Sulfur X-ray Absorption and Emission Spectroscopy of Organic Sulfones.

The sulfones are a widespread group of organo-sulfur compounds, which contain the sulfonyl SO2 group attached to two carbons and have a formal sulfur oxidation state of +2. We have examined the sulfur K near-edge X-ray absorption spectroscopy (XAS) of a range of different sulfones and find substantial spectroscopic variability depending upon the nature of the coordination to the sulfonyl group. We have also examined the sulfur Kß X-ray emission spectroscopy (XES) of selected representative sulfones. Density functional theory simulations show satisfactory reproduction of both absorption and emission spectra while enabling assignment of the various transitions comprising the spectra. The correspondence between observed and simulated spectra shows promise for ab initio prediction of sulfur X-ray absorption and emission spectra of sulfones of any substituent. The absorption spectra and, to a lesser extent, the emission spectra are sensitive to the nature of the organic groups bound to the sulfonyl (SO2) moiety, clearly showing the potential of X-ray spectroscopy as an in situ probe of sulfone chemistry.

Radiochemical, Computational, and Spectroscopic Evaluation of High-Denticity Desferrioxamine Derivatives DFO2 and DFO2p toward an Ideal Zirconium-89 Chelate Platform.

Desferrioxamine (DFO) has long been considered the gold standard chelator for incorporating [89Zr]Zr4+ in radiopharmaceuticals for positron emission tomography (PET) imaging. To improve the stability of DFO with zirconium-89 and to expand its coordination sphere to enable binding of large therapeutic radiometals, we have synthesized the highest denticity DFO derivatives to date: dodecadentate DFO2 and DFO2p. In this study, we describe the synthesis and characterization of a novel DFO-based chelator, DFO2p, which is comprised of two DFO strands connected by an p-NO2-phenyl linker and therefore contains double the chelating moieties of DFO (potential coordination number up to 12 vs 6). The chelator DFO2p offers an optimized synthesis comprised of only a single reaction step and improves water solubility relative to DFO2, but the shorter linker reduces molecular flexibility. Both DFO2 and DFO2p, each with 6 potential hydroxamate ligands, are able to reach a more energetically favorable 8-coordinate environment for Zr(IV) than DFO. The zirconium(IV) coordination environment of these complexes were evaluated by a combination of density functional theory (DFT) calculations and synchrotron spectroscopy (extended X-ray absorption fine structure), which suggest the inner-coordination sphere of zirconium(IV) to be comprised of the outermost four hydroxamate ligands. These results also confirm a single Zr(IV) in each chelator, and the hydroxide ligands which complete the coordination sphere of Zr(IV)-DFO are absent from Zr(IV)-DFO2 and Zr(IV)-DFO2p. Radiochemical stability studies with zirconium-89 revealed the order of real-world stability to be DFO2 > DFO2p ≫ DFO. The zirconium-89 complexes of these new high-denticity chelators were found to be far more stable than DFO, and the decreased molecular flexibility of DFO2p, relative to DFO2, could explain its decreased stability, relative to DFO2.

Molecular Fates of Organometallic Mercury in Human Brain.

Mercury is ubiquitous in the environment, with rising levels due to pollution and climate change being a current global concern. Many mercury compounds are notorious for their toxicity, with the potential of organometallic mercury compounds for devastating effects on the structures and functions of the central nervous system being of particular concern. Chronic exposure of human populations to low levels of methylmercury compounds occurs through consumption of fish and other seafood, although the health consequences, if any, from this exposure remain controversial. We have used high energy resolution fluorescence detected X-ray absorption spectroscopy to determine the speciation of mercury and selenium in human brain tissue. We show that the molecular fate of mercury differs dramatically between individuals who suffered acute organometallic mercury exposure (poisoning) and individuals with chronic low-level exposure from a diet rich in marine fish. For long-term low-level methylmercury exposure from fish consumption, mercury speciation in brain tissue shows methylmercury coordinated to an aliphatic thiolate, resembling the coordination environment observed in marine fish. In marked contrast, for short-term high-level exposure, we observe the presence of biologically less available mercuric selenide deposits, confirmed by X-ray fluorescence imaging, as well as mercury(II)-bis-thiolate complexes, which may be signatures of severe poisoning in humans. These differences between low-level and high-level exposures challenge the relevance of studies involving acute exposure as a proxy for low-level chronic exposure.

Mercury Lα1 High Energy Resolution Fluorescence Detected X-ray Absorption Spectroscopy: A Versatile Speciation Probe for Mercury.

