Cadmium – glutathione solution structures provide new insights into heavy metal detoxification Olivier Delalande 1, *, Herve ´ Desvaux 2 , Emmanuel Godat 1,3 , Alain Valleix 4 , Christophe Junot 3 , Jean Labarre 1 and Yves Boulard 1 1 Laboratoire de Biologie Inte ´ grative ⁄ Service de Biologie Inte ´ grative et Ge ´ ne ´ tique Mole ´ culaire ⁄ Institut de Biologie et de Technologies de Saclay, CEA-Saclay, Gif-sur-Yvette Cedex, France 2 Laboratoire Structure et Dynamique par Re ´ sonance Magne ´ tique ⁄ Service de Chimie Mole ´ culaire, URA CEA-CNRS 331 ⁄ IRAMIS, CEA-Saclay, Gif-sur-Yvette Cedex, France 3 Laboratoire d’Etude du Me ´ tabolisme des Me ´ dicaments ⁄ Service de Pharmacologie et d’Immuno Analyse Mole ´ culaire ⁄ Institut de Biologie et de Technologies de Saclay, CEA-Saclay, Gif-sur-Yvette Cedex, France 4 Service de Chimie Bioorganique et de Marquage ⁄ Institut de Biologie et de Technologies de Saclay, CEA-Saclay, Gif-sur-Yvette Cedex, France Introduction Cadmium is a very toxic metal with mutagenic proper- ties. It also causes oxidative stress, but the mechanisms involved remain unclear [1]. In most eukaryotic cells, the first line of d efence against cadmium is thiol-containing molecules (glutathione, phytochelatin or metallothionein depending on the cell type) that have the property to chelate and sequester the toxic metal. Glutathione is a thiol-containing tripeptide, c-Glu-Cys-Gly, which is ubiquitous and one of the most abundant cellular metabolites in many cell types, such as yeast or Keywords cadmium chelation; glutathione; heavy metal toxicity; NMR; yeast Correspondence Y. Boulard, CEA – Direction des Sciences du Vivant, Institut de Biologie et de Technologies de Saclay, Service de Biologie Inte ´ grative et Ge ´ ne ´ tique Mole ´ culaire, Ba ˆ t.144, 91101 Gif-sur-Yvette Cedex, France Fax: +33 1 69084712 Tel: +33 1 69083584 E-mail: yves.boulard@cea.fr *Present address Centre de Biophysique Mole ´ culaire, CNRS UPR 4301, Rue Charles Sadron, 45071 Orle ´ ans Cedex 2, France (Received 12 July 2010, revised 6 October 2010, accepted 12 October 2010) doi:10.1111/j.1742-4658.2010.07913.x Cadmium is a heavy metal and a pollutant that can be found in large quantities in the environment from industrial waste. Its toxicity for living organisms could arise from its ability to alter thiol-containing cellular com- ponents. Glutathione is an abundant tripeptide (c-Glu-Cys-Gly) that is described as the first line of defence against cadmium in many cell types. NMR experiments for structure and dynamics determination, molecular simulations, competition reactions for metal chelation by different metabo- lites (c-Glu-Cys-Gly, a-Glu-Cys-Gly and c-Glu-Cys) combined with bio- chemical and genetics experiments have been performed to propose a full description of bio-inorganic reactions occurring in the early steps of cad- mium detoxification processes. Our results give unambiguous information about the spontaneous formation, under physiological conditions, of the Cd(GS) 2 complex, about the nature of ligands involved in cadmium chela- tion by glutathione, and provide insights on the structures of Cd(GS) 2 complexes in solution at different pH. We also show that c-Glu-Cys, the precursor of glutathione, forms a stable complex with cadmium, but biological studies of the first steps of cadmium detoxification reveal that this complex does not seem to be relevant for this purpose. Abbreviations GSH, glutathione reduced form; GSSG, glutathione oxidized form. 5086 FEBS Journal 277 (2010) 5086–5096 ª 2010 The Authors Journal compilation ª 2010 FEBS mammalian liver cells, where it is present at millimolar range concentrations [2,3]. In vivo, it has a key role in protecting cells against reactive oxygen species, xenobiotics and heavy metals such as cadmium [4]. Glutathione exists in two forms: the antioxidant reduced form conventionally called glutathione (GSH) and its oxidized form known as glutathione disulfide (GSSG). In vivo, the GSH ⁄ GSSG ratio is in the range of 20–100 depending on the cell type and growth conditions [5,6]. Driven by this biological relevance, numerous spec- troscopic studies of cadmium(II) complexes, in particu- lar of simple thiol-containing ligands [7–11], have been performed, revealing a large diversity of cadmium–pep- tide interactions varying according to pH and metal concentration [7,12–14]. Nevertheless, the nature of the metal binding in the case of GSH remains subject to debate, as cadmium has been proposed to link to amide [15], carboxylates of both glycine [13,14] and glutamate residues [13,14,16] or the amine NH 2 lone pair [11,13,16]. 113 Cd NMR [12] was used to character- ize this interaction, but without success, in contrast to many 113 Cd–protein experiments [17–19]. Simulation of theoretical chemical shifts [20,21] or EXAFS experi- ments [22] were also performed to analyse the cad- mium(II) sphere of co-ordination. From these studies, it appears that the Cd(GS) 2 dimer is the major biologi- cally active form of the complex [15,23], but it is not necessarily the main stable form of the complex in solution at neutral pH [7]. Also, despite the high levels of GSH in cells, the kinetics of the formation of Cd(GS) 2 complexes at physiological pH (6.5–7.0 in the cytosol and 6.0–6.5 in the vacuole) have not been stud- ied. Furthermore, it is not known whether glutathione S-transferase activities are important for the formation of the complex in vivo, as previously suggested [24–26]. Finally, cadmium detoxification in yeast cells is based on export of Cd(GS) 2 complexes outside the cell or into the vacuole compartment. These movements are performed by ABC transporters, respectively Yor1p [27] and Ycf1p (similar to human MRP1) [15,28]. Genetic data unambiguously indicate that the Ycf1p vacuolar transporter has a more important role in vac- uolar sequestration of cadmium compared with the Yor1p transporter [27]. A third efflux recently described is also present in some yeast strains. It con- sists of a P 1B -type ATPase able to directly expulse Cd 2+ ions outside the cells [29]. Here we provide further insights into cadmium com- plexation by metabolites. We considered four glutathi- one-related peptides, GSH, c-Glu-Cys, a-Glu-Cys-Gly (a-GSH) and the free GSSG oxidized form. Despite the wide range of peptides considered for cadmium chelation studies, c-Glu-Cys has never been studied, even though it is a precursor used by glutathione syn- thetase for c-GSH production. Its study seems biologi- cally relevant as this metabolite is overproduced in yeast under cadmium stress conditions [3]. Also, because its cellular concentration is in the range of that of c-GSH, it could compete with glutathione for cadmium chelation in the detoxification process [30]. The choice of the synthetic peptide (a-GSH) was moti- vated by its ability to modulate cadmium complexa- tion. Finally, because the thiolate group is strongly implicated in metal co-ordination, we also considered the free GSSG glutathione oxidized form as a reliable model to validate the solution structure refinement procedure. Indeed, this molecule bearing a disulfide bridge leads to a global structure close to Cd(GS) 2 where the cadmium is bridging sulfur atoms. Because of the absence of a definitive structural model of the Cd(GS) 2 complex, we combined absolute distance determination using off-resonance ROESY experiments with molecular dynamics simulations and biochemical observations to provide insight into the solution struc- ture of GSH complexes of cadmium at different bio- logically relevant pH. We also describe competitive experiments giving indications on the relative affinity in vivo of these different natural peptides for cadmium. The data suggest that the biological importance of Cd(GS) 2 for detoxification is more driven by the selec- tivity of the transporter than by the stability of the complex. Results Co-ordination of cadmium from NMR studies The chelation of cadmium by the thiol-containing pep- tides GSH, a-GSH and c-Glu-Cys (chemical structures are given in Fig. 1) in aqueous solution and physiolog- ical pH can visually be observed and characterized by simple 1D NMR experiments. Indeed, after the addi- tion of cadmium to the GSH sample in a 1 : 2 Cd ⁄ GSH stoichiometry (0.5–5 mm solutions in our experiments), a white precipitate instantaneously formed and the solution became acidic. Integration of NMR signals relative to a reference peak (CH 2 of l-glycine) indicated that the precipitate corresponded to  10% at pH 6.4 to  20% at pH 7.2 of the total amount of GSH. Notably, the precipitate was resolubi- lized after restoring the pH to a neutral value and shaking the sample. These results were confirmed using radioactive 35 S-GSH to quantify both precipitate and soluble forms of complexes (Table S1). Similarly, the addition of cadmium to the a-GSH sample resulted in O. Delalande et al. Cadmium–glutathione complex in solution FEBS Journal 277 (2010) 5086–5096 ª 2010 The Authors Journal compilation ª 2010 FEBS 5087 the formation of a precipitate, but in this case, its dis- solution was impossible, even after changing the pH, vigorous shaking or sonication. Furthermore, analysis of NMR spectra indicated the precipitation of a 1 : 1 stoichiometric complex. A simple pH-dependent analy- sis of the electric charges indicated that because the precipitated complex is necessarily neutral at this pH, the cysteine residue should be in its thiolate S ) form. Indeed, at neutral pH, a-GSH bears two carboxylate groups (COO ) ) and the amino group of the glutamate is protonated (NH þ 3 ). From these simple observations, information about the co-ordination modes of a-GSH and GSH can be deduced. The transient precipitate observed in the case of GSH should have a very similar complexation mode to that of a-GSH, with a 1 : 1 stoichiometry and a glo- bal charge of zero. Restoring the pH to its initial value allows this precipitated form to be transformed to the more stable 1 : 2 complex [13,14,31,32]. In both cases, a-GSH and GSH peptides form a bidentate complex with cadmium where the sulfur of the cysteine and the carboxylate group of the glutamate or the glycine resi- due are implicated. These two carboxylate groups are fully equivalent in terms of metal co-ordination struc- tures. Consequently, the difference observed in cadmium chelation with a-GSH and GSH is due to the different location of the amino group of the glutamate residue in both peptides. The Cd(a-GSH) complex in 1 : 1 stoichiometry is structurally stable, whereas intermo- lecular interactions between GSH chains are necessary to stabilize the 2 : 1 complex of GSH with cadmium. Analyses of 1 H 1D NMR spectra of GSH and c-Glu-Cys in the presence of cadmium are also very informative (Fig. 1) and show that both Cd(GS) 2 and Cd(c-Glu-Cys) 2 complexes have common properties. First, the broadening of both cysteine a and b proton resonances after cadmium addition to the sample sug- gests the existence of an exchange process involving the metal ion and the cysteine residue. Second, we observed that the two cysteine b protons, which are equivalent in the absence of cadmium (only one chemi- cal shift in NMR spectra), are well differentiated (two chemical shifts) after metal addition [see 1D spectra for GSH (Fig. S1) or 2D spectra for c-Glu-Cys (Fig. S2)]. This observation clearly indicates an asym- metry of the final complex due to metal co-ordination and represents a direct probe to follow cadmium chela- tion. Finally, the dependence of NMR spectra on pH values is a way to probe the chemical structure of the complex. In acidic conditions (pH = 5.6), the addition GSH GSH + Cd GSSG α-GSH γ-EC + Cd γ-EC NH 2E NH G NH C ** * # α C α G α E γ E β E β′ C /β′′ C 9.0 8.5 4.5 4.0 3.5 2.53.0 p . p .m. p . p .m. Fig. 1. 