Isabelle Kieffer

X-ray Absorption Spectroscopy Proves the Trigonal-Planar Sulfur-Only Coordination of Copper(I) with High-Affinity Tripodal Pseudopeptides

A series of tripodal ligands L derived from nitrilotriacetic acid (NTA) and extended by three converging metal-binding cysteine chains were previously found to bind selectively copper(I) both in vitro and in vivo. The ligands L(1) (ester) and L(2) (amide) were demonstrated to form copper(I) species with very high affinities, close to that reported for the metal-sequestering metallothioneins (MTs; log K(Cu-MT) ≈ 19). Here, an in-depth study by Cu K-edge X-ray absorption spectroscopy (XAS) was performed to completely characterize the copper(I) coordination sphere in the complexes, previously evidenced by other physicochemical analyses. The X-ray absorption near-edge structure (XANES) spectra shed light on the equilibrium between a mononuclear complex and a cluster for both L(1) (ester) and L(2) (amide). The exclusive symmetric CuS3 geometry adopted in the mononuclear complexes (Cu-S ≈ 2.23 Å) was clearly demonstrated by extended X-ray absorption fine structure (EXAFS) analyses. The EXAFS analyses also proved that the clusters are organized on a symmetric CuS3 core (Cu-S ≈ 2.26 Å) and interact with three nearby copper atoms (Cu---Cu ≈ 2.7 Å), consistent with the Cu6S9-type clusters previously characterized by pulsed gradient spin echo NMR spectroscopy. XAS data obtained for other architectures based on the NTA template (L(3) acid, L(4) without a functionalized carbonyl group, etc.) demonstrated the formation of polymetallic species only, which evidence the necessity of the proximal ester or amide group to stabilize the CuS3 mononuclear species. Finally, XAS was demonstrated to be a powerful method to quantify the equilibrium between the two copper(I) environments evidenced with L(1) and L(2) at different copper concentrations and to determine the equilibrium constants between these two complexes.

Zinc(II) Binding Site to the Amyloid-β Peptide: Insights from Spectroscopic Studies with a Wide Series of Modified Peptides

The Zn(II) ion has been linked to Alzheimer’s disease (AD) due to its ability to modulate the aggregating properties of the amyloid-β (Aβ) peptide, where Aβ aggregation is a central event in the etiology of the disease. Delineating Zn(II) binding properties to Aβ is thus a prerequisite to better grasp its potential role in AD. Because of (i) the flexibility of the Aβ peptide, (ii) the multiplicity of anchoring sites, and (iii) the silent nature of the Zn(II) ion in most classical spectroscopies, this is a difficult task. To overcome these difficulties, we have investigated the impact of peptide alterations (mutations, N-terminal acetylation) on the Zn(Aβ) X-ray absorption spectroscopy fingerprint and on the Zn(II)-induced modifications of the Aβ peptides’ NMR signatures. We propose a tetrahedrally bound Zn(II) ion, in which the coordination sphere is made by two His residues and two carboxylate side chains. Equilibria between equivalent ligands for one Zn(II) binding position have also been observed, the predominant site being made by the side chains of His6, His13 or His14, Glu11, and Asp1 or Glu3 or Asp7, with a slight preference for Asp1.

D-Penicillamine tripodal derivatives as efficient copper(I) chelators.

