XAS Investigation of Silver(I) Coordination in Copper(I) Biological

Dec 3, 2015 - ... UMS 832 CNRS, Université Joseph Fourier, F-38041 Grenoble, France ... Marianne Marchioni , Pierre-Henri Jouneau , Mireille Chevalle...
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XAS Investigation of Silver(I) Coordination in Copper(I) Biological Binding Sites Giulia Veronesi,*,†,‡ Thomas Gallon,†,∥ Aurélien Deniaud,† Bastien Boff,∥ Christelle Gateau,∥ Colette Lebrun,∥ Claude Vidaud,§ Françoise Rollin-Genetet,§ Marie Carrière,∥ Isabelle Kieffer,¶,⊥ Elisabeth Mintz,† Pascale Delangle,*,∥ and Isabelle Michaud-Soret*,† †

CNRS, UMR 5249, CNRS-CEA-UJF; CEA; and University Grenoble Alpes, Laboratoire de Chimie et Biologie des Métaux (LCBM), F-38054 Grenoble, France ‡ ESRF, European Synchrotron Radiation Facility, 71 Avenue des Martyrs, 38043 Grenoble, France ∥ University Grenoble Alpes and CEA, INAC-SCIB, F-38000 Grenoble, France § CEA/DSV/iBEB/SBTN, BP 17171, 30207 Bagnols sur Cèze, France ¶ BM30B/FAME beamline, ESRF, F-38043 Grenoble cedex 9, France ⊥ Observatoire des Sciences de l’Univers de Grenoble, UMS 832 CNRS, Université Joseph Fourier, F-38041 Grenoble, France S Supporting Information *

ABSTRACT: 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 L1 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.

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INTRODUCTION

supporting the hypothesis that Ag(I) interferes with copper homeostasis.12,13 Cu homeostasis is tightly regulated in eukaryotic cells: after specific transporter uptake via Ctr1, Cu(I) ions are chelated by cytoplasmic glutathione (GSH) and Cu-chaperones. The latter transport and deliver Cu(I) to their cellular targets such as P-type ATPases, superoxide dismutase, and cytochrome c oxidase.14 The common structural feature of Cu-chaperones is the presence of a solvent-exposed chelating site composed of the thiolate (R−S−) side-chains of Cys residues, for rapid metal binding and exchange; Ag(I), like Cu(I), is a soft Lewis acid, which shows high binding affinity for soft Lewis basis such as R−S−. Therefore, it is likely that a

The widespread use of silver-based nanomaterials as biocide in dietary, daily care, and medical products has recently led humans to daily exposure to this unphysiological toxic metal.1,2 The toxicity of silver nanoparticles has been mainly ascribed to their enhanced dissolution in cells with respect to the bulk material, and the released Ag(I) ions are regarded as the major source of cellular damage.3,4 Therefore, the physicochemical behavior of silver nanoparticles in cellular models and the interactions of the nonphysiological Ag(I) ion with biomolecules have recently drawn the attention of the scientific community, and the understanding of the molecular mechanisms eliciting toxicity became a major challenge in toxicology.5−8 It has been shown in vivo and in vitro that Ag(I) can replace Cu(I) in its native binding sites in copper proteins,9−11 © XXXX American Chemical Society

Received: July 23, 2015

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DOI: 10.1021/acs.inorgchem.5b01658 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry prime pathway for Ag(I) intracellular trafficking is through complexation with Cu-chaperones. Another family of proteins involved in Cu homeostasis and presenting thiol-only metal binding sites are metallothioneins (MTs): these low molecular weight proteins comprise a high number of Cys residues (∼30% of the total amino acids) that can efficiently bind Zn(II) and/or Cu(I) in multinuclear clusters,15 allowing metal storage and playing a crucial role in intracellular Cu detoxification.14 Ag(I) binds to yeast MT (yMT) in vitro, forming a Ag7MT complex where the 7 Ag(I) atoms are coordinated by 10 Cys residues in a single cluster.16 In rabbit MT (rMT), up to 18 Ag(I) atoms can be sequestered in the two distinct metal binding domains of the protein.9,17 For their ability to bind Ag(I) in vitro, their proven role in toxic metals detoxification,18 and their increased expression in metallic stress conditions, MTs are thought to be privileged targets for Ag(I) ions in cells. Ag(I) binding to biologically relevant thiols in physiological conditions is therefore a key aspect for the understanding of the mechanistic aspects of toxicity. However, very little is known about the coordination chemistry of Ag(I) in the complexes formed in neutral solutions: only recently the coordination of Ag(I) with the R−S− groups of Cys, GSH, and Penicillamine (Pen) was characterized.19 Nevertheless, because this study was performed on alkaline solutions, its conclusions cannot be extended to physiologically relevant conditions. More is known about Ag(I)−thiolate complexes in crystalline form: the numerous structures deposited in the Cambridge Structural Database (CSD)20 reveal that, depending on the steric hindrance of the S donor ligand, AgSR complexes tend to form extended networks or compact ring structures,21−23 where the Ag(I) ions occupy either digonal AgS2 or trigonal AgS3 binding sites. The aim of the present study was to bring insight into the interactions between Ag(I) and biologically relevant thiols in solution and at physiological pH, starting from small molecules and then extending the approach to metalloproteins involved in copper homeostasis in eukaryotic cells. We previously characterized by X-ray absorption fine structure (XAFS) spectroscopy the coordination sphere of Ag(I) in a Ag/GSH = 1:1 solution at pH 7.4.7 The chaperone Atox1 (initially called HAH1 for human Atx1 homologue) is a 68-amino-acid human protein essential for Cu homeostasis.24 The structures of apo- and Cu(I)-Atox1 have been resolved with X-ray diffraction (XRD) and NMR methods.25,26 The MxCxxC loop is a common feature of Cuchaperones (like Atox1 in human, Atx1 in yeast, or CopZ in bacterium)27,28 and Cu-transporting P-type ATPases (such as ATP7A and ATP7B)29 and binds Cu(I) with two Cys residues in digonal coordination (Figure 1a). This motif is thought to play a key role in metal transfer between the two families of proteins. Nonphysiological metals like Cd(II) and Hg(II) can form stable complexes with Atox1 by binding to the two Cys of the MxCxxC motif;25 the formation of Ag(I)Atox1 complexes has been proven with spectroscopic methods,30 while the structure of the Ag(I) binding site has not been resolved to date. Ag(I) binding to MT has been proven, as stated above, and largely investigated over the past 30 years. However, the presence of multinuclear metal binding sites make MT a challenging and paradigmatic system in bioinorganic chemistry, and the details of metal coordination (native Cu(I) and Zn(II) as well as exogenous Ag(I), Cd(II), and Au(I)) are still a matter

