XAS Study of Arsenic Coordination in

Environ. Sci. Technol. 2008, 42, 5342–5347

XAS Study of Arsenic Coordination in Euglena gracilis Exposed to Arsenite J E N N Y F E R M I O T , * ,† G U I L L A U M E M O R I N , † ´ RIEL SKOURI-PANET,† FE ´ LINE FE ´ RARD,† EMMANUEL AUBRY,‡ CE ¨ L BRIAND,§ YUHENG WANG,† JOE GEORGES ONA-NGUEMA,† FRANC ¸ OIS GUYOT,† AND G O R D O N E . B R O W N |,⊥ ´ ´ Institut de Mineralogie et de Physique des Milieux Condenses ´ Paris 6 et Paris 7, et (IMPMC), UMR 7590, CNRS, Universites IPGP., 140, rue de Lourmel. 75 015 Paris, France, ´ Biogeochimie et Ecologie des Milieux Continentaux (Bioemco), UMR 7618 Universite´ Paris 6, INRA, INAPG, CNRS, ENS, ´ ENSCP, Case 120. Tour 56, couloir 56-66. 4e`me etage. 4 place Jussieu. 75252 Paris cedex 05, France, Laboratoire d’Electrophysiologie des Membranes, EA 3514, Universite´ Paris 7, 4, place Jussieu. 75 252 Paris cedex 05, France, Surface and Aqueous Geochemistry Group, Department of Geological and Environmental Sciences, Stanford University, Stanford, California 94305-2115, and Stanford Synchrotron Radiation Laboratory, SLAC, Menlo Park, California 94025

Received December 8, 2007. Revised manuscript received April 2, 2008. Accepted April 10, 2008.

Among the few eukaryotes adapted to the extreme conditions prevailing in acid mine drainage, Euglenae are ubiquitous in these metal(loid)-impacted environments, where they can be exposed to As(III) concentrations up to a few hundreds of mg · L-1. In order to evaluate their resistance to this toxic metalloid and to identify associated detoxification mechanisms, we investigated arsenic coordination in the model photosynthetic protozoan, Euglena gracilis, cultured at pH 3.2 and exposed to As(III) at concentrations ranging from 10 to 500 mg · L-1. E. gracilis is shown to tolerate As(III) concentrations up to 200 mg · L-1, without accumulating this metalloid. X-ray absorption spectroscopy at the As K-edge shows that, in the cells, arsenic mainly binds to sulfur ligands, likely in the form of arsenic-trisglutathione (As-(GS)3) or arsenic-phytochelatin (As-PC) complexes, and to a much lesser extent, to carbon ligands, presumably in the form of methylated As(III)-compounds. The key role of the glutathione pathway in As(III) detoxification is confirmed by the lower growth rate of E. gracilis cultures exposed to arsenic, in the presence of buthionine sulfoximine, an inhibitor of glutathione synthesis. This study provides the first investigation at the molecular scale of intracellular arsenic speciation in E. gracilis and thus contributes to the understanding of arsenic detoxification mechanisms in a eukaryotic microorganism under extreme acid mine drainage conditions. * Corresponding author: [email protected]; Tel: 0033144279832; Fax: 0033144273785 † Institut de Mine´ralogie et de Physique des Milieux Condense´s (IMPMC). ‡ Bioge´ochimie et Ecologie des Milieux Continentaux (Bioemco). § Laboratoire d’Electrophysiologie des Membranes. | Department of Geological and Environmental Sciences. ⊥ Stanford Synchrotron Radiation Laboratory. 5342