1D 1 H NMR spectra of the different glutathione species. Spectra were recorded in H 2 O at 280K and pH 7.2. From bottom to top, GSH, Cd(GS) 2 , GSSG, c-Glu-Cys, Cd(c-Glu-Cys) 2 and a-GSH. The chemical structures from bottom to top of GSH, c-Glu-Cys and a-GSH are indicated on the right. *Corresponds to impurities present in the aGSH sample. # Indicates the resonance of the CH 2 group of L-glycine, which was used as a reference signal for peak integrations. Arrows indicate characteristic resonances of cadmium chelation by GSH or c-Glu-Cys and of the oxidized form of glutathione. Spectra were aligned with respect to L-glycine CH 2 resonance. Cadmium–glutathione complex in solution O. Delalande et al. 5088 FEBS Journal 277 (2010) 5086–5096 ª 2010 The Authors Journal compilation ª 2010 FEBS of cadmium to the sample did not affect the amino group resonance at 7.6 p.p.m. of the GSH peptide (Fig. S3), indicating that this potential ligand is not involved in metal chelation. At neutral pH (pH = 6.4 and pH = 7.2), the exchange rate with the solvent of amide protons (NH) of both cysteine and glycine as a function of temperature was of the same order (Fig. S4), demonstrating that no amide deprotonation occurs after cadmium chelation to GSH or c-Glu-Cys peptides, as has been previously suggested for the cysteine amide nitrogen [15]. The lability of cadmium bound to the GSH mole- cules was assessed by 14 N versus 15 N glutathione competition reactions (Fig. 2). Chelation of cadmium to the 15 N-labelled GSH pool led to the formation of the Cd( 15 N-GS) 2 complex, which induced (despite a partial signal overlap with the cysteinyl proton) the disappearance of the glycine amide cross-peak on the spectra. After the addition of 14 N-GSH in the same proportion to the 15 N-enriched GSH (1 : 1 stoichiom- etry), the glycine 15 N- 1 H cross-peak was restored. This indicates the presence of free 15 N-GSH peptide. The observed reappearance consequently resulted from a chemical exchange process, in the 0.1–10 ms range, between bound and unbound GSH molecules to the cadmium. The relative affinity of cadmium to GSH and other peptides was explored by competitive complexation experiments, as shown in part of the TOCSY spectrum in Fig. S2. The quantification of NMR data allowed the evaluation of the chelation fraction: 42.5 ± 5.5 and 57.5 ± 5.5% for GSH and c-Glu-Cys, respec- tively. These values clearly indicate the similar affinities of both natural metabolites for cadmium. NMR structural models for Cd(GS) 2 and comparison with the GSSG model Because of the small molecular mass of GSH (307.5 Da), NOESY experiments are not appropriate to determine internuclear distances and ROESY-type experiments are also known to lead to quantification problems [33]. To circumvent this major problem, we decided to use an alternative approach based on the off-resonance ROESY pulse sequence [34]. This method allows the determina- tion of absolute internuclear distances and of local corre- lation times. These parameters were used to build initial structural models of both GSSG and Cd(GS) 2 com- plexes. A refinement protocol with an explicit solvent was first performed on GSSG structures and then applied to the Cd(GS) 2 complex. The best structures (shown in Fig. 3) were obtained, in agreement with the co-ordina- tion study, using the protonated N-terminal c-glutamate residue. A total of 26 and 25 NMR constraints per monomer (GSH unit) were respectively used for GSSG and Cd(GS) 2 structure determinations (Table 1). Sur- prisingly, strong differences were observed for the major- ity of the distances recorded for both molecules, despite their similar topological arrangement. Because the exis- tence of a disulfide bond between the two glutathione units in the oxidized form cannot induce those striking variations, this suggests that the tridimensional organiza- tion of the Cd(GS) 2 complex is very different from GSSG. When the pH was varied between 6.4 and 7.2, sig- nificant differences for several distances were observed for the Cd(GS) 2 complex (distances marked in Table 1). Some of them were characterized as effects of inter-GSH interactions. This interpretation resulted from sampling of numerous conformations in molecular dynamic trajec- tories, which indicated that these NMR data could not be due to interproton distances in a unique GSH unit of the dimer (Table S2). This was confirmed by bad refine- ment convergence of simulated annealing calculations parametrized only for intra-GSH distances, in agreement with the fact that we did not observe the 1 : 1 stoichiome- tric complex in the experimental conditions. The pairwise local correlation times (s c ) extracted from off-resonance ROESY experiments were longer for Cd(GS) 2 complexes ( 1.5 · 10 )9 s) than for oxidized p.p.m. p.p.m. 116 117 118 119 120 121 * GSH 15 N + Cd + 14 N-GSH 8.18.28.38.4 Fig. 2. Expanded contour plot of three superimposed 15 N- 1 H heteronuclear single quantum coherence spectra: 15 N-GSH without cadmium (red), after the addition of cadmium and the formation of the Cd( 15 N-GS) 2 complex (green), after the addition of 14 N-GSH in a 1 : 1 stoichiometry with respect to 15 N-GSH (purple). Spectra were recorded in H 2 O at 275 K and pH = 7.0. Because of the overlap of the cross-peaks, the 2D spectra (green and purple) were shifted towards the 15 N dimension (vertical axis). O. Delalande et al. Cadmium–glutathione complex in solution FEBS Journal 277 (2010) 5086–5096 ª 2010 The Authors Journal compilation ª 2010 FEBS 5089 glutathione ( 0.4 · 10 )9 s). A detailed analysis of the local correlation times revealed that pH changes mainly affected inter-GSH unit glutamic acid ⁄ glycine chain interactions with an increase in correlation times for higher pH revealing a greater complex rigidity. Cd(GS) 2 structures determined by simulated anneal- ing calculations were clustered into six families based on minimal penalties induced from NMR distance restraints. 