Characterization of Bisphosphonate Hydrate Crystals by Phosphorus K-Edge X-Ray Absorption Near-Edge Structure Spectroscopy.

X-ray absorption near-edge structure (XANES) spectroscopy is a new method for the characterization of active pharmaceutical ingredients. XANES spectra show unique features depending on the electronic states of the X-ray absorbing elements and provide information about the chemical environment that affects the electronic states. In this study, six bisphosphonate hydrate crystals were used to investigate, for the first time, how the phosphorus K-edge XANES spectra are affected by the interatomic interactions and charged states of phosphonate moieties. Phosphorus K-edge XANES spectra showed several differences among the bisphosphonates. In particular, the chlorine atoms covalently bonded near the phosphonate and the number of electric charges of the phosphonate moieties seemed to have large effects on peak shape in XANES spectra. Unique shapes of the XANES spectra demonstrated that differences in interactions at the oxygen atoms of the phosphonate moieties could change the shapes of the XANES spectrum peaks to the extent that each material was distinguished based on the spectra. Since slight differences in interatomic interactions and charged states lead to variations in the spectra, XANES spectroscopy could be widely applied as the fingerprint method to evaluate active pharmaceutical ingredients.

Multimodal Spectroscopic Analysis of the Fe-S Clusters of the as-Isolated Escherichia coli SufBC2D Complex.

Iron-sulfur (Fe-S) clusters are essential inorganic cofactors dedicated to a wide range of biological functions, including electron transfer and catalysis. Specialized multiprotein machineries present in all types of organisms support their biosynthesis. These machineries encompass a scaffold protein, on which Fe-S clusters are assembled before being transferred to cellular targets. Here, we describe the first characterization of the native Fe-S cluster of the anaerobically purified SufBC2D scaffold from Escherichia coli by XAS and Mössbauer, UV-visible absorption, and EPR spectroscopies. Interestingly, we propose that SufBC2D harbors two iron-sulfur-containing species, a [2Fe-2S] cluster and an as-yet unidentified species. Mutagenesis and biochemistry were used to propose amino acid ligands for the [2Fe-2S] cluster, supporting the hypothesis that both SufB and SufD are involved in the Fe-S cluster ligation. The [2Fe-2S] cluster can be transferred to ferredoxin in agreement with the SufBC2D scaffold function. These results are discussed in the context of Fe-S cluster biogenesis.

Nanostructure and dynamics of N-truncated copper amyloid-ß peptides from advanced X-ray absorption fine structure.

An X-ray absorption spectroscopy (XAS) electrochemical cell was used to collect high-quality XAS measurements of N-truncated Cu:amyloid-ß (Cu:Aß) samples under near-physiological conditions. N-truncated Cu:Aß peptide complexes contribute to oxidative stress and neurotoxicity in Alzheimer's patients' brains. However, the redox properties of copper in different Aß peptide sequences are inconsistent. Therefore, the geometry of binding sites for the copper binding in Aß4-8/12/16 was determined using novel advanced extended X-ray absorption fine structure (EXAFS) analysis. This enables these peptides to perform redox cycles in a manner that might produce toxicity in human brains. Fluorescence XAS measurements were corrected for systematic errors including defective-pixel data, monochromator glitches and dispersion of pixel spectra. Experimental uncertainties at each data point were measured explicitly from the point-wise variance of corrected pixel measurements. The copper-binding environments of Aß4-8/12/16 were precisely determined by fitting XAS measurements with propagated experimental uncertainties, advanced analysis and hypothesis testing, providing a mechanism to pursue many similarly complex questions in bioscience. The low-temperature XAS measurements here determine that CuII is bound to the first amino acids in the high-affinity amino-terminal copper and nickel (ATCUN) binding motif with an oxygen in a tetragonal pyramid geometry in the Aß4-8/12/16 peptides. Room-temperature XAS electrochemical-cell measurements observe metal reduction in the Aß4-16 peptide. Robust investigations of XAS provide structural details of CuII binding with a very different bis-His motif and a water oxygen in a quasi-tetrahedral geometry. Oxidized XAS measurements of Aß4-12/16 imply that both CuII and CuIII are accommodated in an ATCUN-like binding site. Hypotheses for these CuI, CuII and CuIII geometries were proven and disproven using the novel data and statistical analysis including F tests. Structural parameters were determined with an accuracy some tenfold better than literature claims of past work. A new protocol was also developed using EXAFS data analysis for monitoring radiation damage. This gives a template for advanced analysis of complex biosystems.

