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.

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.

Alzheimer's Drug PBT2 Interacts with the Amyloid ß 1-42 Peptide Differently than Other 8-Hydroxyquinoline Chelating Drugs.

Although Alzheimer's disease (AD) was first described over a century ago, it remains the leading cause of age-related dementia. Innumerable changes have been linked to the pathology of AD; however, there remains much discord regarding which might be the initial cause of the disease. The "amyloid cascade hypothesis" proposes that the amyloid ß (Aß) peptide is central to disease pathology, which is supported by elevated Aß levels in the brain before the development of symptoms and correlations of amyloid burden with cognitive impairment. The "metals hypothesis" proposes a role for metal ions such as iron, copper, and zinc in the pathology of AD, which is supported by the accumulation of these metals within amyloid plaques in the brain. Metals have been shown to induce aggregation of Aß, and metal ion chelators have been shown to reverse this reaction in vitro. 8-Hydroxyquinoline-based chelators showed early promise as anti-Alzheimer's drugs. Both 5-chloro-7-iodo-8-hydroxyquinoline (CQ) and 5,7-dichloro-2-[(dimethylamino)methyl]-8-hydroxyquinoline (PBT2) underwent unsuccessful clinical trials for the treatment of AD. To gain insight into the mechanism of action of 8HQs, we have investigated the potential interaction of CQ, PBT2, and 5,7-dibromo-8-hydroxyquinoline (B2Q) with Cu(II)-bound Aß(1-42) using X-ray absorption spectroscopy (XAS), high energy resolution fluorescence detected (HERFD) XAS, and electron paramagnetic resonance (EPR). By XAS, we found CQ and B2Q sequestered ∼83% of the Cu(II) from Aß(1-42), whereas PBT2 sequestered only ∼59% of the Cu(II) from Aß(1-42), suggesting that CQ and B2Q have a higher relative Cu(II) affinity than PBT2. From our EPR, it became clear that PBT2 sequestered Cu(II) from a heterogeneous mixture of Cu(II)Aß(1-42) species in solution, leaving a single Cu(II)Aß(1-42) species. It follows that the Cu(II) site in this Cu(II)Aß(1-42) species is inaccessible to PBT2 and may be less solvent-exposed than in other Cu(II)Aß(1-42) species. We found no evidence to suggest that these 8HQs form ternary complexes with Cu(II)Aß(1-42).

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.

Hg(II) Binding to Thymine Bases in DNA.

The compounds of mercury can be highly toxic and can interfere with a range of biological processes, although many aspects of the mechanism of toxicity are still obscure or unknown. One especially intriguing property of Hg(II) is its ability to bind DNA directly, making interstrand cross-links between thymine nucleobases in AT-rich sequences. We have used a combination of small molecule X-ray diffraction, X-ray spectroscopies, and computational chemistry to study the interactions of Hg(II) with thymine. We find that the energetically preferred mode of thymine binding in DNA is to the N3 and predict only minor distortions of the DNA structure on binding one Hg(II) to two cross-adjacent thymine nucleotides. The preferred geometry is predicted to be twisted away from coplanar through a torsion angle of between 32 and 43°. Using 1-methylthymine as a model, the bis-thymine coordination of Hg(II) is found to give a highly characteristic X-ray spectroscopic signature that is quite distinct from other previously described biological modes of binding of Hg(II). This work enlarges and deepens our view of significant biological targets of Hg(II) and demonstrates tools that can provide a characteristic signature for the binding of Hg(II) to DNA in more complex matrices including intact cells and tissues, laying the foundation for future studies of mechanisms of mercury toxicity.

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.

Solution Chemistry of Copper(II) Binding to Substituted 8-Hydroxyquinolines.