New tripodal metal-chelating agents derived from nitrilotriacetic acid (NTA) and extended by three unnatural amino acids D-penicillamine (D-Pen) are presented. D-Pen is actually the drug most extensively used to treat copper (Cu) overload in Wilsons disease and as such is a very attractive building block for the design of chelating agents. D-Pen is also a bulkier analogue of cysteine, with the β-methylene hydrogen atoms replaced by larger methyl groups. The hindrance of the gem-dimethyl group close to the thiol functions is demonstrated to influence the speciation and stability of the metal complexes. The ligands L(4) (ester) and L(5) (amide) were obtained from NTA and commercial D-Pen synthons in four and five steps with overall yields of 14 and 24%, respectively. Their ability to bind Cu(I), thanks to their three thiolate functions, has been investigated using both spectroscopic and analytical methods. UV, CD, and NMR spectroscopy and mass spectrometry evidence the formation of two Cu(I) complexes with L(5): the mononuclear complex CuL(5) and one cluster (Cu2L(5))2. In contrast, the bulkier ethyl ester derivative L(4) cannot accommodate the mononuclear complex in solution and thus forms exclusively the cluster (Cu2L(4))2. Cu K-edge X-ray absorption spectroscopy (XAS and EXAFS) confirms that Cu(I) is bound in trigonal-planar sulfur-only environments in all of these complexes with Cu- - -S distances ranging from 2.22 to 2.23 Å. Such C3-symmetric CuS3 cores are coordination modes frequently adopted in Cu(I) proteins such as metallothioneins. These two ligands bind Cu(I) tightly and selectively, which makes them promising chelators for intracellular copper detoxification in vivo.

Visualization, quantification and coordination of Ag+ ions released from silver nanoparticles in hepatocytes

Silver nanoparticles (AgNPs) can enter eukaryotic cells and exert toxic effects, most probably as a consequence of the release of Ag+ ions. Due to the elusive nature of Ag+ ionic species, quantitative information concerning AgNP intracellular dissolution is missing. By using a synchrotron nanoprobe, silver is visualized and quantified in hepatocytes (HepG2) exposed to AgNPs; the synergistic use of electron microscopy allows for the discrimination between nanoparticular and ionic forms of silver within a single cell. AgNPs are located in endocytosis vesicles, while the visualized Ag+ ions diffuse in the cell. The averaged NP dissolution rates, measured by X-ray absorption spectroscopy, highlight the faster dissolution of citrate-coated AgNPs with respect to the less toxic PVP-coated AgNPs; these results are confirmed at the single-cell level. The released Ag+ ions recombine with thiol-bearing biomolecules: the Ag-S distances measured in cellulo, and the quantitative evaluation of gene expression, provide independent evidence of the involvement of glutathione and metallothioneins in Ag+ binding. The combined use of cutting-edge imaging techniques, atomic spectroscopy and molecular biology brings insight into the fate of AgNPs in hepatocytes, and more generally into the physicochemical transformations of metallic nanoparticles in biological environments and the resulting disruption of metal homeostasis.

XAS Investigation of Silver(I) Coordination in Copper(I) Biological Binding Sites.

Silver(I) is an unphysiological ion that, as the physiological copper(I) ion, shows high binding affinity for thiolate ligands; its toxicity has been proposed to be due to its capability to replace Cu(I) in the thiolate binding sites of proteins involved in copper homeostasis. Nevertheless, the nature of the Ag(I)-thiolate complexes formed within cells is poorly understood, and the details of Ag(I) coordination in such complexes in physiologically relevant conditions are mostly unknown. By making use of X-ray absorption spectroscopy (XAS), we characterized the Ag(I) binding sites in proteins related to copper homeostasis, such as the chaperone Atox1 and metallothioneins (MTs), as well as in bioinspired thiolate Cu(I) chelators mimicking these proteins, in solution and at physiological pH. Different Ag(I) coordination environments were revealed: the Ag-S bond length was found to correlate to the Ag(I) coordination number, with characteristic values of 2.40 and 2.49 Å in AgS2 and AgS3 sites, respectively, comparable to the values reported for crystalline Ag(I)-thiolate compounds. The bioinspired Cu(I) chelator L(1) is proven to promote the unusual trigonal AgS3 coordination and, therefore, can serve as a reference compound for this environment. In the Cu(I)-chaperone Atox1, Ag(I) binds in digonal coordination to the two Cys residues of the Cu(I) binding loop, with the AgS2 characteristic bond length of 2.40 ± 0.01 Å. In the multinuclear Ag(I) clusters of rabbit and yeast metallothionein, the average Ag-S bond lengths are 2.48 ± 0.01 Å and 2.47 ± 0.01 Å, respectively, both indicative of the predominance of trigonal AgS3 sites. This work lends insight into the coordination chemistry of silver in its most probable intracellular targets and might help in elucidating the mechanistic aspects of Ag(I) toxicity.