In Solution Identification of the Lysine-Cysteine Redox Switch with a NOS Bridge in Transaldolase by Sulfur K-Edge X-ray Absorption Spectroscopy.

A novel covalent post-translational modification (lysine-NOS-cysteine) was discovered in proteins, initially in the enzyme transaldolase of Neisseria gonorrhoeae (NgTAL) [Nature 2021, 593, 460-464], acting as a redox switch. The identification of this novel linkage in solution was unprecedented until now. We present detection of the NOS redox switch in solution using sulfur K-edge X-ray absorption spectroscopy (XAS). The oxidized NgTAL spectrum shows a distinct shoulder on the low-energy side of the rising edge, corresponding to a dipole-allowed transition from the sulfur 1s core to the unoccupied σ* orbital of the S-O group in the NOS bridge. This feature is absent in the XAS spectrum of reduced NgTAL, where Lys-NOS-Cys is absent. Our experimental and calculated XAS data support the presence of a NOS bridge in solution, thus potentially facilitating future studies on enzyme activity regulation mediated by the NOS redox switches, drug discovery, biocatalytic applications, and protein design.

Ultrafast X-ray Absorption Spectroscopy Reveals Excited-State Dynamics of B12 Coenzymes Controlled by the Axial Base.

Polarized time-resolved X-ray absorption spectroscopy at the Co K-edge is used to probe the excited-state dynamics and photolysis of base-off methylcobalamin and the excited-state structure of base-off adenosylcobalamin. For both molecules, the final excited-state minimum shows evidence for an expansion of the cavity around the Co ion by ca. 0.04 to 0.05 Å. The 5-coordinate base-off cob(II)alamin that is formed following photodissociation has a structure similar to that of the 5-coordinate base-on cob(II)alamin, with a ring expansion of 0.03 to 0.04 Å and a contraction of the lower axial bond length relative to that in the 6-coordinate ground state. These data provide insights into the role of the lower axial ligand in modulating the reactivity of B12 coenzymes.

Trace Element Distribution and Arsenic Speciation in Toenails as Affected by External Contamination and Evaluation of a Cleaning Protocol.

Trace element concentrations in toenail clippings have increasingly been used to measure trace element exposure in epidemeological research. Conventional methods such as inductively coupled plasma mass spectrometry (ICP-MS) and high-performance liquid chromatography ICP-MS (HPLC-ICP-MS) are commonly used to measure trace elements and their speciation in toenails. However, the impact of the removal of external contamination on trace element quantification has not been thoroughly studied. In this work, the microdistribution of trace elements (As, Ca, Co, Cu, Fe, K, Mn, Ni, Rb, S, Sr, Ti, and Zn) in dirty and washed toenails and the speciation of As in situ in toenails were investigated using synchrotron X-ray fluorescence microscopy (XFM) and laterally resolved X-ray absorption near edge spectroscopy (XANES). XFM showed different distribution patterns for each trace element, consistent with their binding properties and nail structure. External (terrestrial) contamination was identified and distinguished from the endogenous accumulation of trace elements in toenails─contaminated areas were characterized by the co-occurrence of Co, Fe, and Mn with elements such as Ti and Rb (i.e., indicators of terrestrial contamination). The XANES spectra showed the presence of one As species in washed toenails, corresponding to As bound to sulfhydryl groups. In dirty specimens, a mixed speciation was found in localized areas, containing AsIII-S species and AsV species. ArsenicV is thought to be associated with surface contamination and exogenous As. These findings provide new insights into the speciation of arsenic in toenails, the microdistribution of trace elements, and the effectiveness of a cleaning protocol in removing external contamination.

Exploring uranium bioaccumulation in the brown alga Ascophyllum nodosum: insights from multi-scale spectroscopy and imaging.