Mercury is in some sense an enigmatic element. The element and some of its compounds are a natural part of the biogeochemical cycle; while many of these can be deadly poisons at higher levels, environmental levels in the absence of anthropogenic contributions would generally be below the threshold for concern. However, mercury pollution, particularly from burning fossil fuels such as coal, is providing dramatic and increasing emissions into the environment. Because of this, the environmental chemistry and toxicology of mercury are of growing importance, with the fate of mercury being vitally dependent upon its speciation. X-ray absorption spectroscopy (XAS) provides a powerful tool for in situ chemical speciation, but is severely limited by poor spectroscopic energy resolution. Here, we provide a systematic examination of mercury Lα1 high energy resolution fluorescence detected XAS (HERFD-XAS) as an approach for chemical speciation of mercury, in quantitative comparison with conventional Hg LIII-edge XAS. We show that, unlike some lighter elements, chemical shifts in the Lα1 X-ray fluorescence energy can be safely neglected, so that mercury Lα1 HERFD-XAS can be treated simply as a high-resolution version of conventional XAS. We present spectra of a range of mercury compounds that may be relevant to the environmental and life science research and show that density functional theory can produce adequate simulations of the spectra. We discuss strengths and limitations of the method and quantitatively demonstrate improvements both in speciation for complex mixtures and in background rejection for low concentrations.

Oxygen K-edge X-ray absorption spectra of liquids with minimization of window contamination.

Oxygen K-edge X-ray absorption spectroscopy is used routinely to study a range of solid materials. However, liquid samples are studied less frequently at the oxygen K-edge due to the combined challenges of high-vacuum conditions and oxygen contamination of window materials. A modular sample holder design with a twist-seal sample containment system that provides a simple method to encapsulate liquid samples under high-vacuum conditions is presented. This work shows that pure silicon nitride windows have lower oxygen contamination than both diamond- and silicon-rich nitride windows, that the levels of oxygen contamination are related to the age of the windows, and provides a protocol for minimizing the background oxygen contamination. Acid-washed 100 nm-thick silicon nitride windows were found to give good quality oxygen K-edge data on dilute liquid samples.

High Energy Resolution Fluorescence Detected X-ray Absorption Spectroscopy: An Analytical Method for Selenium Speciation.

Selenium is in many ways an enigmatic element. It is essential for health but toxic in excess, with the difference between the two doses being narrower than for any other element. Environmentally, selenium is of concern due to its toxicity. As the rarest of the essential elements, its low levels often provide challenges to the analytical chemist. X-ray absorption spectroscopy (XAS) provides a powerful tool for in situ chemical speciation but is severely limited by poor spectroscopic resolution arising from core-hole lifetime broadening. Here we explore selenium Kα1 high energy resolution fluorescence detected XAS (HERFD-XAS) as a novel approach for chemical speciation of selenium, in comparison with conventional Se K-edge XAS. We present spectra of a range of selenium species relevant to environmental and life science studies, including spectra of seleno-amino acids, which show strong similarities with S K-edge XAS of their sulfur congeners. We discuss strengths and limitations of HERFD-XAS, showing improvements in both speciation performance and low concentration detection. We also develop a simple method to correct fluorescence self-absorption artifacts, which is generally applicable to any HERFD-XAS experiment.

Sulfur Kß X-ray emission spectroscopy: comparison with sulfur K-edge X-ray absorption spectroscopy for speciation of organosulfur compounds.

Until recently, sulfur was known as a "spectroscopically silent" element because of a paucity of convenient spectroscopic probes suitable for in situ chemical speciation. In recent years the technique of sulfur K-edge X-ray absorption spectroscopy (XAS) has been used extensively in sulfur speciation in a variety of different fields. With an initial focus on reduced forms of organic sulfur, we have explored a complementary X-ray based spectroscopy - sulfur Kß X-ray emission spectroscopy (XES) - as a potential analytical tool for sulfur speciation in complex samples. We compare and contrast the sensitivity of sulfur Kß XES with that of sulfur K-edge XAS, and find differing sensitivities for the two techniques. In some cases an approach involving both sulfur K-edge XAS and sulfur Kß XES may be a powerful combination for deducing sulfur speciation in samples containing complex mixtures.

Structural Characterization of the Solution Chemistry of Zirconium(IV) Desferrioxamine: A Coordination Sphere Completed by Hydroxides.

