Structural evidence for intermediates during O2 formation in photosystem II.

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.

On the simulation and interpretation of substrate-water exchange experiments in photosynthetic water oxidation.

Water oxidation by photosystem II (PSII) sustains most life on Earth, but the molecular mechanism of this unique process remains controversial. The ongoing identification of the binding sites and modes of the two water-derived substrate oxygens ('substrate waters') in the various intermediates (Si states, i = 0, 1, 2, 3, 4) that the water-splitting tetra-manganese calcium penta-oxygen (Mn4CaO5) cluster attains during the reaction cycle provides central information towards resolving the unique chemistry of biological water oxidation. Mass spectrometric measurements of single- and double-labeled dioxygen species after various incubation times of PSII with H218O provide insight into the substrate binding modes and sites via determination of exchange rates. Such experiments have revealed that the two substrate waters exchange with different rates that vary independently with the Si state and are hence referred to as the fast (Wf) and the slow (WS) substrate waters. New insight for the molecular interpretation of these rates arises from our recent finding that in the S2 state, under special experimental conditions, two different rates of WS exchange are observed that appear to correlate with the high spin and low spin conformations of the Mn4CaO5 cluster. Here, we reexamine and unite various proposed methods for extracting and assigning rate constants from this recent data set. The analysis results in a molecular model for substrate-water binding and exchange that reconciles the expected non-exchangeability of the central oxo bridge O5 when located between two Mn(IV) ions with the experimental and theoretical assignment of O5 as WS in all S states. The analysis also excludes other published proposals for explaining the water exchange kinetics.

Untangling the sequence of events during the S2 → S3 transition in photosystem II and implications for the water oxidation mechanism.

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.

Water Oxidation by Pentapyridyl Base Metal Complexes? A Case Study.

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.

Operando tracking of oxidation-state changes by coupling electrochemistry with time-resolved X-ray absorption spectroscopy demonstrated for water oxidation by a cobalt-based catalyst film.

Transition metal oxides are promising electrocatalysts for water oxidation, i.e., the oxygen evolution reaction (OER), which is critical in electrochemical production of non-fossil fuels. The involvement of oxidation state changes of the metal in OER electrocatalysis is increasingly recognized in the literature. Tracing these oxidation states under operation conditions could provide relevant information for performance optimization and development of durable catalysts, but further methodical developments are needed. Here, we propose a strategy to use single-energy X-ray absorption spectroscopy for monitoring metal oxidation-state changes during OER operation with millisecond time resolution. The procedure to obtain time-resolved oxidation state values, using two calibration curves, is explained in detail. We demonstrate the significance of this approach as well as possible sources of data misinterpretation. We conclude that the combination of X-ray absorption spectroscopy with electrochemical techniques allows us to investigate the kinetics of redox transitions and to distinguish the catalytic current from the redox current. Tracking of the oxidation state changes of Co ions in electrodeposited oxide films during cyclic voltammetry in neutral pH electrolyte serves as a proof of principle.

Revisiting Metal-Organic Frameworks for Oxygen Evolution: A Case Study.

Water splitting is a promising reaction for storing sustainable but intermittent energies. In water splitting, water oxidation is a bottleneck, and thus different catalysts have been synthesized for water oxidation. Metal-organic frameworks (MOFs) are among the highly efficient catalysts for water oxidation, and so far, MOF-based catalysts have been divided into two categories: MOF-derived catalysts and direct MOF catalysts. In particular, a nickel/cobalt MOF is reported to be one of the best direct catalysts for water oxidation. For the first-row transition MOF structures in general, a hypothesis is that the harsh conditions of OER could cause the decomposition of organic ligands and the formation of water-oxidizing oxide-based structures. By electrochemical methods, scanning electron microscopy, transmission electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy, Fourier-transform infrared spectroscopy, and X-ray absorption spectroscopy, a nickel/cobalt MOF known to be a highly efficient catalyst for water oxidation is shown to form Ni/Co oxide, making it a candidate catalyst for oxygen evolution. MOFs are interesting precatalysts for metal oxide water-oxidizing catalysts, but control experiments are necessary for determining whether a certain MOF or other MOFs are true catalysts for OER. Thus, finding a true and direct MOF electrocatalyst for OER is a challenge.

