboratories around the world for many years, as well as his research achievements. We also reflect upon his approach to life, science and student supervision, as we perceive it. Tom was not only a thoughtful scientist that inspired many to enter this field of research, but also a wonderful supervisor and friend, who is deeply missed (see footnote*).

Stable and efficient oxygen evolution reaction (OER) catalysts for the oxidation of water to dioxygen in highly acidic media are currently limited to expensive noble metal (Ir and Ru) oxides since ...

Knowledge of the manganese oxidation states of the oxygen-evolving Mn 4 CaO 5 cluster in photosystem II (PSII) is crucial toward understanding the mechanism of biological water oxidation. There is a 4 decade long debate on this topic that historically originates from the observation of a multiline electron paramagnetic resonance (EPR) signal with effective total spin of S = 1/2 in the singly oxidized S 2 state of this cluster. This signal implies an overall oxidation state of either Mn(III) 3 Mn(IV) or Mn(III)Mn(IV) 3 for the S 2 state. These 2 competing assignments are commonly known as “low oxidation (LO)” and “high oxidation (HO)” models of the Mn 4 CaO 5 cluster. Recent advanced EPR and Mn K-edge X-ray spectroscopy studies converge upon the HO model. However, doubts about these assignments have been voiced, fueled especially by studies counting the number of flash-driven electron removals required for the assembly of an active Mn 4 CaO 5 cluster starting from Mn(II) and Mn-free PSII. This process, known as photoactivation, appeared to support the LO model since the first oxygen is reported to evolve already after 7 flashes. In this study, we improved the quantum yield and sensitivity of the photoactivation experiment by employing PSII microcrystals that retained all protein subunits after complete manganese removal and by oxygen detection via a custom built thin-layer cell connected to a membrane inlet mass spectrometer. We demonstrate that 9 flashes by a nanosecond laser are required for the production of the first oxygen, which proves that the HO model provides the correct description of the Mn 4 CaO 5 cluster’s oxidation states.

In this study, we presented a new approach for quantification of bicarbonate (HCO3-) molecules bound to PSII. Our method, which is based on a combination of membrane-inlet mass spectrometry (MIMS) ...

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 K beta 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 K beta XES signal. We report here a systematic study of the effects of XFEL peak power, beam focus, and dose on the Mn K beta(1,3) 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 & SIM;5 x 10(15) to 5 x 10(17) W/cm(2) at a pulse length of & SIM;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.

Layers of amorphous manganese oxides were directly formed on the surfaces of different carbon materials by exposing the carbon to aqueous solutions of permanganate (MnO4- ) followed by sintering at ...

X-ray crystallography at X-ray free-electron laser sources is a powerful method for studying macromolecules at biologically relevant temperatures. Moreover, when combined with complementary techniq ...

The development of efficient, durable, and inexpensive hydrogen evolution electrodes remains a key challenge for realizing a sustainable H2 fuel production via electrocatalytic water splitting. Herein, nickel–iron phosphide porous films with precisely controlled metal content were synthesized on Ti foil using a simple and scalable two-step strategy of spray-pyrolysis deposition followed by low-temperature phosphidation. The nickel–iron phosphide of an optimized Ni:Fe ratio of 1:4 demonstrated excellent overall catalytic activity for hydrogen evolution reaction (HER) in 0.5 M H2SO4, achieving current densities of −10 and −30 mA cm–2 at overpotentials of 101 and 123 mV, respectively, with a Tafel slope of 43 mV dec–1. Detailed analysis obtained by X-ray diffraction, electron microscopy, electrochemistry, and X-ray photoelectron spectroscopy revealed that the superior overall HER activity of nickel–iron phosphide as compared to nickel phosphide and iron phosphide was a combined effect of differences in the m...