Positron emission tomography (PET) using radiolabeled, monoclonal antibodies has become an effective, noninvasive method for tumor detection and is a critical component of targeted radionuclide therapy. Metal ion chelator and bacterial siderophore desferrioxamine (DFO) is the gold standard compound for incorporation of zirconium-89 in radiotracers for PET imaging because it is thought to form a stable chelate with [89Zr]Zr4+. However, DFO may not bind zirconium-89 tightly in vivo, with free zirconium-89 reportedly liberated into the bones of experimental mouse models. Although high bone uptake has not been observed to date in humans, this potential instability has been proposed to be related to the unsaturated coordination sphere of [89Zr]Zr-DFO, which is thought to consist of the 3 hydroxamate groups of DFO and 1 or 2 water molecules. In this study, we have used a combination of X-ray absorption spectroscopy and density functional theory (DFT) geometry optimization calculations to further probe the coordination chemistry of this complex in solution. We find the extended X-ray absorption fine structure (EXAFS) curve fitting of an aqueous solution of Zr(IV)-DFO to be consistent with an 8-coordinate Zr with oxygen ligands. DFT calculations suggest that the most energetically favorable Zr(IV) coordination environment in DFO likely consists of the 3 hydroxamate ligands from DFO, each with bidentate coordination, and 2 hydroxide ligands. Further EXAFS curve fitting provides additional support for this model. Therefore, we propose that the coordination sphere of Zr(IV)-DFO is most likely completed by 2 hydroxide ligands rather than 2 water molecules, forming Zr(DFO)(OH)2.

Kinetic and structural analysis of Escherichia coli phosphoenolpyruvate carboxykinase mutants.

BACKGROUND: Phosphoenolpyruvate carboxykinase (PEPCK) is a metabolic enzyme in the gluconeogenesis pathway, where it catalyzes the reversible conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) and CO2. The substrates for Escherichia coli PEPCK are OAA and MgATP, with Mn2+ acting as a cofactor. Analysis of PEPCK structures have revealed amino acid residues involved in substrate/cofactor coordination during catalysis. METHODS: Key residues involved in coordinating the different substrates and cofactor bound to E. coli PEPCK were mutated. Purified mutant enzymes were used for kinetic assays. The structure of some mutant enzymes were determined using X-ray crystallography. RESULTS: Mutation of residues D269 and H232, which comprise part of the coordination sphere of Mn2+, reduced kcat by 14-fold, and significantly increased the Km values for Mn2+ and OAA. Mutation of K254 a key residue in the P-loop motif that interacts with MgATP, significantly elevated the Km value for MgATP and reduced kcat. R65 and R333 are key residues that interacts with OAA. The R65Q and R333Q mutations significantly increased the Km value for OAA and reduced kcat respectively. CONCLUSIONS: Our results show that mutation of residues involved in coordinating OAA, MgATP and Mn2+ significantly reduce PEPCK activity. K254 plays an important role in phosphoryl transfer, while R333 is involved in both OAA decarboxylation and phosphoryl transfer by E. coli PEPCK. GENERAL SIGNIFICANCE: In higher organisms including humans, PEPCK helps to regulate blood glucose levels, hence PEPCK is a potential drug target for patients with non-insulin dependent diabetes mellitus.

X-ray absorption spectroscopy of organic sulfoxides.

Organic sulfoxides, a group of compounds containing the sulfinyl S[double bond, length as m-dash]O group, are widespread in nature, important in health and disease, and used in a variety of applications in the pharmaceutical industry. We have examined the sulfur K-edge X-ray absorption near-edge spectra of a range of different sulfoxides and find that their spectra are remarkably similar. Spectra show an intense absorption peak that is comprised of two transitions; a S 1s → (S-O)σ* and a S 1s → [(S-O)π* + (S-C)σ*] transition. In most cases these are sufficiently close in energy that they are not properly resolved; however for dimethylsulfoxide the separation between these transitions increases in aqueous solution due to hydrogen bonding to the sulfinyl oxygen. We also examined tetrahydrothiophene sulfoxide using both the sulfur and oxygen K-edge. This compound has a mild degree of ring strain at the sulfur atom, which changes the energies of the two transitions so that the S 1s → [(S-O)π* + (S-C)σ*] is below the S 1s → (S-O)σ*. A comparison of the oxygen K-edge X-ray absorption near-edge spectra of tetrahydrothiophene sulfoxide with that of an unhindered sulfoxide shows little change, indicating that the electronic environment of oxygen is very similar.

Sulfur K-Edge X-ray Absorption Spectroscopy of Aryl and Aryl-Alkyl Sulfides.