H/D Isotope Effects Reveal Factors Controlling Catalytic Activity in Co-Based Oxides for Water Oxidation.

Understanding the mechanism for electrochemical water oxidation is important for the development of more efficient catalysts for artificial photosynthesis. A basic step is the proton-coupled electron transfer, which enables accumulation of oxidizing equivalents without buildup of a charge. We find that substituting deuterium for hydrogen resulted in an 87% decrease in the catalytic activity for water oxidation on Co-based amorphous-oxide catalysts at neutral pH, while 16O-to-18O substitution lead to a 10% decrease. In situ visible and quasi-in situ X-ray absorption spectroscopy reveal that the hydrogen-to-deuterium isotopic substitution induces an equilibrium isotope effect that shifts the oxidation potentials positively by approximately 60 mV for the proton coupled CoII/III and CoIII/IV electron transfer processes. Time-resolved spectroelectrochemical measurements indicate the absence of a kinetic isotope effect, implying that the precatalytic proton-coupled electron transfer happens through a stepwise mechanism in which electron transfer is rate-determining. An observed correlation between Co oxidation states and catalytic current for both isotopic conditions indicates that the applied potential has no direct effect on the catalytic rate, which instead depends exponentially on the average Co oxidation state. These combined results provide evidence that neither proton nor electron transfer is involved in the catalytic rate-determining step. We propose a mechanism with an active species composed by two adjacent CoIV atoms and a rate-determining step that involves oxygen-oxygen bond formation and compare it with models proposed in the literature.

Unequal misses during the flash-induced advancement of photosystem II: effects of the S state and acceptor side cycles.

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 (Si states, i = 0-4) resulting in O2 evolution after each fourth flash, and to the reduction of plastoquinone to plastoquinol on the acceptor side of PSII. However, the Si-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 S2, S3 and S0 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 QA/QA- in PSII is significantly less negative in C. merolae (Em = - 104 mV) than in spinach (Em = - 163 mV). 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 Em of QA-/QA plays only a minor role for the miss parameter. By contrast, the increased energy gap for the backward electron transfer from QA- to Pheo slows down the charge recombination reaction with the S3 and S2 states considerably. In addition, our data support the concept that the S2 → S3 transition is the least efficient step during the oxidation of water to molecular oxygen in the Kok cycle of PSII.

Structural and functional role of anions in electrochemical water oxidation probed by arsenate incorporation into cobalt-oxide materials.

Direct (photo)electrochemical production of non-fossil fuels from water and CO2 requires water-oxidation catalysis at near-neutral pH in the presence of appropriate anions that serve as proton acceptors. We investigate the largely enigmatic structural role of anions in water oxidation for the prominent cobalt-phosphate catalyst (CoCat), an amorphous and hydrated oxide material. Co3([(P/As)O]4)2·8H2O served, in conjunction with phosphate-arsenate exchange, as a synthetic model system. Its structural transformation was induced by prolonged operation at catalytic potentials and probed by X-ray absorption spectroscopy not only at the metal (Co), but for the first time also at the anion (As) K-edge. For initially isostructural microcrystals, anion exchange determined the amorphization process and final structure. Comparison to amorphous electrodeposited Co oxide revealed that in CoCat, the arsenate binds not only at oxide-layer edges, but also arsenic substitutes cobalt positions within the layered-oxide structure in an unusual AsO6 coordination. Our results show that in water oxidation catalysis at near-neutral pH, anion type and exchange dynamics correlate with the catalyst structure and redox properties.

Behavior of Ru-bda Water-Oxidation Catalysts in Low Oxidation States.