Figure 1. Solution structures of (a) the Cu(I)Atox1 complex (PDB entry 1TL4)26 and (b) silver-substituted yeast metallothionein (PDB entry 1AOO).16 Images were made with VMD.31

of study today.18 The Ag7 cluster of yMT has been characterized by NMR, and a solution structure of the complex is deposited in the PDB (entry 1AOO, see Figure 1b).16,32 In contrast, the overall structure of rMT with bonded Ag(I) is still unknown; however, several spectroscopic methods have been used to unravel the structure of the metal clusters and the average Ag(I) coordination.9,33 Chelators inspired from the metal binding sites in the latter Cu proteins were also studied to deepen our knowledge of Ag(I) coordination in Ag−SR complexes. Cyclodecapeptides with two cysteines have been previously demonstrated to mimic the Cu binding loop of metallochaperones by coordinating the metal ion with two sulfur atoms with binding constants (Kd ≈ 10−15.5 to 10−17) in the same range as Atx1 (Kd ≈ 10−17.4).34−36 Therefore, the peptide P2, with a β-sheet structure and two cysteines’ side-chains oriented on the same face of the cycle (Scheme 1) was investigated here for its ability Scheme 1. Bioinspired Ligands Used in This Study

to chelate Ag(I).36 Metallothioneins are obviously the most efficient natural ligands of Cu(I), thanks to the very stable CuS3 coordination. Pseudopeptides such as L1 (Scheme 1) derived from nitrilotriacetic acid and functionalized with three converging cysteines perfectly mimic the coordination properties of MT for Cu(I).37,38 Interestingly, L1 forms a C3symmetric mononuclear Cu(I) complex, which is as stable as Cu(I) complexes with MT (Kd ≈ 10−19). In excess of Cu, this species transforms in a Cu6S9 core, which has also been identified in MT. Importantly, spectroscopic studies including X-ray absorption spectroscopy (XAS) data demonstrated that Cu(I) is exclusively found in a symmetric CuS3 environment.39 L1 also provides a MS3 environment with Hg(II),40 a soft divalent metal ion, and was therefore chosen as a model for the MS3 coordination mode in this study with Ag(I). The present work investigates the structure of Ag(I) binding sites in the Ag(I)−thiolate complexes formed with the two B

DOI: 10.1021/acs.inorgchem.5b01658 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry bioinspired sulfur compounds P2 and L1, with two MTs and, for the first time, with the Cu-chaperone Atox1. We made use of XAFS spectroscopy, which allows selective interrogation of the chosen metal and measurement of the interatomic distances in a radius of ∼5 Å around the absorber with a precision on the order of 0.01 Å; recent applications of XAS to metalloproteins allowed the characterization of Cu binding environment in CusCFBA and CopK, two prokaryotic proteins.41,42 We revealed the existence of a correlation between Ag(I) coordination number and Ag−S bond length; this correlation helped in disclosing the average Ag(I) binding mode in the multinuclear clusters of MTs, including rMT, for which no structure of the Ag−protein complex is deposited in the PDB. Therefore, we present an exhaustive characterization of Ag(I) binding to biological Cu(I)−thiolate sites, which might help in deciphering the intracellular trafficking pathways of the metal ions released from silver nanoparticles, as well as in disclosing the Ag(I) toxicity mechanisms related to the disruption of copper homeostasis.