8-Hydroxyquinolines (8HQs) are a family of lipophilic metal ion chelators that have been used in a range of analytical and pharmaceutical applications over the last 100 years. More recently, CQ (clioquinol; 5-chloro-7-iodo-8-hydroxyquinoline) and PBT2 (5,7-dichloro-2-[(dimethylamino)methyl]-8-hydroxyquinoline) have undergone clinical trials for the treatment of Alzheimer's disease and Huntington's disease. Because CQ and PBT2 appear to redistribute metals into cells, these compounds have been redefined as copper and zinc ionophores. Despite the attention surrounding the clinical trials and the clear link between 8HQs and metals, the fundamental solution chemistry of how these compounds bind divalent metals such as copper and zinc, as well as their mechanism(s) of action in mammalian systems, remains poorly understood. In this study, we used a combination of X-ray absorption spectroscopy (XAS), high-energy resolution fluorescence detected (HERFD) XAS, electron paramagnetic resonance (EPR), and UV-visible absorption spectroscopies to investigate the aqueous solution chemistry of a range of 8HQ derivatives. To circumvent the known solubility issues with 8HQ compounds and their complexes with Cu(II), and to avoid the use of abiological organic solvents, we have devised a surfactant buffer system to investigate these Cu(II) complexes in aqueous solution. Our study comprises the first comprehensive investigation of the Cu(II) complexes formed with many 8HQs of interest in aqueous solution, and it provides the first structural information on some of these complexes. We find that halogen substitutions in 8HQ derivatives appear to have little effect on the Cu(II) coordination environment; 5,7-dihalogenated 8HQ conformers all have a pseudo square planar Cu(II) bound by two quinolin-8-olate anions, in agreement with previous studies. Conversely, substituents in the 2-position of the 8HQ moiety appear to cause significant distortions from the typical square-planar-like coordination of most Cu(II)-bis-8HQ complexes, such that the 8HQ moieties in the Cu(II)-bis-8HQ complex are rotated approximately 30-40° apart in a "propeller-like" arrangement.

Direct Observation of Methylmercury and Auranofin Binding to Selenocysteine in Thioredoxin Reductase.

Selenoenzymes, containing a selenocysteine (Sec) residue, fulfill important roles in biology. The mammalian thioredoxin reductase selenoenzymes are key regulators of antioxidant defense and redox signaling and are inhibited by methylmercury species and by the gold-containing drug auranofin. It has been proposed that such inhibition is mediated by metal binding to Sec in the enzyme. However, direct structural observations of these classes of inhibitors binding to selenoenzymes have been few to date. Here we therefore have used extended X-ray absorption fine structure as a direct structural probe to investigate binding to the selenium site in recombinant rat thioredoxin reductase 1 (TrxR1). The results demonstrate for the first time the direct and complete binding of the metal atom of the inhibitors to the selenium atom in TrxR1 for both methylmercury and auranofin, indicating that TrxR1 inhibition indeed can be attributed to such direct metal-selenium binding.

Rethinking the Minamata Tragedy: What Mercury Species Was Really Responsible?

Industrial release of mercury into the local Minamata environment with consequent poisoning of local communities through contaminated fish and shellfish consumption is considered the classic case of environmental mercury poisoning. However, the mercury species in the factory effluent has proved controversial, originally suggested as inorganic, and more recently as methylmercury species. We used newly available methods to re-examine the cerebellum of historic Cat 717, which was fed factory effluent mixed with food to confirm the source. Synchrotron high-energy-resolution fluorescence detection-X-ray absorption spectroscopy revealed sulfur-bound organometallic mercury with a minor ß-HgS phase. Density functional theory indicated energetic preference for α-mercuri-acetaldehyde as a waste product of aldehyde production. The consequences of this alternative species in the "classic" mercury poisoning should be re-evaluated.

X-ray Absorption Spectroscopy Investigations of Copper(II) Coordination in the Human Amyloid ß Peptide.