The Ru complex [RuII (bda-κ-N2 O2 )(N-NH2 )2 ] (1; bda2- =2,2'-bipyridine-6,6'-dicarboxylate, N-NH2 =4-(pyridin-4-yl)aniline) was used as a synthetic intermediate to prepare new RuII and RuIII bda complexes that contain NO+ , MeCN, or H2 O ligands. In acidic solution complex 1 reacts with an excess of NO+ (generated in situ from sodium nitrite) to form a new Ru complex in which the aryl amine ligand N-NH2 is transformed into a diazonium salt [N-N2+ =4-(pyridin-4-yl)benzenediazonium)] together with the formation of a new Ru(NO) moiety in the equatorial zone, to generate [RuII (bda-κ-N2 O)(NO)(N-N2 )2 ]3+ (23+ ). Here the bda2- ligand binds in a κ-N2 O tridentate manner with a dangling carboxylate group. Similarly, complex 1 can also react with a coordinating solvent, such as MeCN, at room temperature to give [RuII (bda-κ-N2 O)(MeCN)(N-NH2 )2 ] (3). In acidic aqueous solutions, a related reaction occurs in which solvent water coordinates to the Ru center to form {[RuII {bda-κ-(NO)3 }(H2 O)(N-NH3 )2 ](H2 O)n }2+ (42+ ) and is strongly hydrogen-bonded with additional water molecules in the second coordination sphere. Furthermore, under acidic conditions the aniline ligands are also protonated to form the corresponding anilinium cationic ligands N-NH3+ . Additionally, the one-electron oxidized complex {[RuIII {bda-κ-(NO)3.5 }(H2 O)(N-NH3 )2 ](H2 O)n }3+ (53+ ) was characterized, in which the fractional value in the κ notation indicates the presence of an additional contact to the pseudo-octahedral geometry of the Ru center. The coordination modes of the complexes were studied in the solid state and in solution through single-crystal XRD, X-ray absorption spectroscopy, variable-temperature NMR spectroscopy, and DFT calculations. While κ-N2 O is the main coordination mode for 23+ and 3, an equilibrium that involves isomers with κ-N2 O and κ-NO2 coordination modes and neighboring hydrogen-bonded water molecules is observed for 42+ and 53+ .

Kα X-ray Emission Spectroscopy on the Photosynthetic Oxygen-Evolving Complex Supports Manganese Oxidation and Water Binding in the S3 State.

The unique manganese-calcium catalyst in photosystem II (PSII) is the natural paragon for efficient light-driven water oxidation to yield O2. The oxygen-evolving complex (OEC) in the dark-stable state (S1) comprises a Mn4CaO4 core with five metal-bound water species. Binding and modification of the water molecules that are substrates of the water-oxidation reaction is mechanistically crucial but controversially debated. Two recent crystal structures of the OEC in its highest oxidation state (S3) show either a vacant Mn coordination site or a bound peroxide species. For purified PSII at room temperature, we collected Mn Kα X-ray emission spectra of the S0, S1, S2, and S3 intermediates in the OEC cycle, which were analyzed by comparison to synthetic Mn compounds, spectral simulations, and OEC models from density functional theory. Our results contrast both crystallographic structures. They indicate Mn oxidation in three S-transitions and suggest additional water binding at a previously open Mn coordination site. These findings exclude Mn reduction and render peroxide formation in S3 unlikely.

Temperature Dependence of the Catalytic Two- versus Four-Electron Reduction of Dioxygen by a Hexanuclear Cobalt Complex.