Legacy radioactive waste can be defined as the radioactive waste produced during the infancy of the civil nuclear industry's development in the mid-20th Century, a time when, unfortunately, waste storage and treatment were not well planned. The marine environment is one of the environmental compartments worth studying in this regard because of legacy waste in specific locations of the seabed. Comprising nearly 70% of the earth's service, the oceans are the largest and indeed the final destination for contaminated fresh waters. For this reason, long-term studies of the accumulation biochemical mechanisms of metallic radionuclides in the marine ecosystem are required. In this context the brown algal compartment may be ecologically relevant because of forming large and dense algal beds in coastal areas and potential important biomass for contamination. This report presents the first step in the investigation of uranium (U, an element used in the nuclear cycle) bioaccumulation in the brown alga Ascophyllum nodosum using a multi-scale spectroscopic and imaging approach. Contamination of A. nodosum specimens in closed aquaria at 13 °C was performed with a defined quantity of U(VI) (10-5 M). The living algal uptake was quantified by ICP-MS and a localization study in the various algal compartments was carried out by combining electronic microscopy imaging (SEM), X-ray Absorption spectroscopy (XAS) and micro X-ray Florescence (µ-XRF). Data indicate that the brown alga is able to concentrate U(VI) by an active bioaccumulation mechanism, reaching an equilibrium state after 200 h of daily contamination. A comparison between living organisms and dry biomass confirms a stress-response process in the former, with an average bioaccumulation factor (BAF) of 10 ± 2 for living specimens (90% lower compared to dry biomass, 142 ± 5). Also, these results open new perspectives for a potential use of A. nodosum dry biomass as uranium biosorbent. The different partial BAFs (bioaccumulation factors) range from 3 (for thallus) to 49 (for receptacles) leading to a compartmentalization of uranium within the seaweed. This reveals a higher accumulation capacity in the receptacles, the algal reproductive parts. SEM images highlight the different tissue distributions among the compartments with a superficial absorption in the thallus and lateral branches and several hotspots in the oospheres of the female individuals. A preliminary speciation XAS analysis identified a distinct U speciation in the gametes-containing receptacles as a pseudo-autunite phosphate phase. Similarly, XAS measurements on the lateral branches (XANES) were not conclusive with regards to the occurrence of an alginate-U complex in these tissues. Nonetheless, the hypothesis that alginate may play a role in the speciation of U in the algal thallus tissues is still under consideration.

Modulating the Coordination Environment of Carbon-Dot-Supported Fe Single-Atom Nanozymes for Enhanced Tumor Therapy.

Herein, carbon dot (CD)-supported Fe single-atom nanozymes with high content of pyrrolic N and ultrasmall size (ph-CDs-Fe SAzyme) are fabricated by a phenanthroline-mediated ligand-assisted strategy. Compared with phenanthroline-free nanozymes (CDs-Fe SAzyme), ph-CDs-Fe SAzyme exhibit higher peroxidase (POD)-like activity due to their structure similar to that of ferriporphyrin in natural POD. Aberration-corrected high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM) and X-ray absorption fine structure spectroscopy (XAFS) analyses show that metal Fe is dispersed in ph-CDs-Fe SAzyme as single atoms. Steady-state kinetic studies show that the maximum velocity (Vmax ) and turnover number (kcat ) of H2 O2  homolytic cleavage catalyzed by ph-CDs-Fe SAzyme are 3.0 and 6.2 more than those of the reaction catalyzed by CDs-Fe SAzyme. Density functional theory (DFT) calculations show that the energy barrier of the reaction catalyzed by ph-CDs-Fe SAzyme is lower than that catalyzed by CDs-Fe SAzyme. Antitumor efficacy experiments show that ph-CDs-Fe SAzyme can efficiently inhibit the growth of tumor cells both in vitro and in vivo by synergistic chemodynamic and photothermal effects. Here a new paradigm is provided for the development of efficient antitumor therapeutic approaches based on SAzyme with POD-like activity.

An XAS study of Hg(II) sorption to Al-based drinking water treatment residuals.

Drinking water treatment residuals (DWTRs) are produced from the coagulation and flocculation processes in conventional drinking water treatment. The abundant metal oxide content of these materials resulting from the use of coagulants, like alum and ferric chloride, has driven strong research interest into the reuse of DWTRs as sorptive materials. Using a suite of aluminum-based DWTRs, we provide new insights into Hg(II) sorption mechanisms. Experiments performed at circum-neutral pH show that sorption capacities are related to the amount of organic carbon/matter present in DWTRs. We found that carbon rich samples can scavenge about 9000 mg/kg of Hg, in contrast to 2000 mg/kg for lime based DWTRs. X-ray absorption spectroscopy (XAS) at the Hg L3 edge further characterizes mercury coordination. X-ray absorption near edge structure (XANES) and extended x-ray absorption fine structure (EXAFS) results point to a partial association of mercury with sulfur at low mass loadings, transitioning to a full association with oxygen/carbon at higher concentrations of sorbed Hg(II) and in DWTRs with limited sulfur content. These results suggest that sorption of Hg(II) is primarily controlled by the carbon/organic matter fraction of DWTRs, but not by the coagulants.