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Introduction Acid mine drainages, which result from the weathering of sulfide mine wastes, usually exhibit high metal(loid) concentrations. In particular, (bio)leaching of asenic-bearing sulfide minerals (e.g., arsenopyrite) leads to extreme dissolved concentrations of the reduced form of arsenic, As(III) (1, 2). Under the acidic conditions prevailing in these environments, As(III) is present in a highly toxic and acute form, arsenious acid H3AsIIIO3, which is the common dissolved form of arsenite below pH 9.23 (i.e., the first pKa of arsenious acid (3)), and which represents a potential threat for endogenous organisms. Indeed, plants and microorganisms observed in such impacted sites have to cope with extremely elevated arsenic concentrations up to several hundreds of mg · L-1 (2). For instance, Acidithiobacillus ferrooxidans, widespread in AMD environments, is known to be resistant to arsenite by extruding As(III) via an ATP-driven arsenite efflux pump (4–7). In eukaryotes, arsenite detoxification generally relies either on vacuolar storage (8, 9) and/or exportation (8–10) of As(III). In most cases, arsenite-glutathione complexes play a key role in these metabolic pathways (10–13). In spite of these important findings, arsenic resistance mechanisms are still poorly documented in eukaryotic microorganisms observed in extreme AMD environments. Among the few eukaryotes adapted to acidic conditions and high metalloid concentrations, Euglenae are photosynthetic protozoans frequently exposed to elevated As(III) concentrations in AMD. In particular, Euglena mutabilis, phylogenetically close to Euglena gracilis (14), is widespread in AMD environments (15), and was shown to be exposed to arsenite concentrations up to 250 mg · L-1 in the Carnoule`s AMD (16). Although E. gracilis is known to be tolerant to various heavy metals, such as Cd (17, 18), Cr (19), Cu, and Zn (20), resistance mechanisms of Euglenae to As(III) have never been investigated before. Until now, only one study on E. mutabilis has been conducted that evaluated the potential accumulation of arsenic in these organisms, using an indirect chemical technique (16). In this context, we have investigated arsenic speciation in E. gracilis, in order to assess the mechanisms of arsenic detoxification in these organisms, possibly accounting for their adaptation to extreme AMD conditions. We present here the results of an X-ray absorption spectroscopy (XAS) study of E. gracilis cultured under acidic conditions, in the presence of dissolved arsenite, which is the common highly toxic form of arsenic in acid mine drainage.

Materials and Methods Cell Cultures. Chemicals used for culture medium preparation were all of reagent grade from Sigma-Aldrich, but FeCl3 and NaAsO2 from Fluka-Chemika. Euglena gracilis Z (n°1224-5d, Cambridge) was cultured at ambient temperature, under permanent light exposure, in a pH 3.2 culture medium composed of KH2PO4 (0.5 g.L-1), MgSO4, 7H2O (0.5 g.L-1), CaCl2, 2H2O (0.26 g.L-1), (NH4)2HPO4 (0.5 g.L-1), and a complement of vitamins, zinc (as ZnSO4), iron (as FeCl3), and manganese (as MnSO4) (21). Ethanol was supplied as a carbon source. In test cultures and abiotic controls, arsenic was added as NaAsIIIO2, at the following concentrations: 10, 20, 50, 100, 200, and 500 mg · L-1. Growth kinetics of the cultures was monitored by measuring the absorbance of the cultures at 640 nm for 8 days. Standard deviations were calculated from two independent sets of cultures. Abiotic controls were prepared by adding As(III) at 10 to 200 mg · L-1 10.1021/es703072d CCC: $40.75