The synthesis and characterization of a hexanuclear cobalt complex 1 involving a nonheme ligand system, L1, supported on a Sn6O6 stannoxane core are reported. Complex 1 acts as a unique catalyst for dioxygen reduction, whose selectivity can be changed from a preferential 4e-/4H+ dioxygen-reduction (to water) to a 2e-/2H+ process (to hydrogen peroxide) only by increasing the temperature from -50 to 25 °C. A variety of spectroscopic methods (119Sn-NMR, magnetic circular dichroism (MCD), electron paramagnetic resonance (EPR), SQUID, UV-vis absorption, and X-ray absorption spectroscopy (XAS)) coupled with advanced theoretical calculations has been applied for the unambiguous assignment of the geometric and electronic structure of 1. The mechanism of the O2-reduction reaction has been clarified on the basis of kinetic studies on the overall catalytic reaction as well as each step in the catalytic cycle and by low-temperature detection of intermediates. The reason why the same catalyst can act in either the two- or four-electron reduction of O2 can be explained by the constraint provided by the stannoxane core that makes the O2-binding to 1 an entropically unfavorable process. This makes the end-on µ-1,2-peroxodicobalt(III) intermediate 2 unstable against a preferential proton-transfer step at 25 °C leading to the generation of H2O2. In contrast, at -50 °C, the higher thermodynamic stability of 2 leads to the cleavage of the O-O bond in 2 in the presence of electron and proton donors by a proton-coupled electron-transfer (PCET) mechanism to complete the O2-to-2H2O catalytic conversion in an overall 4e-/4H+ step. The present study provides deep mechanistic insights into the dioxygen reduction process that should serve as useful and broadly applicable principles for future design of more efficient catalysts in fuel cells.

Tracking Catalyst Redox States and Reaction Dynamics in Ni-Fe Oxyhydroxide Oxygen Evolution Reaction Electrocatalysts: The Role of Catalyst Support and Electrolyte pH.

Ni-Fe oxyhydroxides are the most active known electrocatalysts for the oxygen evolution reaction (OER) in alkaline electrolytes and are therefore of great scientific and technological importance in the context of electrochemical energy conversion. Here we uncover, investigate, and discuss previously unaddressed effects of conductive supports and the electrolyte pH on the Ni-Fe(OOH) catalyst redox behavior and catalytic OER activity, combining in situ UV-vis spectro-electrochemistry, operando electrochemical mass spectrometry (DEMS), and in situ cryo X-ray absorption spectroscopy (XAS). Supports and pH > 13 strongly enhanced the precatalytic voltammetric charge of the Ni-Fe oxyhydroxide redox peak couple, shifted them more cathodically, and caused a 2-3-fold increase in the catalytic OER activity. Analysis of DEMS-based faradaic oxygen efficiency and electrochemical UV-vis traces consistently confirmed our voltammetric observations, evidencing both a more cathodic O2 release and a more cathodic onset of Ni oxidation at higher pH. Using UV-vis, which can monitor the amount of oxidized Ni+3/+4 in situ, confirmed an earlier onset of the redox process at high electrolyte pH and further provided evidence of a smaller fraction of Ni+3/+4 in mixed Ni-Fe centers, confirming the unresolved paradox of a reduced metal redox activity with increasing Fe content. A nonmonotonic super-Nernstian pH dependence of the redox peaks with increasing Fe content-displaying Pourbaix slopes as steep as -120 mV/pH-suggested a two proton-one electron transfer. We explain and discuss the experimental pH effects using refined coupled (PCET) and decoupled proton transfer-electron transfer (PT/ET) schemes involving negatively charged oxygenate ligands generated at Fe centers. Together, we offer new insight into the catalytic reaction dynamics and associated catalyst redox chemistry of the most important class of alkaline OER catalysts.

Room-Temperature Energy-Sampling Kß X-ray Emission Spectroscopy of the Mn4Ca Complex of Photosynthesis Reveals Three Manganese-Centered Oxidation Steps and Suggests a Coordination Change Prior to O2 Formation.

In oxygenic photosynthesis, water is oxidized and dioxygen is produced at a Mn4Ca complex bound to the proteins of photosystem II (PSII). Valence and coordination changes in its catalytic S-state cycle are of great interest. In room-temperature (in situ) experiments, time-resolved energy-sampling X-ray emission spectroscopy of the Mn Kß1,3 line after laser-flash excitation of PSII membrane particles was applied to characterize the redox transitions in the S-state cycle. The Kß1,3 line energies suggest a high-valence configuration of the Mn4Ca complex with Mn(III)3Mn(IV) in S0, Mn(III)2Mn(IV)2 in S1, Mn(III)Mn(IV)3 in S2, and Mn(IV)4 in S3 and, thus, manganese oxidation in each of the three accessible oxidizing transitions of the water-oxidizing complex. There are no indications of formation of a ligand radical, thus rendering partial water oxidation before reaching the S4 state unlikely. The difference spectra of both manganese Kß1,3 emission and K-edge X-ray absorption display different shapes for Mn(III) oxidation in the S2 → S3 transition when compared to Mn(III) oxidation in the S1 → S2 transition. Comparison to spectra of manganese compounds with known structures and oxidation states and varying metal coordination environments suggests a change in the manganese ligand environment in the S2 → S3 transition, which could be oxidation of five-coordinated Mn(III) to six-coordinated Mn(IV). Conceivable options for the rearrangement of (substrate) water species and metal-ligand bonding patterns at the Mn4Ca complex in the S2 → S3 transition are discussed.