3D models representative of each conforma- tional family and calculated for pH = 6.4 or pH = 7.2 are presented in Fig. 3 and coordinates are given in Tables S3–S4. Those of GSSG model are given in Table S5. Our model clearly indicates that between zero and two water molecules are present and complete the cad- mium co-ordination sphere. This number depends on the pH value and the cadmium charge, which varies from 0 to +2.0 in our calculations. Despite these varia- tions, the final models are very similar. The pH value mainly influences the interactions between the two GSH units, whereas the presence of a positively charged cadmium clearly favours the bonding of the glycine carboxylate group. It should be noted that symmetric structural models are severely penalized because of their deviation from imposed NMR restraints. Discussion Cadmium glutathione complexes When cadmium is complexed by glutathione, different species exist in solution in equilibrium: a mixture of the Cd(GS) 2 1 : 2 complex and the Cd(GS) 1 : 1 complex. A recent study [7] suggested that the 1 : 1 monochelate is one of the major complexes formed at low glutathi- one concentration. On the other hand, at physiological pH and higher glutathione concentration, which are the relevant in cellulo conditions, the 1 : 2 complex is pre- dominant, as shown by speciation studies [7,14]. As a consequence, in this work, we focused on the Cd(GS) 2 complex, as apart from its biological relevance, it is the transport-active complex [15]. To this end, we per- formed NMR experiments at the optimal conditions for the formation of the Cd(GS) 2 complex, considering that the 1 : 1 complex that precipitates is almost totally transformed into the water-soluble Cd(GS) 2 1 : 2 com- plex after restoring the pH and shaking. Consequently, as shown by NMR, we observed only the major and the most stable soluble Cd(GS) 2 complexes formed under the conditions of the study (pH 6.4 and 7.2, a temperature of 17 °C, concentration over 1 mm and at a favourable 1 : 2 stoichiometry). Eventual disturbing effects on spectra arising from additional minor species could not be excluded, but the absence of such NMR spectral signatures substantiates the assumption that these forms are negligible in our experimental condi- tions. Finally, the characterization of unambiguous inter-GSH unit cross-peaks was also in accordance with the hypothesis of the predominance of the dimeric form in solution. On the other hand, interproton distance variations observed after pH changes may result from local conformational changes in the dimeric Cd(GS) 2 complex. 88% 12% 66% pH 6.4 pH 7.2 Cd charge = 1.0 Cd charge = 0.0 71% 25% 59% Fig. 3. Representation of the best 3D con- formational families obtained for Cd(GS) 2 NMR-refined structures at pH = 6.4 and pH = 7.2. The first two rows present two different rotated views of the major confor- mations (population in %) obtained for the Cd(GS) 2 complex model refinement at both pH 6.4 and 7.2 and using noncharged cadmium. The bottom row presents two views of the major conformation of refine- ments carried out with a +1 charged cadmium. In these structures, the sphere of co-ordination of cadmium (green sphere) is completed with two water molecules in the case of the Cd(GS) 2 models with noncharged metal. For models with charged cadmium there is one water molecule in the case of pH = 6.4 (bottom left) and no water molecule for pH = 7.2 (bottom right). Cadmium–glutathione complex in solution O. Delalande et al. 5090 FEBS Journal 277 (2010) 5086–5096 ª 2010 The Authors Journal compilation ª 2010 FEBS Other cadmium sulfur–metabolite complexes NMR experiments performed on complexation of cad- mium with other sulfur metabolites revealed some characteristics of their chelation with glutathione. First, at biologically relevant concentrations, GSSG does not chelate cadmium, demonstrating that the thiol group is essential. Second, a-GSH and c-GSH have different co-ordination modes with cadmium (1 : 1 stoichiometry for Cd ⁄ a-GSH with implication of glutamate and 1 : 2 stoichiometry for Cd ⁄ c-GSH) showing that c-glutamate is not implicated in the che- lation of GSH. Third, GSH and c-Glu-Cys have the same co-ordination mode for cadmium with nearly the same affinity, indicating that the carboxylate group of glycine is not a key cadmium ligand. Finally, NMR experiments demonstrated unambiguously that the chelation is a spontaneous and rapid (millisecond time scale) phenomenon. Dynamic co-ordination effects In the complex formed in aqueous solution with both glutathione and c-Glu-Cys at biologically relevant con- centrations and pH, cadmium is mainly linked by thiols from two distinct GSH or c-Glu-Cys units. Cysteine sulfur affinity for cadmium provides a strong anchoring site for glutathione derivatives [8–10,13]. Nevertheless, we observed during our 14 N-GSH versus 15 N-GSH com- petition experiments, a significant lability of cadmium, suggesting that we need to reconsider the strength of cadmium chelation by glutathione in a biological con- text. Based on the disappearance of the 1 H- 15 N cysteine resonance, this exchange rate typically occurs in the millisecond time range. This phenomenon could explain our inability to directly observe the 113 Cd resonance at high magnetic field (data not shown). An obvious con- clusion of the in vitro part of the present study is that the formation of Cd(GS) 2 complexes is spontane- ous and rapid in the tested conditions, which were close to physiological (ambient temperature, pH tested from 5.6 to 7.2, GSH concentration in the millimolar range). Consequently, it is unlikely that glutathione S-transfer- ases are required to catalyse complex formation [24–26]. In our structural models, cadmium always has a tet- rahedral co-ordination sphere in which two ligands are thiolate groups. Precipitation observations and NMR hydrogen exchange data between amide protons of GSH and bulk water show that cadmium ligation by nitrogen (amino group of glutamic acid or amide of cysteine) does not seem to occur, conversely to what has been previously proposed for cadmium [15] as derived from zinc studies [35]. This was confirmed by numerical simulations, which never led to a structure where nitrogen was involved in cadmium complexation. Moreover, it is in agreement with the pKa measured for the amino group of the N-terminal c-glutamate resi- due (9.42–9.48 from references [14,16]). In most of our structural models, metal completes its co-ordination with two water molecules. Furthermore, at neutral pH, for the best calculated NMR structures, no carboxylate group is involved. Our results demonstrate that the complexation of cadmium by glutathione primarily involves the deprotonated sulfhydryl groups from cysteine residues and two water molecules. Dimerization effects Although cadmium is only complexed through the thio- late groups, the relevance of glutathione for cadmium Table 1. NMR interproton distances. Distances were calculated from build-up curves measured at different h angles for oxidized glutathione GSSG (pH = 7.0) and Cd(c-GS) 2 complexes at pH = 6.4 and 7.2, respectively. Glutathione state GSSG pH = 7.0 Cd(c-GS) 2 pH = 6.4 Cd(c-GS) 2 pH = 7.2 Interproton distances r (A ˚ ) ± 0.7 r (A ˚ ) ± 0.2 r (A ˚ ) ± 0.3 NH (CYS) – b (GLU) 5.2 4.2 4.3 NH (CYS) – c (GLU) 5.0 2.5 2.5 NH (CYS) – b¢¢ (CYS) 4.6 2.3 2.3 NH (CYS) – b¢ (CYS) 5.7 2.6 2.7 NH (CYS) – a (GLU) 5.0 4.1 a 4.5 a NH (GLY) – b (GLU) 5.9 4.7 a 5.0 a NH (GLY) – c (GLU) 4.7 3.6 3.7 NH (GLY) – b¢¢ (CYS) 3.8 2.8 b 2.8 b NH (GLY) – b¢ (CYS) 3.7 2.8 b 2.8 b NH (GLY) – a (GLY) 2.9 2.8 3.0 NH (GLY) – a (CYS) 2.6 2.6 2.7 a (CYS) – b (GLU) 6.8 3.6 3.5 a (CYS) – c (GLU) 4.6 3.6 a 3.3 a a (CYS) – b¢¢ (CYS) 3.1 2.1 2.1 a (CYS) – b¢ (CYS) 3.1 2.0 2.1 a (CYS) – a (GLY) 3.5 4.0 a 3.6 a a (GLU) – b (GLU) 2.8 2.7 2.7 a (GLU) – c (GLU) 3.2 3.0 2.9 a (GLY) – b¢¢ (CYS) 3.1 Not observed a 3.2 a a (GLY) – b¢ (CYS) 3.4 Not observed Not observed b¢¢ (CYS) – b (GLU) 3.7 3.4 b 3.3 b b¢¢ (CYS) – c (GLU) 3.6 4.6 b 4.5 b b¢ (CYS) – b (GLU) 3.3 3.6 a,b 4.2 a,b b¢ (CYS) – c (GLU) 3.1 Not observed a 4.0 a,b b¢ (CYS) – b¢¢ (CYS) 1.7 1.8 1.8 b (GLU) – c (GLU) 2.5 2.6 2.5 a Significant distance differences when pH varied from 6.4 to 7.2 for Cd(GS) 2 . b Inter-GSH unit distance for the Cd(c-GS) 2 complexes. O. Delalande et al. Cadmium–glutathione complex in solution FEBS Journal 277 (2010) 5086–5096 ª 2010 The Authors Journal compilation ª 2010 FEBS 5091 detoxification clearly depends on the whole structure and not just the co-ordination modes. Indeed, strong interactions between two GSH units are directly observed on the off-resonance ROESY spectra, leading to clear structural constraints. Based on most struc- tural models of the dimer complex, the driving force for these peptide interactions seems to involve gluta- mate side chains of both GSH units, which form a hydrophobic core and electrostatic interactions between glycine and glutamate side chains. Under these conditions, the atypical c-configuration of the N-terminal glutamate seems to lock the structure of the Cd(GS) 2 complex and decrease the accessibility to the metal on one side. This breaks the symmetry of the complex, explaining 1D NMR spectral modifications after metal addition. Off-resonance ROESY experiments also provided a reliable description of flexibility occurring in the com- plex. Pairwise correlation times collected for Cd(GS) 2 complexes are much longer (about 1.5 ns) than those derived from GSSG (0.4 ns). Even if this is partially expected due to the increase in the relative molecular mass, the observed difference, almost a factor of 4, cannot be ascribed to this sole effect. This result thus reveals the appearance of a significant structural rigidi- fication after metal co-ordination. Furthermore, local correlation times also confirmed that the side chain of glutamate is more rigid in Cd(GS) 2 compared with GSSG, substantiating the previous comment on the importance of the GSH side chains in dimer stabilization. Cadmium–glutathione complex and the detoxification process The protonation state of the cadmium–glutathione com- plex is strongly dependent on the pH, which can signifi- cantly differ in the different subcellular compartments. Intracellular pH values are in the range from 6.5 to 7.2 in the cytosol and from 6.0 to 6.5 in the vacuole [36–38]. The complex should thus be stabilized in the cytosol, favouring specific recognition and efficient transport by Ycf1p. In vacuolar acidic conditions, the equilibria should be displaced to protonated forms with enhance- ment of inter-GSH interactions and destabilization of Cd(GS) 2 leading to possible ligand substitution. In this schema, thiolate reprotonation could be the first chemi- cal event in the cadmium releasing process by glutathi- one and so a key step in the detoxification process. The competition experiment showing similar effi- ciency in the formation of Cd(GS) 2 and Cd(c-Glu- Cys) 2 complexes suggests that the latter complex can also be formed in vivo,asc-Glu-Cys pools can reach high concentrations in the range of GSH levels following cadmium exposure [3]. In addition, the het- erologous complex involving the two metabolites Cd(GS)(c-Glu-Cys) is also expected. Interestingly the mutant strain Dgsh2, devoid of glutathione synthase activity and unable to produce glutathione, accumu- lates c-Glu-Cys at high intracellular levels (Fig. 4). This strain has a high chelating capacity, demon- strated by a global level of free thiols (GSH + c-Glu-Cys) higher than the wild-type (Fig. 4A). In addition, although our data indicate that Cd(c-Glu- Cys) 2 complexes are efficiently formed, this strain was shown to be hypersensitive to cadmium [39]. This phenotype suggests an impaired detoxification of cadmium in this strain due to a decreased rate of transport of Cd(c -Glu-Cys) 2 compared with Cd(GS) 2 complexes. This defect may concern the transport into the vacuole through Ycf1 or the export outside the cell through Yor1 or both. Using wild-type cells labelled with 35 S-GSH, we observed that the total export of glutathione [including free GSH and Cd(GS) 2 complexes] outside the cells is not significant (5.9–8.7% in cells treated for 3 h with 0.1 mm cad- mium compared with 4.4% in untreated cells; Table 2). This very low level of Cd(GS) 2 export is consistent with the very slight cadmium-sensitive phe- notype of the yor1D strain [27]. Thus, considering the low contribution of Cd(GS) 2 complex export to cad- mium resistance, we assume that the gsh2D pheno- type is caused by a low efficiency transport into the vacuole of Cd(c-Glu-Cys) 2 compared with Cd(GS) 2 complexes. Consistent with this interpretation is the observation that, even under standard conditions, c-Glu-Cys is far less efficiently transported than GSH into purified vacu oles overexpressing YCF1 (M. Lazard γ -Glu-Cys 0 5 10 15 20 Concentration (mM) WT Dgsh2 Concentration (mM) GSH 0 5 10 15 WT Dgsh2 Dgsh2 Cysteine γ -glutamyl-cysteine Glutathione Gsh1 Gsh2 Glutamate Glycine A B Fig. 4. c-Glu-Cys concentration is strongly increased in Dgsh2 cells. (A) Wild-type and Dgsh2 cells grown in minimum medium supple- mented with 400 l M glutathione were treated with 200 lM cad- mium for 3 h. The intracellular metabolites were extracted and analysed by LC ⁄ MS as previously described [3]. (B) Representation of the glutathione biosynthesis