Aryl and mixed aryl-alkyl organic sulfides are important species in a variety of fields, including the drug and food industries. They also are present in fossil fuels, where they contribute to the range of sulfur compounds that must be removed by the fuel industry. We have used sulfur K-edge X-ray absorption spectroscopy, in combination with density functional theory calculations, to study the aryl sulfide diphenyl sulfide and two different aryl-alkyl sulfides. The sulfur K near-edge X-ray absorption spectra are strongly affected by the coordination of the phenyl ring and are distinct from spectra of the alkyl sulfides. For diphenyl sulfide the spectra are predicted to be sensitive to rotation about the S-C bonds, with experimental spectra corresponding to a sum of thermally accessible conformations. We also have investigated the vapor-phase spectrum of diphenyl sulfide, which is found to be very similar to that of toluene solutions of the compound.

Ajothiolanes: 3,4-Dimethylthiolane Natural Products from Garlic ( Allium sativum).

Stereoisomers of 5-(2-allylsulfinyl)-3,4-dimethylthiolane-2-ol, a family of 3,4-dimethylthiolanes of formula C9H16O2S2 we name ajothiolanes, were isolated from garlic ( Allium sativum) macerates and characterized by a variety of analytical and spectroscopic techniques, including ultraperformance liquid chromatography (UPLC), direct analysis in real time-mass spectrometry (DART-MS), and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Ajothiolanes were found to be spectroscopically identical to a family of previously described compounds named garlicnins B1-4 (C9H16O2S2), whose structures we demonstrate have been misassigned. 2D 13C-13C NMR incredible natural abundance double quantum transfer experiments (INADEQUATE) were used to disprove the claim of nine contiguous carbons in these compounds, while X-ray absorption spectroscopy (XAS) along with computational modeling was used to disprove the claim that these compounds were thiolanesulfenic acids. On the basis of the similarity of their NMR spectra to those of the ajothiolanes, we propose that the structures of previously described, biologically active onionins A1-3 (C9H16O2S2), from extracts of onion ( Allium cepa) and Allium fistulosum, and garlicnin A (C12H20O2S4), from garlic extracts, should also be reassigned, in each case as isomeric mixtures of 5-substituted-3,4-dimethylthiolane-2-ols. We conclude that 3,4-dimethylthiolanes may be a common motif in Allium chemistry. Finally, we show that another garlic extract component, garlicnin D (C7H12O2S3), claimed to have an unprecedented structure, is in fact a known compound from garlic with a structure different from that proposed, namely, 2( E)-3-(methylsulfinyl)-2-propenyl 2-propenyl disulfide.

A Photochemically Generated Selenyl Free Radical Observed by High Energy Resolution Fluorescence Detected X-ray Absorption Spectroscopy.

Selenium-based selenyl free radicals are chemical entities that may be involved in a range of biochemical processes. We report the first X-ray spectroscopic observation of a selenyl radical species generated photochemically by X-ray irradiation of low-temperature solutions of l-selenocysteine. We have employed high energy resolution fluorescence detected X-ray absorption spectroscopy (HERFD-XAS) and electron paramagnetic resonance (EPR) spectroscopy, coupled with density functional theory calculations, to characterize and understand the species. The HERFD-XAS spectrum of the selenyl radical is distinguished by a uniquely low-energy transition with a peak energy at 12 659.0 eV, which corresponds to a 1s → 4p transition to the singly occupied molecular orbital of the free radical. The EPR spectrum shows the broad features and highly anisotropic g-values that are expected for a selenium free radical species. The availability of spectroscopic probes for selenyl radicals may assist in understanding why life chooses selenium over sulfur in selected biochemical processes.

X-ray-Induced Photoreduction of Hg(II) in Aqueous Frozen Solution Yields Nearly Monatomic Hg(0).

We use X-ray-induced photochemistry, which is well known to cause changes in a number of systems, to reduce Hg(II) to Hg(0) in frozen aqueous solution with added glycerol maintained at 10 K. X-ray absorption spectroscopy was used to monitor the extent of the reaction and to characterize the species. An analysis of the extended X-ray absorption fine structure (EXAFS) of the photochemical product indicated a nearly monatomic Hg(0) species bound only by long, weak bonds to oxygens at ∼3.5 Å. The results of the EXAFS analysis agree quantitatively with the results of density functional theory calculations using the meta-GGA approximation with the M11-L functional. This is the first structural characterization of nearly monatomic Hg(0) bound by hard ligands similar to those expected in aqueous environmental systems. We conclude that Hg(0) is expected to exist in solution as a nearly monatomic entity.

Cryoprotectants Severely Exacerbate X-ray-Induced Photoreduction.