Alzheimer's disease (AD) is the main cause of age-related dementia and currently affects approximately 5.7 million Americans. Major brain changes associated with AD pathology include accumulation of amyloid beta (Aß) protein fragments and formation of extracellular amyloid plaques. Redox-active metals mediate oligomerization of Aß, and the resultant metal-bound oligomers have been implicated in the putative formation of harmful, reactive species that could contribute to observed oxidative damage. In isolated plaque cores, Cu(II) is bound to Aß via histidine residues. Despite numerous structural studies of Cu(II) binding to synthetic Aß in vitro, there is still uncertainty surrounding Cu(II) coordination in Aß. In this study, we used X-ray absorption spectroscopy (XAS) and high energy resolution fluorescence detected (HERFD) XAS to investigate Cu(II) coordination in Aß(1-42) under various solution conditions. We found that the average coordination environment in Cu(II)Aß(1-42) is sensitive to X-ray photoreduction, changes in buffer composition, peptide concentration, and solution pH. Fitting of the extended X-ray absorption fine structure (EXAFS) suggests Cu(II) is bound in a mixture of coordination environments in monomeric Aß(1-42) under all conditions studied. However, it was evident that on average only a single histidine residue coordinates Cu(II) in monomeric Aß(1-42) at pH 6.1, in addition to 3 other oxygen or nitrogen ligands. Cu(II) coordination in Aß(1-42) at pH 7.4 is similarly 4-coordinate with oxygen and nitrogen ligands, although an average of 2 histidine residues appear to coordinate at this pH. At pH 9.0, the average Cu(II) coordination environment in Aß(1-42) appears to be 5-coordinate with oxygen and nitrogen ligands, including two histidine residues.

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.

The metal chaperone Atox1 regulates the activity of the human copper transporter ATP7B by modulating domain dynamics.

The human transporter ATP7B delivers copper to the biosynthetic pathways and maintains copper homeostasis in the liver. Mutations in ATP7B cause the potentially fatal hepatoneurological disorder Wilson disease. The activity and intracellular localization of ATP7B are regulated by copper, but the molecular mechanism of this regulation is largely unknown. We show that the copper chaperone Atox1, which delivers copper to ATP7B, and the group of the first three metal-binding domains (MBD1-3) are central to the activity regulation of ATP7B. Atox1-Cu binding to ATP7B changes domain dynamics and interactions within the MBD1-3 group and activates ATP hydrolysis. To understand the mechanism linking Atox1-MBD interactions and enzyme activity, we have determined the MBD1-3 conformational space using small angle X-ray scattering and identified changes in MBD dynamics caused by apo-Atox1 and Atox1-Cu by solution NMR. The results show that copper transfer from Atox1 decreases domain interactions within the MBD1-3 group and increases the mobility of the individual domains. The N-terminal segment of MBD1-3 was found to interact with the nucleotide-binding domain of ATP7B, thus physically coupling the domains involved in copper binding and those involved in ATP hydrolysis. Taken together, the data suggest a regulatory mechanism in which Atox1-mediated copper transfer activates ATP7B by releasing inhibitory constraints through increased freedom of MBD1-3 motions.

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.

Binding of Copper and Cisplatin to Atox1 Is Mediated by Glutathione through the Formation of Metal-Sulfur Clusters.

Copper is an essential nutrient required for many biological processes involved in primary metabolism, but free copper is toxic due to its ability to catalyze formation of free radicals. To prevent toxic effects, in the cell copper is bound to proteins and low molecular weight compounds, such as glutathione, at all times. The widely used chemotherapy agent cisplatin is known to bind to copper-transporting proteins, including copper chaperone Atox1. Cisplatin interactions with Atox1 and other copper transporters are linked to cancer resistance to platinum-based chemotherapy. Here we analyze the binding of copper and cisplatin to Atox1 in the presence of glutathione under redox conditions that mimic intracellular environment. We show that copper(I) and glutathione form large polymers with a molecular mass of approximately 8 kDa, which can transfer copper to Atox1. Cisplatin also can form polymers with glutathione, albeit at a slower rate. Analysis of simultaneous binding of copper and cisplatin to Atox1 under physiological conditions shows that both metals are bound to the protein through copper-sulfur-platinum bridges.

Dynamics of the metal binding domains and regulation of the human copper transporters ATP7B and ATP7A.

Copper transporters ATP7A and ATP7B regulate copper levels in the human cells and deliver copper to the biosynthetic pathways. ATP7A and ATP7B belong to the P-type ATPases and share much of the domain architecture and the mechanism of ATP hydrolysis with the other, well-studied, enzymes of this type. A unique structural feature of the copper ATPases is the chain of six cytosolic metal-binding domains (MBDs), which are believed to be involved in copper-dependent regulation of the activity and intracellular localization of these enzymes. Although the structures of all the MBDs have been solved, the mechanism of copper-dependent regulation of ATP7B and ATP7A, the roles of individual MBDs, and the relationship between the regulatory and catalytic copper binding are still unknown. We describe the structure and dynamics of the MBDs, review the current knowledge about their functional roles and propose a mechanism of regulation of ATP7B by copper-dependent changes in the dynamics and conformation of the MBD chain. Transient interactions between the MBDs, rather than transitions between distinct static conformations are likely to form the structural basis of regulation of the ATP-dependent copper transporters in human cells. © 2016 IUBMB Life, 69(4):226-235, 2017.