pathway. Gsh1, c-glutamyl-cysteine synthetase; Gsh2, glutathione synthetase. Cadmium–glutathione complex in solution O. Delalande et al. 5092 FEBS Journal 277 (2010) 5086–5096 ª 2010 The Authors Journal compilation ª 2010 FEBS & P. Plateau, personal communication). The absence of cadmium co-o rdination vi a c arboxy late gr oups of glycine residues in structur al models supports the idea that detoxification differences observed for GSH and c-Glu-Cys only occur at the recognition step of cad- mium–metabolite c omplexes by the t ra nsporter. We thus sugg est that the gl yci ne residue may b e involved in metal-complex recognition by the Ycf1p transporter and no t in direct interaction with cadmium. The transporter Ycf1 is a key element in cadmium detoxification, as shown by the cadmium-sensitive phenotype of the ycf1D mutant strain and the cad- mium-resistant phenotype of strains overexpressing YCF1 [40]. Our data suggest that under physiological conditions, the formation of Cd(GS) 2 complexes is spontaneous and should not constitute a bottleneck in the detoxification process. The next steps, transport into the vacuole and the metabolism of the complexes in the vacuole, remain to be fully understood. Experimental procedures Strains and culture conditions The Saccharomyces cerevisiae strain used for the production of 15 N-GSH was S288C (Mata SUC2 mal mel gal2 CUP1). The strain used for the production of 15 N-c-Glu-Cys was Dgsh2 previously constructed in the BY4742 genetic back- ground (MATa, ura3D0, his3D1, lys2D0, leu2D0) [41] by EUROSCARF (Intitute of Molecular Biosciences, Johann Wolfgang Goethe-University Frankfurt, Germany). This mutant has the KanMX4 marker inserted into the GSH2 locus. Cells were grown at 30 °C in minimal yeast nitrogen base medium (0.67%) supplemented with 2% glucose as a carbon source and with auxotrophic requirements (uracil, histidine, lysine, leucine and 30 lm glutathione) when nec- essary (strain Dgsh2). The standard yeast nitrogen base medium contains 30 m m 14 N-ammonium sulfate. For 15 N labelling, 30 mm 14 N-ammonium sulfate was replaced by 10 mm 15 N-ammonium sulfate (Eurisotop, Gif-sur-Yvette, France) as the sole source of nitrogen. Preparation of 15 N-enriched metabolites After growth for at least 25 generations in 15 N-ammonium sulfate, the cell culture (400 mL corresponding to  10 10 cells) was treated with 50 lm Cd 2+ to induce an overproduction of 15 N-GSH [3]. After 4 h of treatment, the cells were collected by centrifugation, washed quickly with cold water and resuspended in 3 mL of 0.1% perchloric acid. Cells were transferred to boiling water for 5 min, cen- trifuged and the supernatant was collected. This extract contained soluble yeast metabolites, including 15 N-GSH (S288C strain) and 15 N- c-Glu-Cys (Dgsh2 strain). The extracts containing 15 N-GSH and 15 N-c-Glu-Cys were purified on a carbohydrate analysis column (4.6 · 250 mm, 5 lm) from Waters (Saint-Quentin en Yvelines, France). Chromatographic separations were performed using a Surveyor pump and a Surveyor auto- sampler (ThermoFisher Scientifics, Les Ulis, France), under isocratic conditions with a flow rate of 0.8 mLÆmin )1 . The mobile phase consisted of water containing acetic acid at 0.4%. The effluent from the liquid chromatography was split by a factor of 1 ⁄ 20 before its introduction into the MS. ESI MS was performed using an LCQ-Duo ion trap MS fitted with an electrospray source (ThermoFisher Scientifics) operated in the positive mode. The mass spec- trometer was operated with the capillary temperature at 250 °C, sheath gas at 80 (arbitrary units) and the auxiliary gas at 20 (arbitrary units). The target was fixed at 2 · 10 7 ions and the automatic gain control was turned on. The electrospray voltage was 4.5 kV, the capillary voltage 10.6 V and the tube lens offset )6 V. The injection time was 50 ms. MS were recorded at unit mass resolution with- out in-source fragmentation using the single ion recording detection mode. The signals for 15 N-GSH and 15 N- c-Glu- Cys were monitored at m ⁄ z 311 and 253, with retention times of 23 and 40 min, respectively. The fractions corre- sponding to these retention time ranges were collected and finally lyophilized before NMR experiments. Sample preparation In the case of samples used for distance extraction, chelation or competition reaction experiments, GSH or GSSG were dissolved in 500 mL (90% H 2 O 10% D 2 O) resulting in 1 mm minimal ionic strength samples, with a final 1 : 2 Cd ⁄ GSH stoichiometry. To decrease oxidation processes, all samples were sealed after bubbling with dry nitrogen gas for a few minutes. Classical peptides were purchased from Sigma-Aldrich (St Louis, MO, USA) and a-GSH was synthesized and purified for NMR quality by Eurogentec (Seraing, Belgium). NMR spectroscopy All NMR experiments were performed on Bruker Advance DRX spectrometers (Bruker, Ettlingen, Germany). 1D and 2D 1 H spectra in H 2 O were recorded at 500 MHz by using a Watergate [42] or an excitation sculpting sequence [43] to suppress the water signal. Peak assignments were carried out using classical techniques, in particular for proton Table 2. Total export of 35 S-glutathione by wild-type cells. The values reported in the Table are the ratio S ⁄ T (see Radioactive experiments section in Experimental procedures). Strain No treatment 100 l M cadmium S288C 4.4 ± 0.3% 5.9 ± 0.8% BY4742 4.4 ± 0.8% 8.7 ± 0.3% O. Delalande et al. Cadmium–glutathione complex in solution FEBS Journal 277 (2010) 5086–5096 ª 2010 The Authors Journal compilation ª 2010 FEBS 5093 resonances through TOCSY and off-resonance ROESY experiments. Our attribution agreed with those obtained in other studies [16,44]. l-glycine at a final concentration of 2.5 mm was added to the sample before recording 1D NMR spectra because the CH 2 resonance was not affected by the addition of cadmium and this resonance did not overlap with the other signals (see Fig. 1). It was used as an internal reference for peak integration and species quantification. Proton–proton distances were extracted from off-reso- nance ROESY build-up curves using a procedure already described [45]. The off-resonance ROESY pulse sequence [46] was adapted for the excitation sculpting water suppres- sion method. Seven h angles between the effective and static magnetic field directions (5, 15, 25, 35, 45 and 54.7°) and six mixing times (25, 50, 75, 100, 150 and 200 ms) were used. For metal-reduced glutathione, the spectra were recorded at three different pH values: 5.6, 6.4 and 7.2. For the samples with c-Glu-Cys and metal-free oxidized gluta- thione, the experiments were performed at pH = 7.0. All off-resonance ROESY spectra were collected at 500 MHz at 274.3K with TXI or BBI probes. 