 2008 American Chemical Society

Published on Web 06/10/2008

in the culture medium. To evaluate the potential role of DLbuthionine (S,R)-sulfoximine (BSO) on arsenic detoxification, additional cultures of E. gracilis were grown under conditions similar to those above, but in the presence of 0.5 mM BSO. Sample Preparation. Cells were harvested by centrifugation (6500 rpm, 10 min) after 10 days of culture, rinsed twice in MilliQ water, dried at ambient temperature under vacuum, and ground in an agate mortar. Powders were stored under nitrogen atmosphere before XAS analysis. For total arsenic analysis, 10-20 mg of dry powders were weighed (precision: 0.1 mg) and batch digested in 1 mL 15 M HNO3 at 100 °C for 24 h, sonicated for 5 min and diluted to 5 mL in MilliQ water. Supernatants of the 10 day old cultures and of the abiotic controls were 0.22 µm-filtered. A first fraction of filtered supernatants was stored at -20 °C until XAS analysis. For one of the samples (Eg-S-As10), glycerol was added to the solution at 10% to avoid solute segregation effect (22) during XAS data collection at 10 K. A second fraction was acidified with 1 M HNO3 for analysis of total As concentration in solution. A third faction was used to separate As(III) and As(V) on an anionic resin (Varian Bondelut JR-SAX 500 mg) after a method modified from Ficklin (1982, ref 23). Briefly, the resin was humidified with 5 mL of MilliQ water. Then, 5 mL of the 0.22 µm-filtered supernatant of the culture and 15 mL of MilliQ water were successively eluted through the resin to obtain the As(III)-extract. Finally, 15 mL of 0.12 M HCl were eluted to obtain the As(V)-extract. Arsenic Analysis in Solution. Total As concentrations in the digests, in the culture supernatants and abiotic controls, as well as in the As(III)-extracts from the culture supernatants were determined by flame atomic absorption spectrometry (Solaar S, Thermo electron corporation). Arsenic concentrations in the As(V)-extracts from the culture supernatants were measured by furnace atomic absorption spectrometry (Unicam 989 QZ, Thermo electron corporation). Model Compounds. Arsenobetaine (AB, Sigma-Aldrich) and As(III)-tris-glutathione (As-(GS)3) were used as model compounds for XAS experiments. As-(GS)3 was synthesized under dry nitrogen atmosphere by adding an excess of reduced glutathione (GSH, Sigma-Aldrich) to NaAsO2 (Fluka Chemika): 1 mg of NaAsO2 was allowed to react overnight with 100 mg of GSH, in 5 mL of degassed MilliQ water. The solution was evaporated to dryness at ambient temperature under nitrogen atmosphere and the resulting white precipitate was collected as a powder. XAS Data Collection. Data were recorded at the As K-edge (11859 eV), using a Si(220) double-crystal monochromator on beamline 11-2 at the Stanford Synchrotron Radiation Laboratory (SSRL). Data were collected in fluorescence detection mode using a 30-element Ge-array detector and 6 ∆µ Ge filters to attenuate elastic scattering. Energy resolution was around 0.4-0.5 eV, with a spot size of 500 × 250 µm2. Energy was calibrated by using a double-transmission setup in which the As K-edge spectrum of the samples and that of a reference sample (crystalline scorodite, FeAsO4 · 2H2O with an absorption maximum set at 11875.0 eV) were simultaneously recorded. In order to limit As(III) oxidation under the X-ray beam (24), all data were recorded at 10-15 K using a modified Oxford liquid He cryostat. Between 6 and 14 EXAFS scans were accumulated for each sample in order to obtain a reliable signal-to-noise ratio up to k ) 13 Å-1. Samples were automatically moved 1 mm between each EXAFS scan to avoid any As(III) oxidation during beam exposure. Nevertheless, no measurable change in the XANES spectrum was observed after two EXAFS scans recorded on the same position. XAS Data Analysis. X-ray absorption spectra were averaged using the SixPack software (25). EXAFS data were extracted using the XAFS program (26) following the procedure detailed previously (24). Radial distribution functions

FIGURE 1. Growth curves of E. gracilis cultures determined by measuring the absorbance at 640 nm. Cells were cultured at As(III) concentrations ranging from 0 (As0) to 200 mg · L-1 (As200), in the presence (BSO+) or in the absence (BSO-) of buthionine sulfoximine. Error bars were estimated from two independent sets of cultures. Growth kinetics are slowed down with increasing As(III) concentrations in the culture medium. BSO reduces the final cell densities. around the As absorber were obtained by calculating the Fourier transform (FT) of the k3χ(k) EXAFS functions using a Kaiser-Bessel window within the 2.7-13 Å-1 k-range with a Bessel weight of 2.5. Least-squares fitting of the unfiltered EXAFS functions was performed with the plane-wave formalism, using a Levenberg-Marquard minimization algorithm. Ab initio phase shifts and total amplitude functions employed in this fitting procedure were calculated with the curved-wave formalism using the ab initio FEFF 8 code (27) and the crystallographic parameters of phenyldichloroarsineBritish Anti-Lewisite (28). The fit quality was estimated within the 2.7-13 Å-1 k-range, using a reduced χ2 of the following form: χ2 ) Nind/(Nind - p)Σ(k4χ(k)exp - k4χ(k)calc)2 with Nind (the number of independent parameters) ) (2∆k∆R)/π), p the number of free fit parameters. The fitting procedure was performed on k4-weighted χ(k) functions in order to emphasize the signal at high k values, which improves the accuracy of the fitted distances for light backscattering atoms. Data are however displayed as k3-weighted χ(k) functions. For sample Eg-As10, XANES data were fit by linear lest-squares fitting (LSF) within the 11860-11890 eV energy range, using linear combinations of XANES spectra of the As-(GS)3 and AB model compounds. This LSF procedure is detailed in a previous work by Morin et al. (2003, ref (29)).