Structural differences of oxidized iron-sulfur and nickel-iron cofactors in O2-tolerant and O2-sensitive hydrogenases studied by X-ray absorption spectroscopy.

The class of [NiFe]-hydrogenases comprises oxygen-sensitive periplasmic (PH) and oxygen-tolerant membrane-bound (MBH) enzymes. For three PHs and four MBHs from six bacterial species, structural features of the nickel-iron active site of hydrogen turnover and of the iron-sulfur clusters functioning in electron transfer were determined using X-ray absorption spectroscopy (XAS). Fe-XAS indicated surplus oxidized iron and a lower number of ~2.7 Å Fe-Fe distances plus additional shorter and longer distances in the oxidized MBHs compared to the oxidized PHs. This supported a double-oxidized and modified proximal FeS cluster in all MBHs with an apparent trimer-plus-monomer arrangement of its four iron atoms, in agreement with crystal data showing a [4Fe3S] cluster instead of a [4Fe4S] cubane as in the PHs. Ni-XAS indicated coordination of the nickel by the thiol group sulfurs of four conserved cysteines and at least one iron-oxygen bond in both MBH and PH proteins. Structural differences of the oxidized inactive [NiFe] cofactor of MBHs in the Ni-B state compared to PHs in the Ni-A state included a ~0.05 Å longer Ni-O bond, a two times larger spread of the Ni-S bond lengths, and a ~0.1 Å shorter Ni-Fe distance. The modified proximal [4Fe3S] cluster, weaker binding of the Ni-Fe bridging oxygen species, and an altered localization of reduced oxygen species at the active site may each contribute to O2 tolerance.

Oxygen Evolution Reaction Dynamics, Faradaic Charge Efficiency, and the Active Metal Redox States of Ni-Fe Oxide Water Splitting Electrocatalysts.

Mixed Ni-Fe oxides are attractive anode catalysts for efficient water splitting in solar fuels reactors. Because of conflicting past reports, the catalytically active metal redox state of the catalyst has remained under debate. Here, we report an in operando quantitative deconvolution of the charge injected into the nanostructured Ni-Fe oxyhydroxide OER catalysts or into reaction product molecules. To achieve this, we explore the oxygen evolution reaction dynamics and the individual faradaic charge efficiencies using operando differential electrochemical mass spectrometry (DEMS). We further use X-ray absorption spectroscopy (XAS) under OER conditions at the Ni and Fe K-edges of the electrocatalysts to evaluate oxidation states and local atomic structure motifs. DEMS and XAS data consistently reveal that up to 75% of the Ni centers increase their oxidation state from +2 to +3, while up to 25% arrive in the +4 state for the NiOOH catalyst under OER catalysis. The Fe centers consistently remain in the +3 state, regardless of potential and composition. For mixed Ni100-xFex catalysts, where x exceeds 9 atomic %, the faradaic efficiency of O2 sharply increases from ∼30% to 90%, suggesting that Ni atoms largely remain in the oxidation state +2 under catalytic conditions. To reconcile the apparent low level of oxidized Ni in mixed Ni-Fe catalysts, we hypothesize that a kinetic competition between the (i) metal oxidation process and the (ii) metal reduction step during O2 release may account for an insignificant accumulation of detectable high-valent metal states if the reaction rate of process (ii) outweighs that of (i). We conclude that a discussion of the superior catalytic OER activity of Ni-FeOOH electrocatalysts in terms of surface catalysis and redox-inactive metal sites likely represents an oversimplification that fails to capture essential aspects of the synergisms at highly active Ni-Fe sites.