A combination dual-sized microparticle system modulates dendritic cells and prevents type 1 diabetes in prediabetic NOD mice.

We developed a novel poly(lactic-co-glycolic acid)-based, microparticle (MP) system providing concurrent delivery of multiple encapsulated immuno-suppressive factors and antigen, for in vivo conditioning of dendritic cells (DCs) toward a tolerance promoting pathway. Subcutaneous administration prevents onset of type 1 diabetes (T1D) in NOD mice. Two MP sizes were made: phagocytosable MPs were fabricated encapsulating vitamin D3 or insulin B(9-23) peptide, while unphagocytosable MPs were fabricated encapsulating TGF-ß1 or GM-CSF. The combination of Vit D3/TGF-ß1 MPs confers an immature and LPS activation-resistant phenotype to DCs, and MP-delivered antigen is efficiently and functionally presented. Notably, two subcutaneous injections into 4week old NOD mice using the combination of MPs encapsulating Vit D3, Ins B, TGF-ß1 and GM-CSF protected 40% of mice from T1D development, significant in comparison to the control. This work represents one of the first applications of a biomaterial-based, MP vaccine system to successfully prevent autoimmune diabetes.

Synchrotron speciation of umbilical cord mercury and selenium after environmental exposure in Niigata.

The insidious and deadly nature of mercury's organometallic compounds is informed by two large scale poisonings due to industrial mercury pollution that occurred decades ago in Minamata and Niigata, Japan. The present study examined chemical speciation for both mercury and selenium in a historic umbilical cord sample from a child born to a mother who lived near the Agano River in Niigata. The mother had experienced mercury exposure leading to more than 50 ppm mercury measured in her hair and was symptomatic 9 years prior to the birth. We sought to determine the mercury and selenium speciation in the child's cord using Hg Lα1 and Se Kα1 high-energy resolution fluorescence detected X-ray absorption spectroscopy, the chemical speciation of mercury was found to be predominantly organometallic and coordinated to a thiolate. The selenium was found to be primarily in an organic form and at levels higher than those of mercury, with no evidence of mercury-selenium chemical species. Our results are consistent with mercury exposure at Niigata being due to exposure to organometallic mercury species.

Antibacterial effects of zinc oxide nanorod surfaces.

Antibacterial coating approaches are being investigated to modify implants to reduce bacterial adhesion and viability in order to reduce implant-associated infection. Nanostructured materials possess unique surface properties, and nanotopographic surfaces have been reported to modulate bacterial adhesion. Zinc oxide (ZnO) films presenting well-controlled nanorod surface structures have recently been developed. To assess the efficacy of ZnO nanorod surfaces as an anti-bacterial coating, we evaluated bacterial adhesion and viability, compared to sputtered ZnO substrates (a relatively flat control) and glass substrates (as a reference). Common implant-associated pathogens, Pseudomonas aeruginosa and Staphylococcus epidermidis were investigated. The number of adherent P. aeruginosa on ZnO nanorod surfaces was found to be reduced compared to glass and sputtered ZnO, while the adherent number of S. epidermidis on the ZnO nanorods was equivalent to glass. Regarding bacteria viability, the ZnO nanorod and sputtered ZnO surfaces demonstrated a modest, but significant bactericidal effect on adherent P. aeruginosa, killing 2.5-fold and 1.7-fold more over the number of dead P. aeruginosa on glass, respectively. A greater bactericidal effect of ZnO substrates on S. epidermidis was found, with sputtered ZnO and ZnO nanorod substrates killing -20-fold and 30-fold more over the number of dead S. epidermidis on glass, respectively. These data support the further investigation and optimization of ZnO nanorod coatings with potential for bacterial adhesion resistance and bactericidal properties.