This work has demonstrated that X-ray absorption spectroscopy (XAS), both Mn XANES and EXAFS, of solutions with millimolar concentrations of metal is possible using the femtosecond X-ray pulses from XFELs. Mn XAS data were collected using two different sample delivery methods, a Rayleigh jet and a drop-on-demand setup, with varying concentrations of Mn. Here, a new method for normalization of XAS spectra based on solvent scattering that is compatible with data collection from a highly variable pulsed source is described. The measured XANES and EXAFS spectra of such dilute solution samples are in good agreement with data collected at synchrotron sources using traditional scanning protocols. The procedures described here will enable XFEL-based XAS on dilute biological samples, especially metalloproteins, with low sample consumption. Details of the experimental setup and data analysis methods used in this XANES and EXAFS study are presented. This method will also benefit XAS performed at high-repetition-rate XFELs such as the European XFEL, LCLS-II and LCLS-II-HE.

Photosynthetic water oxidation is catalyzed by the oxygen-evolving complex (OEC) in photosystem II (PSII). This process is energetically driven by light-induced charge separation in the reaction center of PSII, which leads to a stepwise accumulation of oxidizing equivalents in the OEC (S-i states, i=0-4) resulting in O-2 evolution after each fourth flash, and to the reduction of plastoquinone to plastoquinol on the acceptor side of PSII. However, the S-i-state advancement is not perfect, which according to the Kok model is described by miss-hits (misses). These may be caused by redox equilibria or kinetic limitations on the donor (OEC) or the acceptor side. In this study, we investigate the effects of individual S state transitions and of the quinone acceptor side on the miss parameter by analyzing the flash-induced oxygen evolution patterns and the S-2, S-3 and S-0 state lifetimes in thylakoid samples of the extremophilic red alga Cyanidioschyzon merolae. The data are analyzed employing a global fit analysis and the results are compared to the data obtained previously for spinach thylakoids. These two organisms were selected, because the redox potential of Q(A)/Q(A)(-) in PSII is significantly less negative in C. merolae (E-m=-104mV) than in spinach (E-m=-163mV). This significant difference in redox potential was expected to allow the disentanglement of acceptor and donor side effects on the miss parameter. Our data indicate that, at slightly acidic and neutral pH values, the E-m of Q(A)(-)/Q(A) plays only a minor role for the miss parameter. By contrast, the increased energy gap for the backward electron transfer from Q(A)(-) to Pheo slows down the charge recombination reaction with the S-3 and S-2 states considerably. In addition, our data support the concept that the S-2 S-3 transition is the least efficient step during the oxidation of water to molecular oxygen in the Kok cycle of PSII.

International audience; Herein, we communicate about an Earth-abundant semiconductor photocathode (p-Si/TiO2/NiOx) as an alternative for the rare and expensive Pt as a counter electrode for overall photoelectrochemical water splitting. The proposed photoelectrochemical (PEC) water-splitting device mimics the Z-scheme observed in natural photosynthesis by combining two photoelectrodes in a parallel-illumination mode. A nearly 60% increase in the photocurrent density (J(ph)) for pristine -Fe2O3 and a 77% increase in the applied bias photocurrent efficiency (ABPE) were achieved by replacing the conventionally used Pt cathode with an efficient, cost effective p-Si/TiO2/NiOx photocathode under parallel illumination. The resulting photocurrent density of 1.26 mA cm(-2) at 1.23V(RHE) represents a new record performance for hydrothermally grown pristine -Fe2O3 nanorod photoanodes in combination with a photocathode, which opens the prospect for further improvement by doping -Fe2O3 or by its decoration with co-catalysts. Electrochemical impedance spectroscopy measurements suggest that this significant performance increase is due to the enhancement of the space-charge field in -Fe2O3.

Earth-abundant cobalt-doped hematite thin-film electrocatalysts were explored for acidic water oxidation. The strategically doped hematite produced a stable geometric current density of 10 mA cm(-2) for up to 50 h at pH 0.3, as a result of Co-enhanced intrinsic catalytic activity and charge transport properties across the film matrix.