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Mass spectra were acquired on a LXQ-linear ion trap (THERMO Scientific, San Jose, U.S.A.) equipped with an electrospray source. Electrospray full scan spectra in the range m/z = 150−2000 amu were obtained by infusion through a fused silica tubing at 2−10 μL/min. The solutions were analyzed in the negative and positive modes. The LXQ calibration (m/z = 50−2000) was achieved according to the standard calibration procedure from the manufacturer (mixture of caffeine, MRFA, and Ultramark 1621). The temperature of the heated capillary for the LXQ was set to 180−200 °C, the ion-spray voltage was in the range 2−4 kV, and the injection time was 10−100 ms. Atox1 Preparation and Characterization. pET21b-Atox1 plasmid was kindly provided by Prof. David Huffman (Western Michigan University). Human Atox1 was expressed as described by Wernimont et al.44 The cell pellet was resuspended in 50 mM phosphate buffer, pH 6, containing 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethanesulfonyl fluoride (PMSF), and complete protease inhibitor (Roche). After sonication the cell extract was centrifuged for 20 min at 50 000g. The supernatant was kept on ice with constant stirring, and its pH was decreased to 3.25 using H3PO4. The suspension was stirred 30 more minutes on ice and then centrifuged for 30 min at 75 000g. The supernatant was diluted by 2, and tris(2-carboxyethyl)phosphine (TCEP) was added in order to obtain a final concentration of 1 mM. The pH was increased to 6 with NaOH, and the solution was loaded on a SP-Hiload column (GEHealthcare). Atox1 was eluted with a gradient between 0 and 500 mM NaCl in 20 mM MES pH 6. Atox1 was incubated for 1 h with 10 mM EDTA and 10 mM TCEP and then further purified on a superdex75 Hiload and a Superdex peptide HR in 20 mM HEPES, pH 7.4, and 100 mM Na2SO4. Final Atox1 fractions were pooled, concentrated using a Vivaspin6 (Sartorius) with 5 kDa cutoff, aliquoted, flash-frozen in liquid nitrogen, and stored at −80 °C. The Atox1 oligomeric state in the presence of various amounts of silver was studied by size-exclusion chromatography coupled to multiple angle laser light scattering (SEC-MALLS). SEC was performed with a Superdex peptide column (GE Healthcare) equilibrated with 20 mM HEPES, pH 7.4, and 100 mM Na2SO4. The column was calibrated with globular standard proteins. Separations were performed at 30 °C with a 0.5 mL·min−1 flow rate. Online multiangle laser light scattering (MALLS) detection was performed with a DAWN-HELEOS II detector (Wyatt Technology Corp.) using a laser emitting at 690 nm, and protein concentration was measured online by the use of differential refractive index measurements using an Optilab T-rEX detector (Wyatt Technology Corp.) and a refractive index increment, dn/dc, of 0.185 mL·g−1. Weightaveraged molar masses were calculated using the ASTRA software (Wyatt Technology Corp.). For XAFS experiments, Atox1 buffer was exchanged to 20 mM HEPES, pH 7.4, on a Nap5 column (GE-Healthcare). The protein concentration was determined by measuring the 280 nm absorbance of the solution (ε280 (Atox1) = 3 884 M−1·cm−1) with a Hitachi U-3010 spectrophotometer. 0.9 Ag(I) equiv was added to 400 μL of Atox1 1.5 mM. MT Metalation. Ag(I)−rMT was prepared by replacement of Zn(II) and Cd(II) starting from 1 mg of rMT (Metallothionein-1 from rabbit liver, Enzo Life Sciences, Villeurbanne, France) and corresponding to 0.75 mg of protein. All experiments were performed at room temperature. All the eliminations of metal and buffer changes were performed by successive dilutions and concentrations (Vivaspin ultrafiltration units, 3000 Da cutoff, GE HealthCare, Vélizy Villacoublay, France) followed by centrifugation at 13 000g. First the lyophilized protein was solubilized in 200 μL of pure water (Milli-Q, Merck Millipore, Molsheim, France). The pH of the protein solution was lowered to 2 with 10 mM HCl for Zn and Cd removal. The apoprotein concentration was evaluated in this solution from its absorbance at 220 nm (ε220 = 47 300 M−1 cm−1)45 with a Varian Cary50 spectrophotometer (Agilent Technology, Les Ulis, France). Then the pH was raised to 3 with multiple additions of 10 mM sodium acetate/acetic acid, pH 3, and concentrations by centrifugation for chloride elimination. Preparation of Ag(I)−rMT was carried out by successive additions of Ag(I) as a form of AgNO3 calculated to reach

EXPERIMENTAL SECTION

Bioinspired LigandsSample Preparation. The syntheses of the bioinspired ligands P2 and L1 were described elsewhere.36,37 The list of samples analyzed by XAFS spectroscopy is reported in Table 1.

Table 1. List of Samples Analyzed by XAFS Spectroscopy ligand P2

L1

Atox1 yMT rMT

samples

[L] (mM)

[Ag(I)] (mM)