Approximately 11% of enzymes contain a transition metal ion that is essential for catalytic function. Such metalloenzymes catalyze much of the most chemically challenging and biologically essential chemistry carried out by life. X-ray-based methods, predominantly macromolecular crystallography (MX) and also X-ray absorption spectroscopy (XAS), have proved essential for determining structures of transition metal ion-containing active sites in order to deduce enzyme catalytic mechanisms. However, X-ray irradiation can induce change in both the oxidation state and structure of the metal, which is problematic in structure determination. We present an XAS study of whether cryoprotectants such as polyethylene glycol (PEG) or glycerol, routinely added to MX or XAS samples to improve data quality, affect photoreduction. Our data demonstrate a remarkable 10-fold exacerbation in rate of photoreduction of Cu(II) to Cu(I) when alcohol or ether cryoprotectants are present. Our results suggest that widespread use of cryoprotectants may increase the potential for erroneous structures.

X-ray Absorption Spectroscopy of Aliphatic Organic Sulfides.

Organic sulfides, sometimes called thioethers, are important in a variety of materials with diverse roles in biology and the environment. They also contribute a significant proportion of the sulfur in fossil fuels. We have studied a range of aliphatic sulfides using a combination of sulfur K-edge X-ray absorption spectroscopy and density functional theory calculations. We show that the sulfur K-edge near-edge X-ray absorption spectra of aliphatic organic sulfides comprise two intense transitions in the near-edge spectrum, which can be assigned as 1s → (S-C)σ* and 1s → (S-C)π* transitions. These transitions are found to change in a systematic manner in sterically hindered sulfides composed of four-, five- and six-membered rings. Both the 1s → (S-C)σ* and 1s → (S-C)π* transitions are sensitive to the presence of strain in the C-S-C angle, shifting to lower values with more strained ring systems. Steric effects can give obtuse C-S-C angles, which are predicted to cause the two transitions to converge to the same energy and even cross over at very obtuse angles.

Photochemically Generated Thiyl Free Radicals Observed by X-ray Absorption Spectroscopy.

Sulfur-based thiyl radicals are known to be involved in a wide range of chemical and biological processes, but they are often highly reactive, which makes them difficult to observe directly. We report herein X-ray absorption spectra and analysis that support the direct observation of two different thiyl species generated photochemically by X-ray irradiation. The thiyl radical sulfur K-edge X-ray absorption spectra of both species are characterized by a uniquely low energy transition at about 2465 eV, which occurs at a lower energy than any previously observed feature at the sulfur K-edge and corresponds to a 1s→3p transition to the singly occupied molecular orbital of the free radical. Our results constitute the first observation of substantial levels of thiyl radicals generated by X-ray irradiation and detected by sulfur K-edge X-ray absorption spectroscopy.

Biological iron-sulfur storage in a thioferrate-protein nanoparticle.

Iron-sulfur clusters are ubiquitous in biology and function in electron transfer and catalysis. They are assembled from iron and cysteine sulfur on protein scaffolds. Iron is typically stored as iron oxyhydroxide, ferrihydrite, encapsulated in 12 nm shells of ferritin, which buffers cellular iron availability. Here we have characterized IssA, a protein that stores iron and sulfur as thioferrate, an inorganic anionic polymer previously unknown in biology. IssA forms nanoparticles reaching 300 nm in diameter and is the largest natural metalloprotein complex known. It is a member of a widely distributed protein family that includes nitrogenase maturation factors, NifB and NifX. IssA nanoparticles are visible by electron microscopy as electron-dense bodies in the cytoplasm. Purified nanoparticles appear to be generated from 20 nm units containing ∼6,400 Fe atoms and ∼170 IssA monomers. In support of roles in both iron-sulfur storage and cluster biosynthesis, IssA reconstitutes the [4Fe-4S] cluster in ferredoxin in vitro.

The active site structure and catalytic mechanism of arsenite oxidase.

Arsenite oxidase is thought to be an ancient enzyme, originating before the divergence of the Archaea and the Bacteria. We have investigated the nature of the molybdenum active site of the arsenite oxidase from the Alphaproteobacterium Rhizobium sp. str. NT-26 using a combination of X-ray absorption spectroscopy and computational chemistry. Our analysis indicates an oxidized Mo(VI) active site with a structure that is far from equilibrium. We propose that this is an entatic state imposed by the protein on the active site through relative orientation of the two molybdopterin cofactors, in a variant of the Rây-Dutt twist of classical coordination chemistry, which we call the pterin twist hypothesis. We discuss the implications of this hypothesis for other putatively ancient molybdopterin-based enzymes.