In nature, an oxo-bridged Mn(4)CaO(5) cluster embedded in Photosystem II (PSII), a membrane-bound multi-subunit pigment protein complex, catalyzes the water oxidation reaction that is driven by light-induced charge separations in the reaction center of PSII. The Mn(4)CaO(5) cluster accumulates four oxidizing equivalents to enable the four-electron four-proton catalysis of two water molecules to one dioxygen molecule and cycles through five intermediate S-states, S(0) – S(4) in the Kok cycle. One important question related to the catalytic mechanism of the oxygen-evolving complex (OEC) that remains is, whether structural isomers are present in some of the intermediate S-states and if such equilibria are essential for the mechanism of the O-O bond formation. Here we compare results from electron paramagnetic resonance (EPR) and X-ray absorption spectroscopy (XAS) obtained at cryogenic temperatures for the S(2) state of PSII with structural data collected of the S(1), S(2) and S(3) states by serial crystallography at neutral pH (~6.5) using an X-ray free electron laser at room temperature. While the cryogenic data demonstrate the presence of at least two structural forms of the S(2) state, the room temperature crystallography data can be well-described by just one S(2) structure. We discuss the deviating results and outline experimental strategies for clarifying this mechanistically important question.

The oxygen-evolving complex (OEC) in photosystem II catalyzes the oxidation of water to molecular oxygen. Four decades ago, measurements of flash-induced oxygen evolution have shown that the OEC steps through oxidation states S(0), S(1), S(2), S(3) and S(4) before O(2) is released and the S(0) state is reformed. The light-induced transitions between these states involve misses and double hits. While it is widely accepted that the miss parameter is S state dependent and may be further modulated by the oxidation state of the acceptor side, the traditional way of analyzing each flash-induced oxygen evolution pattern (FIOP) individually did not allow using enough free parameters to thoroughly test this proposal. Furthermore, this approach does not allow assessing whether the presently known recombination processes in photosystem II fully explain all measured oxygen yields during Si state lifetime measurements. Here we present a global fit program that simultaneously fits all flash-induced oxygen yields of a standard FIOP (2 Hz flash frequency) and of 11-18 FIOPs each obtained while probing the S(0), S(2) and S(3) state lifetimes in spinach thylakoids at neutral pH. This comprehensive data treatment demonstrates the presence of a very slow phase of S(2) decay, in addition to the commonly discussed fast and slow reduction of S(2) by YD and QB(-), respectively. Our data support previous suggestions that the S(0)→S(1) and S(1)→S(2) transitions involve low or no misses, while high misses occur in the S(2)→S(3) or S(3)→S(0) transitions.

Oxygen evolution catalysts (OEC) are often employed on the surface of photoactive, semiconducting photoanodes to boost their kinetics and stability during photoelectrochemical water oxidation. However, the necessity of using optically transparent OEC to avoid parasitic light absorption by the OEC under front-side illumination is often neglected. Here, we show that furnishing the surface of a WO3 photoanode with suitable loading of FeOOH as a transparent OEC improved the photocurrent density by 300% at 1 V versus RHE and the initial photocurrent-to-O2 Faradaic efficiency from ∼70 to ∼100%. The data from the photovoltammetry, electrochemical impedance, and gas evolution measurements show that these improvements were a combined result of reduced hole-transfer resistance for water oxidation, minimized surface recombination of charge carriers, and improved stability against photocorrosion of WO3. We demonstrate the utility of transparent FeOOH-coated WO3 in a solar-powered, tandem water-splitting device by com...