Ag/L

Ag0.3P2 Ag1P2 Ag1.5P2 Ag0.3L1 Ag1L1 Ag2L1 Ag(I)−Atox1 Ag(I)−yMT Ag(I)−rMT

2.3 2.3 2.3 2.4 2.4 2.3 10 0.44 0.2

0.7 2.3 3.4 0.7 2.4 4.6 9 3.0 2.1

0.3 1.0 1.5 0.3 1.0 2.0 0.9 7.0 10.5

All water solutions were prepared from ultrapure, laboratory-grade water that has been filtered and purified by reverse osmosis using Millipore Milli-Q reverse-osmosis cartridge system (resistivity 18 MΩ cm). 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (10 mM, pH 7.4) was used to prepare the ligands’ solutions for UV and CD titrations and ammonium acetate buffer (10 mM, pH 6.9) for electrospray ionization mass spectrometry (ESI-MS) experiments. Acetonitrile (10% v/v) was added in experiments with L1 for solubility reasons. The XAS samples were prepared in a similar way but using a 8/2 v/v mixture of HEPES buffer (100 mM, pH 7.4) and glycerol. The final concentration of the ligand solution was determined by measuring the free thiol concentration following the Ellman’s procedure.43 This procedure uses 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) as an indicator. Each free thiol group present in the compound yields 1 equiv of TNB2− (ε412 (TNB2−) = 14 150 M−1 cm−1). The silver solution was prepared by dissolving solid AgNO3 into water. Titrations. The UV−visible spectra were recorded with a Varian Cary50 spectrophotometer equipped with optical fibers connected to an external cell holder in the glovebox. The circular dichroism (CD) spectra were acquired with an Applied Photophysics Chirascan spectrometer. CD spectra are reported in molar ellipticity ([Θ] in units of deg cm2 dmol−1). [Θ] = θobs/(10lc), where θobs is the observed ellipticity in millidegrees, l is the optical path length of the cell in centimeters, and c is the ligand concentration in moles per liter. A volume of 2−2.5 mL of the ligand solution (∼50 μM) was transferred in a cell (1 cm path), and aliquots of the metal solution were then added. C

DOI: 10.1021/acs.inorgchem.5b01658 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry the stoichiometry of 12 Ag/rMT, corresponding to 6 Ag(I)/site. Metal binding was monitored by circular dichroism (JASCO J810, Bouguenais, France) to monitor cluster formation. Any metal excess was carefully removed by multiple additions/concentrations in 10 mM sodium acetate/acetic acid (pH 3) solution. Then the protein was concentrated to reach 200 μM in 10 mM HEPES, pH 7.4, for XAFS experiments. The final Ag content was measured by mass spectrometry (Agilent 7700 ICP-MS, Agilent Technology, Les Ullis, France) and estimated at ∼10.5 Ag/rMT. A peptide mimicking yeast MT (yMT) was purchased from Genecust (Dudelange, Luxembourg); its 36-amino-acid sequence (HECQCQCGSCKNNEQCQKSCSCPTGCNSDDKCPCGN) includes the 10 cysteines involved in the 7 metal ions binding sites previously mentioned. Because the synthetic peptide is metal-free and does not require demetalation, it was prepared as the bioinspired ligands (see earlier). XAFS Data Acquisition and Analysis. The Ag K-edge XAFS experiments were carried out at the beamline CRG-FAME-BM30B46 at the European Synchrotron Radiation Facility (ESRF, France). The Ag K absorption edge was scanned in the energy range 25.200−26.260 keV with a nitrogen-cooled Si(220) double-crystal monochromator.47 The incoming photon energy was calibrated with an Ag metallic foil, by defining the first inflection point of its XAFS spectrum at 25.514 keV. Spectra were recorded in fluorescence mode, with a 30-element Ge solid-state detector (Canberra). The number of scans per sample varied with the sample concentration; it was chosen to provide ∼106 photon counts above the absorption edge in the sum spectrum. The experiment was performed at 16 K in a liquid He cryostat. The absence of contamination from metallic silver or silver chloride in the protein samples was checked by comparing the X-ray absorption nearedge structure (XANES) spectra with the ones of reference Ag(0) and AgCl (Figure S1); the details of the preparation of reference compounds are described elsewhere.7 Extended X-ray absorption fine structure (EXAFS) spectra were extracted from the raw data using the Autobk algorithm.48 Theoretical scattering amplitudes and phase shifts were calculated by means of the ab initio codes FEFF6 and FEFF9.49,50 The amplitude reduction factor S02 was calculated by the program from atomic overlap integrals; the calculated value of 0.92 was fixed during the analysis. Data were analyzed using the Athena and Artemis graphic interfaces running the IFEFFIT programs suite.51 The EXAFS spectra were Fouriertransformed over the [2.9, 11] Å−1 k-range, then fitted in the R space in the range [1, 3.4] Å with multiple k-weights, using as minimization algorithm a modified Levenberg−Marquardt method. Two atomic shells around the absorber were included in the model: a first shell of S and a second shell of Ag neighbors. For each atomic shell i, the free parameters were the number of atoms (Ni), the distance to the absorber (Ri), and the Debye−Waller factor (σi2); a shift in the threshold energy (ΔE0) was always set as a global variable. The chosen k and R ranges provide 14 independent points,52 while the total number of variables was 7.

Figure 2. UV spectrophotometric titration of P2 47 μM in HEPES buffer (10 mM, pH 7.4) (top) and L1 47 μM in HEPES buffer (10 mM, pH 7.4)/acetonitrile, 9/1 v/v (bottom) with Ag(I) (0−2.6 equiv).