Synchrotron X-ray methods in the study of mercury neurotoxicology.

In situ methods are valuable in all fields of research. In toxicology, the importance of dose is well known, elevating the need for in situ techniques to measure levels of toxicants and their byproducts in precise anatomically identifiable locations. More recently, additional emphasis has been placed on the value of techniques which can detect chemical form or speciation, which is equally important in the toxicology of a chemical compound. Many important but conventional methods risk losing valuable information due to extractions, digestions, or the general reliance on mobile phases. Few analytical tools possess the power and diversity of X-ray methods as in-situ methods. Here we present an overview, intended for toxicologists and pathologists, of a variety of synchrotron X-ray methods for determining in situ chemical form and distribution of heavier elements. The versatility and range of these synchrotron techniques, which are both established and emerging, is demonstrated in the context of the study of neurotoxicology of mercury, a global pollutant with the ability to harm both human health and the environment.

Characterization of Ambroxol and Its Hydrochloride Salt Crystals by Bromine K-Edge X-Ray Absorption Near-Edge Structure Spectroscopy and X-Ray Crystal Structure Analysis.

Polymorphic crystals of ambroxol, forms I and II, and form A ambroxol hydrochloride crystals were characterized with bromine K-edge X-ray absorption near-edge structure (XANES) spectroscopy and single-crystal X-ray structure analysis. The XANES spectra had unique shapes depending on the crystal forms. Refined single-crystal structures revealed different interatomic interactions around bromine atoms, such as C-H…Br and N-H…Br hydrogen bonds, Br…O halogen bonds, and N-H…π interactions. Differences in these weak interactions could affect the electronic states of the bromines, resulting in differences in the XANES spectra. The results demonstrated that weak non-conventional interatomic interactions could alter the shape of XANES spectra. Hence, the spectra could be used for evaluating polymorphs of active pharmaceutical ingredients.

Unexpected Cu and Zn speciation patterns in the broiler feed-animal-excreta system revealed by XAS spectroscopy.

Trace minerals such as copper (Cu) and zinc (Zn) are animal nutrition supplements necessary for livestock health and breeding performance, yet they also have environmental impacts via animal excretion. Here we investigated changes in Cu and Zn speciation from the feed additive to the broiler excreta stages. The aim of this study was to assess whether different Cu and Zn feed additives induce different Cu and Zn speciation patterns, and to determine the extent to which this speciation is preserved throughout the feed-animal-excreta system. Synchrotron-based X-ray absorption spectroscopy (XAS) was used for this investigation. The principal findings were: (i) in feed, Cu and Zn speciation changed rapidly from the feed additive signature (Cu and Zn oxides or Cu and Zn sulfates) to Cu and Zn organic complexes (Cu phytate and Zn phytate). (ii) in the digestive tract, we showed that Cu and Zn phytate were major Cu and Zn species; Cu sulfide and Zn amorphous phosphate species were detected but remained minor species. (iii) in fresh excreta, Cu sulfide and Zn amorphous phosphate were major species. These results should help to: (i) enhance the design of future research studies comparing different feed additive performances; (ii) assess Cu and Zn bioavailability in the digestive tract; (iii) gain further insight into the fate of Cu and Zn in cultivated soils when poultry manure is used as fertilizer.

X-ray Spectroscopic Study of the Electronic Structure of a Trigonal High-Spin Fe(IV)═O Complex Modeling Non-Heme Enzyme Intermediates and Their Reactivity.

Fe K-edge X-ray absorption spectroscopy (XAS) has long been used for the study of high-valent iron intermediates in biological and artificial catalysts. 4p-mixing into the 3d orbitals complicates the pre-edge analysis but when correctly understood via 1s2p resonant inelastic X-ray scattering and Fe L-edge XAS, it enables deeper insight into the geometric structure and correlates with the electronic structure and reactivity. This study shows that in addition to the 4p-mixing into the 3dz2 orbital due to the short iron-oxo bond, the loss of inversion in the equatorial plane leads to 4p mixing into the 3dx2-y2,xy, providing structural insight and allowing the distinction of 6- vs 5-coordinate active sites as shown through application to the Fe(IV)═O intermediate of taurine dioxygenase. Combined with O K-edge XAS, this study gives an unprecedented experimental insight into the electronic structure of Fe(IV)═O active sites and their selectivity for reactivity enabled by the π-pathway involving the 3dxz/yz orbitals. Finally, the large effect of spin polarization is experimentally assigned in the pre-edge (i.e., the α/ß splitting) and found to be better modeled by multiplet simulations rather than by commonly used time-dependent density functional theory.