Zinc Speciation in the Suspended Particulate Matter of an Urban River (Orge, France): Influence of Seasonality and Urbanization Gradient

Among trace metal pollutants, zinc is the major one in the rivers from the Paris urban area, such as the Orge River, where Zn concentration in the suspended particulate matter (SPM) can reach 2000 mg/kg in the most urbanized areas. In order to better understand Zn cycling in such urban rivers, we have determined Zn speciation in SPM as a function of both the seasonal water flow variations and the urbanization gradient along the Orge River. Using TEM/SEM-EDX and linear combination fitting (LCF) of EXAFS data at the Zn K-edge, we show that Zn mainly occurs as tetrahedrally coordinated Zn(2+) sorbed to ferrihydrite (37-46%), calcite (0-37%), amorphous SiO2 (0-21%), and organic-P (0-30%) and as octahedrally coordinated Zn(2+) in the octahedral layer of phyllosilicates (18-25%). Moreover, the Zn speciation pattern depends on the river flow rate. At low water flow, Zn speciation changes along the urbanization gradient: geogenic forms of Zn inherited from soil erosion decrease relative to Zn bound to organic-phosphates and amorphous SiO2. At high water flow, Zn speciation is dominated by soil-borne forms of Zn regardless the degree of urbanization, indicating that erosion of Zn-bearing minerals dominates the Zn contribution to SPM under such conditions.

Evidence that Soil Properties and Organic Coating Drive the Phytoavailability of Cerium Oxide Nanoparticles

The ISO-standardized RHIZOtest is used here for the first time to decipher how plant species, soil properties, and physical-chemical properties of the nanoparticles and their transformation regulate the phytoavailability of nanoparticles. Two plants, tomato and fescue, were exposed to two soils with contrasted properties: a sandy soil poor in organic matter and a clay soil rich in organic matter, both contaminated with 1, 15, and 50 mg·kg-1 of dissolved Ce2(SO4)3, bare and citrate-coated CeO2 nanoparticles. All the results demonstrate that two antagonistic soil properties controlled Ce uptake. The clay fraction enhanced the retention of the CeO2 nanoparticles and hence reduced Ce uptake, whereas the organic matter content enhanced Ce uptake. Moreover, in the soil poor in organic matter, the organic citrate coating significantly enhanced the phytoavailability of the cerium by forming smaller aggregates thereby facilitating the transport of nanoparticles to the roots. By getting rid of the dissimilarities between the root systems of the different plants and the normalizing the surfaces exposed to nanoparticles, the RHIZOtest demonstrated that the species of plant did not drive the phytoavailability, and provided evidence for soil-plant transfers at concentrations lower than those usually cited in the literature and closer to predicted environmental concentrations.

Non-linear release dynamics for a CeO 2 nanomaterial embedded in a protective wood stain, due to matrix photo-degradation

The release of CeO2-bearing residues during the weathering of an acrylic stain enriched with CeO2 nanomaterial designed for wood protection (Nanobyk brand additive) was studied under two different scenarios: (i) a standard 12-weeks weathering protocol in climate chamber, that combined condensation, water spraying and UV-visible irradiation and (ii) an alternative accelerated 2-weeks leaching batch assay relying on the same weathering factors (water and UV), but with a higher intensity of radiation and immersion phases. Similar Ce released amounts were evidenced for both scenarios following two phases: one related to the removal of loosely bound material with a relatively limited release, and the other resulting from the degradation of the stain, where major release occurred. A non-linear evolution of the release with the UV dose was evidenced for the second phase. No stabilization of Ce emissions was reached at the end of the experiments. The two weathering tests led to different estimates of long-term Ce releases, and different degradations of the stain. Finally, the photo-degradations of the nanocomposite, the pure acrylic stains and the Nanobyk additive were compared. The incorporation of Nanobyk into the acrylic matrix significantly modified the response of the acrylic stain to weathering.