LMCT bands are 3000 and 8000 M−1 cm−1 per Ag(I) for P2 and L1, respectively. These significantly different values are characteristic of different coordination environments in the two systems. Although these features resemble those previously observed with the same ligands coordinating Cu(I), the CD titrations (Figures S2−S5) are indicative of several Ag(I) complexes that coexist during the titrations. Electrospray mass spectrometric titrations point to the same conclusions. Silver complexes with P2 (AgP2 and Ag2P2 stoichiometries) are barely detected as shown in Figure S6. The signals evidenced for the complexes with L1 are much more intense with the total disappearance of the free ligand for 2 Ag(I) equiv (Figure S7). The major ions detected are AgL1 and Ag2L1 with some minor peaks corresponding to cluster species. All together these data are consistent with a complicated speciation of the Ag(I) complexes, with multiple polymetallic species, which strongly contrasts with previous observations obtained with Cu(I) complexes with the same ligands. Therefore, XAFS spectroscopy was used to investigate the Ag(I) coordination in these complexes. Highly diluted samples with small amounts of Cu(I) (0.2 equiv) previously allowed us to evidence the mononuclear complexes; therefore, similar conditions (Ag0.3P2 and Ag0.3L1) were first investigated. Unfortunately, these low Ag concentrations ( 0.03 Å2). This confirms that no Ag atom is present around the Ag absorber in the radius probed by EXAFS spectroscopy. Therefore, the ripples visible above 2.4 Å in the FT spectra of the Ag1L1 complex (Figure 3b) are most probably due to the multiple scattering photoelectron paths involving the C atoms of Cys residues and depend on the relative spatial arrangement of the side chains of the donors; because the determination of this structural information is not trivial and is beyond the scope of this work, the fitting range for the Ag1L1 complex was restricted to [1, 2.4] Å (the same holds for the Ag2L1 complex). When the Ag/L1 mole ratio is increased from 1 to 2, the EXAFS spectrum (Figure 3a) does not undergo major modifications; as expected, the fit results (Table 2) of both structural and dynamical parameters of the two AgL1 complexes are unchanged within the error. The chemical environment of Ag(I) is therefore the same in the two complexes, and no Ag··· Ag interactions appear when Ag stoichiometry is increased; the only possible explanation for this phenomenon is that the Ag2L1 complex forms networks of trigonal AgS3 units where S atoms can bridge between two metal centers. To verify this hypothesis, a model of Ag2S5 cluster was built: it consisted of two trigonal AgS3 units bridged by a S atom, with a S−Ag−S angle of 120°, resulting in a Ag−Ag distance of 4.31 Å. The EXAFS spectrum generated for the Ag2S5 cluster does not show

Figure 3. (a) Experimental Ag K-edge EXAFS spectra (black dots) of Ag(I)−thiolate complexes in solution and the relative best-fitting curves (red traces, obtained from back-transformation into the k space of fits performed in the R space). (b) Fourier-transformed experimental EXAFS spectra (black dots) and the relative best-fitting curves (red traces) obtained from least-squares minimization of spectra generated with ab initio calculations.

models of the different Ag(I)−thiolate coordination geometries in solution and at physiological pH and then to make use of these models to predict the average Ag(I) chemical environment in complex multinuclear clusters as the ones of metallothioneins (MTs); therefore, we focused first of all on Ag(I)-bioinspired ligand complexes in 1:1 stoichiometry. The Fourier-transformed spectra are reported in Figure 3b: a shift in the R-position of the maximum of the first shell peak is

Table 2. Structural and Dynamical Parameters of the Ag(I) Binding Site in AgL Complexes (Where L is a Thiol-Bearing Biomolecule or Bioinspired Compound), Derived from Least-Squares Fitting of Ag K-edge EXAFS Spectra; The One Standard Deviation Error on the Last Digit Is Reported in Brackets Ag−S ligand L

1

P2 GSHa Atox1 yMT rMT a

Ag/L

N

R (Å)

1 2 1 1.5 1 0.9 7 10.5

3.0 (3) 2.9 (3) 2.23 (18) 2.24 (21) 2.0 (3) 2.06 (16) 2.7b 2.7b

2.489 (7) 2.484 (7) 2.441 (7) 2.447 (6) 2.400 (5) 2.400 (10) 2.471 (4) 2.483 (4)

Ag−Ag σ (10 2

5.8 6.1 7.4 8.2 5.5 5.9 7.1 8.5

−3

2

Å)

(5) (5) (3) (4) (3) (2) (2) (3)

N

R (Å)

σ2 (10−3 Å2)

1.1 (6) 1.2 (7) 0.8 (5)

2.992 (18) 3.017 (19) 2.969 (13)

15 (2) 16 (2) 9.9 (12)

0.5 (2) 0.5 (2)

2.948 (7) 3.000 (10)

6.0 (7) 7.1 (9)

Rfit (%) 0.7 0.9 0.7 0.8 0.8 1.0 0.3 0.3

From ref 7. bThese values were fixed to the ones estimated by LCF of the XANES spectra. E