Theoretical Description of Attosecond X-ray Absorption Spectroscopy of Frenkel Exciton Dynamics.

Frenkel excitons are responsible for the transport of light energy in many molecular systems. Coherent electron dynamics govern the initial stage of Frenkel-exciton transfer. Capability to follow coherent exciton dynamics in real time will help to reveal their actual contribution to the efficiency of light-harvesting. Attosecond X-ray pulses are the tool with the necessary temporal resolution to resolve pure electronic processes with atomic sensitivity. We describe how attosecond X-ray pulses can probe coherent electronic processes during Frenkel-exciton transport in molecular aggregates. We analyze time-resolved absorption cross section taking broad spectral bandwidth of an attosecond pulse into account. We demonstrate that attosecond X-ray absorption spectra can reveal delocalization degree of coherent exciton transfer dynamics.

Underpinning the use of indium as a neutron absorbing additive in zirconolite by X-ray absorption spectroscopy.

Indium (In) is a neutron absorbing additive that could feasibly be used to mitigate criticality in ceramic wasteforms containing Pu in the immobilised form, for which zirconolite (nominally CaZrTi2O7) is a candidate host phase. Herein, the solid solutions Ca1-xZr1-xIn2xTi2O7 (0.10 ≤ x ≤ 1.00; air synthesis) and Ca1-xUxZrTi2-2xIn2xO7 (x = 0.05, 0.10; air and argon synthesis) were investigated by conventional solid state sintering at a temperature of 1350 °C maintained for 20 h, with a view to characterise In3+ substitution behaviour in the zirconolite phase across the Ca2+, Zr4+ and Ti4+ sites. When targeting Ca1-xZr1-xIn2xTi2O7, single phase zirconolite-2M was formed at In concentrations of 0.10 ≤ x ≤ 0.20; beyond x ≥ 0.20, a number of secondary In-containing phases were stabilised. Zirconolite-2M remained a constituent of the phase assemblage up to a concentration of x = 0.80, albeit at relatively low concentration beyond x ≥ 0.40. It was not possible to synthesise the In2Ti2O7 end member compound using a solid state route. Analysis of the In K-edge XANES spectra in the single phase zirconolite-2M compounds confirmed that the In inventory was speciated as trivalent In3+, consistent with targeted oxidation state. However, fitting of the EXAFS region using the zirconolite-2M structural model was consistent with In3+ cations accommodated within the Ti4+ site, contrary to the targeted substitution scheme. When deploying U as a surrogate for immobilised Pu in the Ca1-xUxZrTi2-2xIn2xO7 solid solution, it was demonstrated that, for both x = 0.05 and 0.10, In3+ was successfully able to stabilise zirconolite-2M when U was distributed predominantly as both U4+ and average U5+, when synthesised under argon and air, respectively, determined by U L3-edge XANES analysis.

Revealing core-valence interactions in solution with femtosecond X-ray pump X-ray probe spectroscopy.

Femtosecond pump-probe spectroscopy using ultrafast optical and infrared pulses has become an essential tool to discover and understand complex electronic and structural dynamics in solvated molecular, biological, and material systems. Here we report the experimental realization of an ultrafast two-color X-ray pump X-ray probe transient absorption experiment performed in solution. A 10 fs X-ray pump pulse creates a localized excitation by removing a 1s electron from an Fe atom in solvated ferro- and ferricyanide complexes. Following the ensuing Auger-Meitner cascade, the second X-ray pulse probes the Fe 1s → 3p transitions in resultant novel core-excited electronic states. Careful comparison of the experimental spectra with theory, extracts +2 eV shifts in transition energies per valence hole, providing insight into correlated interactions of valence 3d with 3p and deeper-lying electrons. Such information is essential for accurate modeling and predictive synthesis of transition metal complexes relevant for applications ranging from catalysis to information storage technology. This study demonstrates the experimental realization of the scientific opportunities possible with the continued development of multicolor multi-pulse X-ray spectroscopy to study electronic correlations in complex condensed phase systems.

Taking a look at the surface: µ-XRF mapping and fluorine K-edge µ-XANES spectroscopy of organofluorinated compounds in environmental samples and consumer products.