Structure and mechanism leading to formation of the cysteine sulfinate product complex of a biomimetic cysteine dioxygenase model.

Cysteine dioxygenase is a unique nonheme iron enzyme that is involved in the metabolism of cysteine in the body. It contains an iron active site with an unusual 3-His ligation to the protein, which contrasts with the structural features of common nonheme iron dioxygenases. Recently, some of us reported a truly biomimetic model for this enzyme, namely a trispyrazolylborato iron(II) cysteinato complex, which not only has a structure very similar to the enzyme-substrate complex but also represents a functional model: Treatment of the model with dioxygen leads to cysteine dioxygenation, as shown by isolating the cysteine part of the product in the course of the work-up. However, little is known on the conversion mechanism and, so far, not even the structure of the actual product complex had been characterised, which is also unknown in case of the enzyme. In a multidisciplinary approach including density functional theory calculations and X-ray absorption spectroscopy, we have now determined the structure of the actual sulfinato complex for the first time. The Cys-SO2 (-) functional group was found to be bound in an η(2) -O,O-coordination mode, which, based on the excellent resemblance between model and enzyme, also provides the first support for a corresponding binding mode within the enzymatic product complex. Indeed, this is again confirmed by theory, which had predicted a η(2) -O,O-binding mode for synthetic as well as the natural enzyme.

Heterogeneous water oxidation: surface activity versus amorphization activation in cobalt phosphate catalysts.

Is water oxidation catalyzed at the surface or within the bulk volume of solid oxide materials? This question is addressed for cobalt phosphate catalysts deposited on inert electrodes, namely crystallites of pakhomovskyite (Co3(PO4)2⋅8 H2O, Pak) and phosphate-containing Co oxide (CoCat). X-ray spectroscopy reveals that oxidizing potentials transform the crystalline Pak slowly (5-8 h) but completely into the amorphous CoCat. Electrochemical analysis supports high-TOF surface activity in Pak, whereas its amorphization results in dominating volume activity of the thereby formed CoCat material. In the directly electrodeposited CoCat, volume catalysis prevails, but not at very low levels of the amorphous material, implying high-TOF catalysis at surface sites. A complete picture of heterogeneous water oxidation requires insight in catalysis at the electrolyte-exposed "outer surface", within a hydrated, amorphous volume phase, and modes and kinetics of restructuring upon operation.

Rapid X-ray photoreduction of dimetal-oxygen cofactors in ribonucleotide reductase.

Prototypic dinuclear metal cofactors with varying metallation constitute a class of O2-activating catalysts in numerous enzymes such as ribonucleotide reductase. Reliable structures are required to unravel the reaction mechanisms. However, protein crystallography data may be compromised by x-ray photoreduction (XRP). We studied XPR of Fe(III)Fe(III) and Mn(III)Fe(III) sites in the R2 subunit of Chlamydia trachomatis ribonucleotide reductase using x-ray absorption spectroscopy. Rapid and biphasic x-ray photoreduction kinetics at 20 and 80 K for both cofactor types suggested sequential formation of (III,II) and (II,II) species and similar redox potentials of iron and manganese sites. Comparing with typical x-ray doses in crystallography implies that (II,II) states are reached in <1 s in such studies. First-sphere metal coordination and metal-metal distances differed after chemical reduction at room temperature and after XPR at cryogenic temperatures, as corroborated by model structures from density functional theory calculations. The inter-metal distances in the XPR-induced (II,II) states, however, are similar to R2 crystal structures. Therefore, crystal data of initially oxidized R2-type proteins mostly contain photoreduced (II,II) cofactors, which deviate from the native structures functional in O2 activation, explaining observed variable metal ligation motifs. This situation may be remedied by novel femtosecond free electron-laser protein crystallography techniques.