DOI: 10.1021/acs.inorgchem.5b01658 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry additional features with respect to the one of an isolated AgS3 unit, and only minor amplitude modulations are observed (Figure S8); when fitted to the experimental spectrum, the Ag2S5 model does not provide a significant improvement with respect to simple AgS3, which proves that the technique is not sensitive to Ag atoms eventually present at ∼4 Å from the absorber. This is compatible with the hypothesis that when the Ag/L1 mole ratio is increased, the local coordination of Ag(I) remains unchanged, i.e., trigonal planar, and the AgS3 units organize in networks in such a way that excess Ag(I) can be accommodated. This demonstrates that AgL 1 can be considered as a model for trigonal AgS3 coordination. Similarly, when the Ag/P2 mole ratio is increased from 1 to 1.5, the EXAFS spectrum (Figure 3a) does not undergo major modifications and the quantitative fit results (Table 2) do not vary significantly. This proves that the local chemical environment of Ag(I) is the same regardless of Ag stoichiometry, and suggests that S-bridges between AgSx units are formed whatever the Ag/P2 mole ratio. However, in both AgP2 solutions the average number of S neighbors of Ag estimated by EXAFS analysis is slightly higher than 2 (Table 2): this does not allow us to consider AgP2 as a model for digonal AgS2 coordination, because a small fraction of Ag is expected to populate trigonal AgS3 sites. In contrast, the characterization of the Ag(I) coordination sphere in a Ag/GSH = 1:1 solution that we recently published yielded evidence of pure AgS 2 coordination: Ag was found to bind 2.0 ± 0.3 S atoms at 2.400 ± 0.005 Å distance (the fit results, previously published,7 are reported in Table 2 for comparison with this study). A correlation between coordination number and Ag−S bond length is expected: a survey of crystalline Ag(I)−thiolate compounds revealed that the average Ag−S distance is 2.39 ± 0.03 Å in digonal AgS2 sites and 2.51 ± 0.05 Å in trigonal AgS3 sites.19 However, this correlation cannot be extended to Ag(I)− thiolate complexes in solution, for which very little is known. The average Cu−S distance in Cu(I)−thiolate synthetic compounds and protein solutions has been probed by EXAFS spectroscopy53 and was found to be indicative of the fraction of digonal/trigonal sites in multinuclear clusters. Our results highlight that a correlation between bond length and coordination number exists also in Ag(I)−thiolate complexes in solution at physiological pH: when the Ag−S distance extracted from EXAFS analysis (Table 2) is plotted as a function of the estimated number of S (Figure 4, black squares), a trend is clearly defined. The coordination chemistry of Ag is comparable between Ag(I)−thiolate complexes in solution and in crystalline structures: the complexes showing AgS2 and AgS3 sites only, AgGSH and AgL1, respectively, show Ag−S distances (see Table 2) that fall into the ranges calculated from a survey of crystalline Ag(I)−thiolate compounds.19 Therefore, we used these two complexes as reference samples to predict the fraction of digonal/trigonal sites in the multinuclear Ag(I) clusters of metallothioneins. Before moving to multinuclear Ag(I) clusters, we characterized the mononuclear Ag(I) binding site of Ag-loaded Atox1, in order to check if the correlation between Ag coordination number and Ag−S distance highlighted in small compounds applies also to biological macromolecules. Ag(I)−Atox1. The EXAFS spectrum of Ag(I)−Atox1 and its Fourier transform (FT) are reported in Figure 3; the FT presents a first-shell peak and no features above 2.5 Å, which suggests that only first-shell atoms contribute to the signal and no metal−metal interaction is present. The fit of the FT signal

Figure 4. Correlation between Ag(I) coordination number and average Ag−S bond length in Ag(I)−thiolate complexes formed in solution with small biological and bioinspired molecules (black squares), with the Cu-chaperone Atox1 (blue triangle), and with yeast and rabbit metallothioneins (red circles).

in the region [1, 3.4] Å confirms that Ag is bonded to 2 S atoms at 2.400 ± 0.010 Å and that no Ag atom is present in the vicinity of the absorber (see Table 2); this suggests that Ag binds to the two Cys residues of the conserved metal-binding loop (MxCxxC) of the chaperone and that no metal-bridged dimer is formed under our experimental conditions. The crystal structure of Atox1 bound to Cu(I), Cd(II), or Hg(II) has been previously resolved25 and revealed that the different metal ions all bind to the Cys residues of the MxCxxC loop. This binding domain has been proposed to play a crucial role in Cu(I) delivery to ATP7A and ATP7B, which both display six repeats of the MxCxxC motif, allowing them to bind one Cu per motif:29 docking of the metal-bound chaperone to its target and subsequent ligand exchange is expected to occur between the MxCxxC sites, through the formation of an intermediate complex where the metal is simultaneously coordinated by the two proteins.27 A Cu K-edge XAFS study revealed that, when the preparation and reconstitution protocol of Cu(I)−Atox1 is carried out in such a way to minimize the use of thiol reagents, Cu(I) occupies a digonal CuS2 site capable of forming CuS3 adducts with exogenous ligands;54 finally, the solution structure of Cu(I)−Atox1 resolved by NMR methods suggested that, in physiologically relevant conditions, the monomeric form of Cu(I)−Atox1 is obtained, where Cu(I) binds to the two Cys of the MxCxxC loop in digonal CuS2 sites only.26 Less is known about Ag(I)−Atox1, for which no structure has been deposited so far in the PDB. SEC-MALLS analysis showed that, upon metalation up to a 1:1 stoichiometry, Atox1 is monomeric (see Figure S9). In the Ag0.9Atox1 sample, the Ag−S distance measured by EXAFS analysis is 2.40 ± 0.01 Å, with this value being indicative of digonal coordination (see Figure 4). Given the monomeric form of the metal−protein complex and the coordination number of Ag, we are allowed to conclude unambiguously that Ag(I) binds to the two converging Cys of the MxCxxC loop of Atox1. Multinuclear Ag(I) Sites in Metallothioneins. Once the coordination chemistry of Ag in mononuclear Ag(I)−thiolate complexes has been unraveled, this information can be used to predict the average Ag environment in the multinuclear clusters formed with yeast and rabbit metallothionein (yMT and rMT, F