1 H, 15 N-heteronuclear single quantum coherence experi- ments were carried out using gradient coherence selection and sensitivity enhancement. Backbone 15 N amide reso- nances were observed on a 600 MHz spectrometer equipped with a TCI cryoprobe (Bruker). Natural abundance 13 C NMR experiments were also performed, using heteronucle- ar multiple bond correlation sequences and a TCI cryo- probe. All chemical shifts for 1 H were referenced to an internal TSP signal. Molecular modelling Molecular mechanics calculations and molecular dynamic simulation methods were used for model construction using the amber 9 suite programs. Parameters for the c-gluta- mate residue were developed from the Gaussian03 DFT charge calculations method and adapted to the Parm99 force-field [47] using the Resp module and a standard charge fitting protocol. Both protonated and nonprotonat- ed states for the amino group of the c-glutamate residue were implemented and simulated. Cadmium was considered as a hard sphere with a modulated charge varying between 0.0 and +2.0 and a Cd-S distance of 2.46 A ˚ [8]. Other main parameters for angles and dihedrals were adapted from Amber force-field data previously depicted for zinc ion in the four-cysteine tetrahedral environment [8,48]. An explicit solvation model (TIP3P water model and +1.0 dielectric constant) was used in all simulations. Structure refinements were performed using NMR interproton dis- tances as restraints implemented as harmonic functions into a simulated annealing protocol with 5000 final structures collected. Free and restraint molecular dynamic simulations were used to analyse characteristic key distances for co-ordination-type discrimination. Cd-N (2.3 ± 0.1 A ˚ [8]) or C-O (2.2 ± 0.1 A ˚ [49]) restraint distances were imposed between cadmium and potential ligand atoms during the production period. Structures obtained from simulated annealing calculations were sorted and divided into homog- enous conformational groups leading to the best NMR refined models. Radioactive experiments Cells (3 ml at D = 0.4) grown in minimum medium were labelled with 2 lCi of 35 S-GSH (PerkinElmer) for 40 min at 30 °C. Cells were washed and re-suspended in the same medium (with or without 100 lm cadmium). Total 35 S-GSH pools were counted (T). After 3h incubation, the cultures were centrifuged and the amount of radioactivity present in the supernatant was measured (S). Acknowledgements This work was supported by the Commissariat a ` l’Energie Atomique (grant from the Programme de Toxicologie Nucle ´ aire Environnementale). We thank R. Genet for kindly providing 15 N-ammonium sulfate. We thank Dr Carl Mann for careful reading and helpful comments on the manuscript. References 1 Tamas M, Labarre J, Toledano MB & Wysocki R (2006) Mechanisms of Toxic Metal Tolerance in Yeast. Springer, Berlin. 2 Lu SC (1999) Regulation of hepatic glutathione synthe- sis: current concepts and controversies. FASEB J 13, 1169–1183. 3 Lafaye A, Junot C, Pereira Y, Lagniel G, Tabet JC, Ezan E & Labarre J (2005) Combined proteome and metabolite-profiling analyses reveal surprising insights into yeast sulfur metabolism. J Biol Chem 280, 24723– 24730. 4 Singhal RK, Anderson ME & Meister A (1987) Gluta- thione, a first line of defense against cadmium toxicity. FASEB J 1, 220–223. 5 Muller EG (1996) A glutathione reductase mutant of yeast accumulates high levels of oxidized glutathione and requires thioredoxin for growth. Mol Biol Cell 7, 1805–1813. 6 Grant CM, Perrone G & Dawes IW (1998) Glutathione and catalase provide overlapping defenses for protection against hydrogen peroxide in the yeast Saccharomyces cerevisiae. Biochem Biophys Res Commun 253, 893–898. 7 Leverrier P, Montigny C, Garrigos M & Champeil P (2007) Metal binding to ligands: cadmium complexes with glutathione revisited. Anal Biochem 371, 215–228. Cadmium–glutathione complex in solution O. Delalande et al. 5094 FEBS Journal 277 (2010) 5086–5096 ª 2010 The Authors Journal compilation ª 2010 FEBS 8 Zaima H, Ueyama N, Adachi H & Nakamura A (1995) 1H-, 13C-, and 113Cd-NMR study of the Cd(II) complex of a blocked peptide, Z-Cys-Ala-Pro-His-OMe, in organic solvents. Biopolymers 35 , 319–329. 9 Cherifi K, Decock-Le Reverend B, Varnagy K, Kiss T, Sovago I, Locheux C & Kozlowski H (1990) Transition metal complexes of L-cysteine containing di- and tripep- tides. J Inorg Biochem 38, 69–80. 10 Kozlowski H, Urbanska J, Sovago I, Varnagy K, Kiss A, Spychala J & Cherifi K (1990) Cadmium ion interaction with sulphur containing amino acid and peptide ligands. Polyhedron 9, 831–837. 11 Li NC & Manning RA (1955) Some metal complexes of sulfur-containing amino acids. J Am Chem Soc 77 , 5225–5228. 12 Birgerssonn B, Carter RE & Drakenberg T (1977) A cadmium-113 NMR study of cadmium-glutathione complexes. J Magn Reson 28, 299–302. 13 Fuhr BJ & Rabenstein DL (1973) Nuclear magnetic resonance studies of the solution chemistry of metal complexes. IX. The binding of cadmium, zinc, lead, and mercury by glutathione. J Am Chem Soc 95, 6944– 6950. 14 Perrin DD & Watt AE (1971) Complex formation of zinc and cadmium with glutathione. Biochim Biophys Acta 230, 96–104. 15 Li ZS, Lu YP, Zhen RG, Szczypka M, Thiele DJ & Rea PA (1997) A new pathway for vacuolar cadmium sequestration in Saccharomyces cerevisiae: YCF1-cata- lyzed transport of bis(glutathionato)cadmium. Proc Natl Acad Sci USA 94, 42–47. 16 Kadima W & Rabenstein DL (1990) Nuclear magnetic resonance studies of the solution chemistry of metal complexes. 26. Mixed ligand complexes of cadmium, nitrilotriacetic acid, glutathione, and related ligands. J Inorg Biochem 38, 277–288. 