Etioplasts lack thylakoid membranes and photosystem complexes. Light triggers differentiation of etioplasts into mature chloroplasts, and photosystem complexes assemble in parallel with thylakoid membrane development. Plastids isolated at various time points of de-etiolation are ideal to study the kinetic biogenesis of photosystem complexes during chloroplast development. Here, we investigated the chronology of photosystem II (PSII) biogenesis by monitoring assembly status of chlorophyll-binding protein complexes and development of water splitting via O2 production in plastids (etiochloroplasts) isolated during de-etiolation of barley (Hordeum vulgare L.). Assembly of PSII monomers, dimers and complexes binding outer light-harvesting antenna [PSII-light-harvesting complex II (LHCII) supercomplexes] was identified after 1, 2 and 4 h of de-etiolation, respectively. Water splitting was detected in parallel with assembly of PSII monomers, and its development correlated with an increase of bound Mn in the samples. After 4 h of de-etiolation, etiochloroplasts revealed the same water-splitting efficiency as mature chloroplasts. We conclude that the capability of PSII to split water during de-etiolation precedes assembly of the PSII-LHCII supercomplexes. Taken together, data show a rapid establishment of water-splitting activity during etioplast-to-chloroplast transition and emphasize that assembly of the functional water-splitting site of PSII is not the rate-limiting step in the formation of photoactive thylakoid membranes.

This journal is © The Royal Society of Chemistry. The ultra-bright femtosecond X-ray pulses provided by X-ray Free Electron Lasers (XFELs) open capabilities for studying the structure and dynamics of a wide variety of biological and inorganic systems beyond what is possible at synchrotron sources. Although the structure and chemistry at the catalytic sites have been studied intensively in both biological and inorganic systems, a full understanding of the atomic-scale chemistry requires new approaches beyond the steady state X-ray crystallography and X-ray spectroscopy at cryogenic temperatures. Following the dynamic changes in the geometric and electronic structure at ambient conditions, while overcoming X-ray damage to the redox active catalytic center, is key for deriving reaction mechanisms. Such studies become possible by using the intense and ultra-short femtosecond X-ray pulses from an XFEL, where sample is probed before it is damaged. We have developed methodology for simultaneously collecting X-ray diffraction data and X-ray emission spectra, using an energy dispersive spectrometer, at ambient conditions, and used this approach to study the room temperature structure and intermediate states of the photosynthetic water oxidizing metallo-protein, photosystem II. Moreover, we have also used this setup to simultaneously collect the X-ray emission spectra from multiple metals to follow the ultrafast dynamics of light-induced charge transfer between multiple metal sites. A Mn-Ti containing system was studied at an XFEL to demonstrate the efficacy and potential of this method.

A hybrid catalyst 3D electrode for electrochemical water oxidation to molecular oxygen is presented. The electrode comprises needle shaped maghemite nanorods firmly anchored to nitrogen doped carbo ...

Inspired by the period-four oscillation in flash-induced oxygen evolution of photosystem II discovered by Joliot in 1969, Kok performed additional experiments and proposed a five-state kinetic model for photosynthetic oxygen evolution, known as Kok’s S-state clock or cycle(1,2). The model comprises four (meta)stable intermediates (S(0), S(1), S(2) and S(3)) and one transient S(4) state, which precedes dioxygen formation occurring in a concerted reaction from two water-derived oxygens bound at an oxo-bridged tetra manganese calcium (Mn(4)CaO(5)) cluster in the oxygen-evolving complex(3–7). This reaction is coupled to the two-step reduction and protonation of the mobile plastoquinone Q(B) at the acceptor side of PSII. Here, using serial femtosecond X-ray crystallography and simultaneous X-ray emission spectroscopy with multi-flash visible laser excitation at room temperature, we visualize all (meta)stable states of Kok’s cycle as high-resolution structures (2.04–2.08 Å). In addition, we report structures of two transient states at 150 and 400 μs, revealing notable structural changes including the binding of one additional ‘water’, Ox, during the S(2)→S(3) state transition. Our results suggest that one water ligand to calcium (W3) is directly involved in substrate delivery. The binding of the additional oxygen Ox in the S(3) state between Ca and Mn1 supports O–O bond formation mechanisms involving O5 as one substrate, where Ox is either the other substrate oxygen or is perfectly positioned to refill the O5 position during O(2) release. Thus, our results exclude peroxo-bond formation in the S(3) state, and the nucleophilic attack of W3 onto W2 is unlikely.

This chapter compares two different techniques for monitoring photosynthetic O