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Inorganic Chemistry respectively). It has been previously demonstrated that truncated yMT binds 7 Ag(I) ions in a single cluster lying in a cleft interior to the protein backbone where the side-chains of 10 Cys residues converge (PDB entry 1AOO),16 and that rMT binds up to 18 Ag(I) ions in two clusters located in its α and β domains, respectively;17 however, no structure of the Ag(I)− rMT complex has been deposited in the PDB so far, and the structure of the Ag clusters is still debated. Therefore, we made use of XAS to analyze both systems: the simpler yMT and the more complex rMT; this allowed us to compare the results obtained on yMT with the data available in the literature and provide a proof of concept for the analysis protocol, which could therefore be extended to the rMT sample. To measure the fraction of AgS2 and AgS3 sites in the Ag(I)− MT complexes, we fitted their X-ray absorption spectra as linear combinations of the AgGSH and Ag1L1 compounds, considered as the best models of digonal and trigonal Ag(I)− thiolate compounds, respectively (see Figure 4). The X-ray absorption near-edge structure (XANES) spectra of MT and reference compounds are reported in Figure 5a: clearly, the

Table 3. Fraction of Digonal AgS2 and Trigonal AgS3 Sites in the Multinuclear Ag(I) Clusters of Two Metallothioneins (MT), Derived from Fitting of the X-ray Absorption Spectra As Linear Combinations of Reference Compoundsa AgS2 fraction (%) yMT rMT

XANES EXAFS XANES EXAFS

32 30 31 31

(2) (2) (3) (2)

AgS3 fraction (%) 68 70 69 69

(2) (2) (3) (2)

Rfit 1.3 × 10−5 8 × 10−2 2.5 × 10−5 14 × 10−2

a

The near-edge (XANES) and extended (EXAFS) regions of the spectra were fitted separately; the reference compounds used are a Ag/ GSH = 1:1 and a Ag/L1 = 1:1 solution for digonal and trigonal coordination, respectively. The one standard deviation error on the last digit is reported in brackets.

retrieved by LCF of XANES spectra, ensuring the reliability of the results. In both MTs Ag occupies trigonal sites mainly (∼70%); in yMT this means that 5 of the 7 Ag atoms are in AgS3 binding mode while 2 are in AgS2. Remarkably, this finding agrees with the results obtained from a 1H NMR study by Narula et al.32 To measure the average Ag−S bond length in the multinuclear Ag(I) clusters of MT, the EXAFS spectra were fitted based on ab initio models: the average coordination number of Ag was set to the one estimated by LCF analysis (70% AgS3 and 30% AgS2 results in an average number of 2.7 S per Ag), while the other parameters relative to the first Ag−S and second Ag−Ag shells were allowed to vary; the best fitting curves are reported in Figure 3, and the quantitative results are reported in Table 2. We found that the average Ag−S distance is 2.471 ± 0.004 Å in yMT and 2.483 ± 0.004 Å in rMT: when these values are plotted versus the average Ag coordination number (Figure 4, red circles), they nicely fit into the trend defined by mononuclear Ag(I)−thiolate complexes. These Ag− S distances slightly differ from the ones of 2.45 ± 0.02 and 2.44 ± 0.03 Å found by Gui et al. in Ag12−rMT and Ag17−rMT, respectively, calculated by means of sulfur K-edge EXAFS spectroscopy.33 Moreover, in this study the authors concluded that Ag(I) is digonally coordinated in rMT, which is in contrast with the experimental evidence provided in the present work. This is not surprising if we consider that the conclusion of the work by Gui et al. was drawn by comparing the Ag−S distance in Ag(I)−rMT with a digonal Ag(I)−thiolate inorganic complex showing a broad spectrum of Ag−S distances.22 A recently published CSD survey of Ag−S distances in Ag(I)− thiolate compounds highlights that the range of distances in AgS2 sites is wide (2.34−2.53 Å); nevertheless, their average is calculated as 2.39 ± 0.03 Å;19 according to the same survey, for AgS3 sites the average Ag−S distance is 2.51 ± 0.05 Å. On the basis of these observations, and thanks to the large amount of structures deposited in the CSD over the past 20 years, the conclusions of the study from Gui et al. can be reconsidered, because the average Ag−S distance they found in rMT cannot be uniquely assigned to either AgS2 or AgS3 coordination. Our data highlight also that the average chemical environment of Ag is the same in Ag(I)−yMT and in Ag(I)−rMT: the coordination number of Ag and the Ag−S bond lengths are the same within the error, while the first shell Debye−Waller factor is slightly higher in Ag(I)−rMT (Table 2). This is reasonable if we consider that Ag(I)−rMT binds a larger number of Ag atoms in two distinct clusters, with a larger variability in Ag−S distances, which gives rise to a higher static disorder.

Figure 5. Experimental XANES (a) and EXAFS (b) spectra of yeast and rabbit metallothioneins (open circles) and the relative best-fitting curves (red traces) obtained as linear combinations of the experimental XANES (a) and EXAFS (b) spectra of Ag1L1 and AgGSH complexes (black continuous lines), chosen as models of AgS3 and AgS2 coordination, respectively. Linear combination analysis of XANES and EXAFS spectra were performed separately.