17 Hemmingsen L, Damblon C, Antony J, Jensen M, Adolph HW, Wommer S, Roberts GC & Bauer R (2001) Dynamics of mononuclear cadmium beta-lac- tamase revealed by the combination of NMR and PAC spectroscopy. J Am Chem Soc 123, 10329–10335. 18 Goodfellow BJ, Rusnak F, Moura I, Domke T & Moura JJ (1998) NMR determination of the global structure of the 113Cd derivative of desulforedoxin: investigation of the hydrogen bonding pattern at the metal center. Protein Sci 7, 928–937. 19 Pan T, Freedman LP & Coleman JE (1990) Cadmium- 113 NMR studies of the DNA binding domain of the mammalian glucocorticoid receptor. Biochemistry 29, 9218–9225. 20 Hemmingsen L, Olsen L, Antony J & Sauer SP (2004) First principle calculations of (113)Cd chemical shifts for proteins and model systems. J Biol Inorg Chem 9, 591–599. 21 Kidambi SS & Ramamoorthy A (2003) Characteriza- tion of metal centers in bioinorganic complexes using ab initio calculations of 113Cd chemical shifts. Inorg Chem 42, 2200–2202. 22 Abrahams IL & Garner CD (1985) Nature of the cad- mium sites in rat liver metallothionein 1 from Cd K- Edge EXAFS. J Am Chem Soc 107, 4596–4597. 23 Kadima W & Rabenstein DL (1990) A quantitative study of the complexation of cadmium in hemolyzed human erythrocytes by 1H NMR spectroscopy. J Inorg Biochem 40, 141–149. 24 Rai R, Tate JJ & Cooper TG (2003) Ure2, a prion precur- sor with homology to glutathione S-transferase, protects Saccharomyces cerevisiae cells from heavy metal ion and oxidant toxicity. J Biol Chem 278, 12826–12833. 25 Adamis PD, Gomes DS, Pinto ML, Panek AD & Eleutherio EC (2004) The role of glutathione transfer- ases in cadmium stress. Toxicol Lett 154, 81–88. 26 Lewinska A & Bartosz G (2007) Protection of yeast lacking the Ure2 protein against the toxicity of heavy metals and hydroperoxides by antioxidants. Free Radic Res 41, 580–590. 27 Nagy Z, Montigny C, Leverrier P, Yeh S, Goffeau A, Garrigos M & Falson P (2006) Role of the yeast ABC transporter Yor1p in cadmium detoxification. Biochimie 88, 1665–1671. 28 Mendoza-Cozatl D, Loza-Tavera H, Hernandez-Navar- ro A & Moreno-Sanchez R (2005) Sulfur assimilation and glutathione metabolism under cadmium stress in yeast, protists and plants. FEMS Microbiol Rev 29, 653–671. 29 Adle DJ, Sinani D, Kim H & Lee J (2007) A cadmium- transporting P1B-type ATPase in yeast Saccharomyces cerevisiae. J Biol Chem 282, 947–955. 30 Dameron CT, Smith BR & Winge DR (1989) Glutathi- one-coated cadmium-sulfide crystallites in Candida glab- rata. J Biol Chem 264, 17355–17360. 31 Williams DR, Corrie AM & Walker MD (1976) Ther- modynamic considerations in co-ordination, Part XXII. Sequestering ligands for improving the treatment of plumbism and cadmiumism. J Chem Soc Dalton Trans, 1012–1015. 32 Krezel A & Bal W (1999) Coordination chemistry of glutathione. Acta Biochim Pol 46, 567–580. 33 Desvaux H, Berthault P, Birlirakis N & Goldman M (1994) Off-resonance ROESY for the study of dynamic processes. J Magn Reson A 108, 219–229. 34 Esposito V, Das R & Melacini G (2005) Mapping poly- peptide self-recognition through (1)H off-resonance relaxation. J Am Chem Soc 127, 9358–9359. 35 Martin RB & Edsall JT (1959) The association of divalent cations with glutathione. J Am Chem Soc 81, 4044–4047. 36 Gillies RJ, Ugurbil K, den Hollander JA & Shulman RG (1981) 31P NMR studies of intracellular pH and O. Delalande et al. Cadmium–glutathione complex in solution FEBS Journal 277 (2010) 5086–5096 ª 2010 The Authors Journal compilation ª 2010 FEBS 5095 [...].. .Cadmium glutathione complex in solution 37 38 39 40 41 42 43 44 45 46 47 O Delalande et al phosphate metabolism during cell division cycle of Saccharomyces cerevisiae Proc Natl Acad Sci USA 78, 212 5–2 129 Preston RA, Murphy RF & Jones EW (1989) Assay of vacuolar pH in yeast and identification of acidificationdefective mutants Proc Natl Acad Sci USA 86, 702 7– 7031 Chattopadhyay S,... biological molecules J Comput Chem 21, 104 9–1 074 48 Stote RH & Karplus M (1995) Zinc binding in proteins and solution: a simple but accurate nonbonded representation Proteins 23, 1 2–3 1 49 Siripornadulsil S, Traina S, Verma DP & Sayre RT (2002) Molecular mechanisms of proline-mediated tolerance to toxic heavy metals in transgenic microalgae Plant Cell 14, 283 7–2 847 Supporting information The following... gene disruption and other applications Yeast 14, 11 5–1 32 Piotto M, Saudek V & Sklenar V (1992) Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions J Biomol NMR 2, 66 1–6 65 Hwang TL & Shaka AJ (1995) Water suppression that works Excitation sculpting using arbitrary wave-forms and pulsed-field gradients J Magn Reson 112, 27 5– 279 Rabenstein DL, Isab AA, Kadima W & Mohanakrishnan... resonance study of the interaction of cadmium with human erythrocytes Biochim Biophys Acta 762, 53 1–5 41 Desvaux H, Berthault P & Birlirakis N (1995) Dipolar spectral densities from off-resonance 1H NMR relaxation measurements Chem Phys Lett 233, 54 5–5 49 Desvaux H, Berthault P, Birlirakis N, Goldman M & Piotto M (1995) Improved versions of off-resonance ROESY J Magn Reson 113, 4 7–5 2 Wang J, Cieplak P & Kollman... 641 8–6 423 Jin YH, Dunlap PE, McBride SJ, Al-Refai H, Bushel PR & Freedman JH (2008) Global transcriptome and deletome profiles of yeast exposed to transition metals PLoS Genet 4, e1000053 Wemmie JA, Szczypka MS, Thiele DJ & Moye-Rowley WS (1994) Cadmium tolerance mediated by the yeast AP-1 protein requires the presence of an ATP-binding cassette transporter encoding gene, YCF1 J Biol Chem 269, 3259 2–3 2597... GSH in the presence of increased quantities of cadmium Fig S2 An expanded contour plot of the 2D TOCSY spectrum Fig S3 1D NMR spectra of GSH in the presence or not of cadmium at different pH levels Fig S4 Proton exchange of the Cd(GS)2 complex in H2O Table S1 Proportion of GSH precipitated during CdGSH2 complex preparation Table S2 Inter- and intraproton–proton distances whether dimeric or monomeric... to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 277 (2010) 508 6–5 096 ª 2010 The Authors Journal compilation . Cadmium – glutathione solution structures provide new insights into heavy metal detoxification Olivier Delalande 1, *,. chains in dimer stabilization. Cadmium glutathione complex and the detoxification process The protonation state of the cadmium glutathione com- plex is strongly