For the first time, µ-X-ray fluorescence (µ-XRF) mapping combined with fluorine K-edge µ-X-ray absorption near-edge structure (µ-XANES) spectroscopy was applied to depict per- and polyfluoroalkyl substance (PFAS) contamination and inorganic fluoride in sample concentrations down to 100 µg kg-1 fluoride. To demonstrate the matrix tolerance of the method, several PFAS contaminated soil and sludge samples as well as selected consumer product samples (textiles, food contact paper and permanent baking sheets) were investigated. µ-XRF mapping allows for a unique element-specific visualization at the sample surface and enables localization of fluorine containing compounds to a depth of 1 µm. Manually selected fluorine rich spots were subsequently analyzed via fluorine K-edge µ-XANES spectroscopy. To support spectral interpretation with respect to inorganic and organic chemical distribution and compound class determination, linear combination (LC) fitting was applied to all recorded µ-XANES spectra. Complementarily, solvent extracts of all samples were target-analyzed via LC-MS/MS spectrometry. The detected PFAS sum values range from 20 to 1136 µg kg-1 dry weight (dw). All environmentally exposed samples revealed a higher concentration of PFAS with a chain length > C8 (e.g. 580 µg kg-1 dw PFOS for Soil1), whereas the consumer product samples showed a more uniform distribution with regard to chain lengths from C4 to C8. Independent of quantified PFAS amounts via target analysis, µ-XRF mapping combined with µ-XANES spectroscopy was successfully applied to detect both point-specific concentration maxima and evenly distributed surface coatings of fluorinated organic contaminants in the corresponding samples.

Femtosecond proton transfer in urea solutions probed by X-ray spectroscopy.

Proton transfer is one of the most fundamental events in aqueous-phase chemistry and an emblematic case of coupled ultrafast electronic and structural dynamics1,2. Disentangling electronic and nuclear dynamics on the femtosecond timescales remains a formidable challenge, especially in the liquid phase, the natural environment of biochemical processes. Here we exploit the unique features of table-top water-window X-ray absorption spectroscopy3-6 to reveal femtosecond proton-transfer dynamics in ionized urea dimers in aqueous solution. Harnessing the element specificity and the site selectivity of X-ray absorption spectroscopy with the aid of ab initio quantum-mechanical and molecular-mechanics calculations, we show how, in addition to the proton transfer, the subsequent rearrangement of the urea dimer and the associated change of the electronic structure can be identified with site selectivity. These results establish the considerable potential of flat-jet, table-top X-ray absorption spectroscopy7,8 in elucidating solution-phase ultrafast dynamics in biomolecular systems.

Ni-anchored g-C3N4for improved hydrogen evolution in photocatalysis.

In this study, we present a facile wet chemical method for synthesizing Ni-modified polymeric carbon nitride (g-C3N4) nanosheets. X-ray absorption fine structure spectroscopy reveals the formation of a unique Ni-N structure, resulting from Ni atoms anchoring in cavities of g-C3N4. The Ni anchoring on the surface N sites modifies the electronic structure of g-C3N4, demonstrating remarkable effectiveness even at low anchoring amounts. The as-prepared Ni/g-C3N4catalysts show robust performance for photocatalytic hydrogen evolution under visible light irradiation, attributed to the unique Ni-N interactions. Specifically, the photocatalytic H2production rate of the Ni/CN-45 catalyst reached 8482.14µmol·g-1·h-1with an apparent quantum efficiency of 0.75% under light irradiation at 427 nm. This rate surpasses most of the previously reported g-C3N4based photocatalysts and is nearly 8 times higher than that of the pure g-C3N4catalyst (1116.07µmol·g-1·h-1).

Chasing the Elusive "In-Between" State of the Copper-Amyloid ß Complex by X-ray Absorption through Partial Thermal Relaxation after Photoreduction.

The redox activity of Cu ions bound to the amyloid-ß (Aß) peptide is implicated as a source of oxidative stress in the context of Alzheimer's disease. In order to explain the efficient redox cycling between CuII -Aß (distorted square-pyramidal) and CuI -Aß (digonal) resting states, the existence of a low-populated "in-between" state, prone to bind Cu in both oxidation states, has been postulated. Here, we exploited the partial X-ray induced photoreduction at 10 K, followed by a thermal relaxation at 200 K, to trap and characterize by X-ray Absorption Spectroscopy (XAS) a partially reduced Cu-Aß1-16 species different from the resting states. Remarkably, the XAS spectrum is well-fitted by a previously proposed model of the "in-between" state, hence providing the first direct spectroscopic characterization of an intermediate state. The present approach could be used to explore and identify the catalytic intermediates of other relevant metal complexes.