XANES region is poorly sensitive to the coordination geometry, and weak changes in the spectral features are observed between the two reference compounds. It can be noticed that the trigonal complex (Ag1L1) shows a slightly more prominent feature at ∼25 525 eV and that its postedge minimum appears at lower energy (∼25 557 eV) with respect to the digonal complex (AgGSH); the linear combination fitting (LCF) results are reported in Table 3. Considering the low variability between reference compounds in the near-edge region, we performed as well an LCF analysis of the EXAFS spectra in order to corroborate our results. The EXAFS spectra of MTs and of the samples used as references are reported in Figure 5b: the oscillations generated by trigonal and digonal Ag(I) sites differ both in frequency and amplitude, which suggests that LCF of EXAFS spectra is a suitable approach for the estimation of the fraction of each binding mode. The best fits of MT samples are reported in Figure 5b (red curves), and the estimated fractions of digonal/trigonal Ag(I) are reported in Table 3: they correspond within the errors to the values G

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CONCLUSIONS We characterized the coordination environment of Ag(I) in Ag−thiolate complexes formed in solution at pH 7.4, i.e., in physiologically relevant conditions. The investigated thiolate compounds, chosen for their proven efficiency in Cu(I) chelation, were found to efficiently bind Ag(I) as well, as expected on the basis of the similarity between the electronic properties of the two metals. The speciation of Ag(I) in the complexes formed with bioinspired thiolate compounds is more complex than the one of Cu(I) and is indicative of the presence of multiple polymetallic species coexisting during the titration. However, the average local coordination of the Ag(I) atoms remains unchanged regardless of the Ag/ligand mole ratio, as highlighted by XAS data. Remarkably, the pseudopeptide L1 mimicking the coordination properties of MT for Cu(I) was found to bind Ag(I) in trigonal AgS3 geometry, with a Ag−S bond length comparable with the average calculated from a survey of crystalline trigonal Ag(I)−thiolate compounds;19 therefore, L1 can be regarded as a robust model of local AgS3 coordination in Ag−SR complexes. In contrast, we found that the peptide P2, which comprises two Cys residues that bind Cu(I) in digonal CuS2 sites, is not a perfect model of AgS2 coordination. The Ag−glutathione 1:1 complex that we previously characterized is more representative of digonal AgS2 geometry.7 XAS data relative to these complexes highlight the existence of a correlation between Ag(I) coordination number and the Ag−S distance, which is established for Ag(I)− thiolate crystalline compounds but has never been experimentally observed in solution at physiological pH. Indeed, the characteristic Ag−S distances of AgS2 and AgS3 sites in crystallized molecules and in solution correspond within the error. In the silver-loaded Cu-chaperone Atox1, Ag(I) binds to the two Cys residues of the Cu(I)-binding domain, with an average Ag−S distance of 2.40 Å that matches the characteristic AgS2 value measured in the Ag−glutathione digonal complex; therefore, we expect that the correlation found for small compounds can be extended to Ag(I)−thiolate sites of higher molecular weight biomolecules. The exhaustive characterization of mononuclear sites, together with the identification of models of AgS2 and AgS3 coordination, laid the basis for the investigation of the multinuclear Ag(I)−thiolate sites as the ones of metallothioneins: in both silver-loaded yeast MT and in silver-substituted rabbit MT, Ag(I) is found mainly in trigonal coordination geometry (70% of the total Ag(I) atoms bonded to MT, in agreement with previous NMR studies).32 Again, the average Ag−S distance of 2.47−2.48 Å reflects the geometry of the binding site, mainly trigonal, and is markedly longer than the distance measured in the digonal site of Ag(I)−Atox1. Atox1 and MT are two key proteins involved in Cu(I) homeostasis in eukaryotic cells, and they are known to bind also nonphysiological metals in their thiolate chelating sites. Ag(I) is a toxic metal that shows high affinity for these proteins. This property is likely to elicit Ag(I) toxicity: when cells are exposed to Ag(I), e.g., through internalization and in cellulo dissolution of Ag nanoparticles, the free metal ions could displace Cu(I) from its native binding sites and induce a deregulation of Cu(I) homeostasis. The identification of the complexes formed in cells upon exposure to Ag(I) is therefore crucial to elucidate the chemical basis of Ag(I) toxicity; however, this information is often elusive due to the scarce present knowledge concerning the chemistry of Ag(I) in physiologically relevant conditions. Here we demonstrate that

the Ag−S bond length is indicative of the coordination of Ag(I), itself depending on the nature of the complex formed: this information can pave the way to the understanding of intracellular Ag(I) trafficking pathways through substitution of Cu(I) in thiolate sites.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.5b01658. CD and ESI-MS titrations of Ag/bioinspired ligands, SEC-MALLS-RI characterization of Ag(I)-loaded Atox1, and the simulated EXAFS spectrum of an Ag2S5 complex (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. Funding

This work was founded by the CEA-Toxicology Transversal Program through the NanoSilverSol grant and by the CEA Transversal Programs Toxicology and Nanoscience through the NanoTox-RX grant. This research is part of the LabEx SERENADE (Grant ANR-11-LABX-0064) and the LabEx ARCANE (Grant ANR-11-LABX-0003-01). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the ESRF and the French CRG for providing access to beamtime on the beamline BM30B (experiments LS-2331 and 30-02-1087, respectively). The authors thank the iRTSV SEC-MALLS platform and Martine Cuillel, Roger Miras, Mireille Chevallet, and Isabelle Worms for their help and for useful discussion.

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REFERENCES

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