Results E. gracilis Growth in the Presence of Arsenite. Growth kinetics of E. gracilis cells exposed to dissolved As(III) concentrations ranging from 0 to 200 mg · L-1 are presented in Figure 1. In the presence of arsenite, the cell population reaches a stationary phase within 7 days and final cell density ranges from 42 to 100% of that measured in control cultures performed in the absence of arsenic. For cultures exposed to the highest As(III) concentration, 500 mg · L-1, growth is completely inhibited (data not shown). Analyses of the culture supernatants and of the aciddigested pellets (Table 1) indicate that dry cells contain 315 mg · kg-1 As at an initial As(III) concentration of 200 mg · L-1 in the culture medium. Assuming an approximate water content of 90 wt% in the cells, the arsenic concentration in the living cells would thus not exceed 31 mg · L-1, which is about 7 times lower than the arsenic concentration in the culture medium. Using a similar procedure, estimates can VOL. 42, NO. 14, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Total Arsenic Concentrations Measured by Atomic Absorption Spectrometry in Abiotic Controls, In the Filtered Supernatants and in the Dry Cell Mass of E. gracilis Cultures, With Initial As(III) Concentrations Ranging from 10 to 200 mg · L-1, after 10 Days of Culturea

a

initial [AsIII] in the culture medium, mg · L-1

total [As] in abiotic controls, mg · L-1

total [As] in the supernatant, mg · L-1

total [As] in dry cells, mg · kg-1

10 20 50 100 200

9.4 ( 0.1 20 ( 0.1 51 ( 0.1 98 ( 0.1 190 ( 0.1

9.7 ( 0.1 19.4 ( 0.1 48 ( 0.1 95 ( 0.1 194 ( 0.1

14 ( 5 17 ( 5 61 ( 5 163 ( 10 315 ( 10

All arsenic was shown to be in the As(III) state in the supernatants as well as in the dry cells.

also be drawn for the other initial arsenic concentrations, and they indicate that arsenic is not accumulated within the cells. In addition, arsenic was not detected in the cells either by EDX (Energy Dispersive X-ray Spectroscopy) performed in a TEM (Transmission Electron Microscope) on E. gracilis thin sections, or by synchrotron-based µ-XRF (micro X-ray fluorescence) analyses of single fresh cells (data not shown). Accordingly, final arsenic concentrations in the supernatants are only 3-4% lower than the initial ones. These results indicate that E. gracilis is tolerant to arsenite, for initial As(III) concentrations up to 200 mg · L-1, but does not accumulate significant amounts of arsenic. Buthionine sulfoximine (BSO) is an inhibitor of γ-glutamylcysteine-transferase, an enzyme involved in the metabolic pathway of glutathione and phytochelatins (30). In the absence of arsenic, 0.5 mM BSO has no detectable effect on the growth of E. gracilis (Figure 1). In contrast, in the presence of As(III), 0.5 mM BSO significantly decreases the final cell densities at 7 days in a dose-dependent way (Figure 1). Indeed, at 8 days the BSO treatment reduces the cell density by 15 and 50%, for initial As(III) concentrations of 100 and 200 mg · L-1, respectively. These results suggest that the metabolic pathway of glutathione is involved in arsenite detoxification in E. gracilis. Arsenic Speciation in E. gracilis Cultures. X-ray absorption near edge spectroscopy (XANES) of dry cells exhibit an absorption edge maximum at 11869.8 eV, which is characteristic of As(III) coordinated to three thiol groups, such as in AsIII-tris-glutathione (As-(GS)3) (Figure 2) (31). In cells cultivated at As(III) concentrations lower than 100 mg · L-1, we observe a shoulder at 11872.4 eV, consistent with Asmethylated compounds as arsenobetaine (AB), whose intensity rapidly decreases with increasing initial arsenite concentration in the culture medium (Figure 2). Best fits of the extended X-ray absorption fine structure (EXAFS) region of these spectra are listed in Table 2 and plotted in Figure 3. Within the range of initial arsenite concentrations used in our experiments, the first-neighbor shell around arsenic consists mainly of sulfur ligands at 2.25 ( 0.03 Å, as in As-(GS)3. In cells cultured at initial arsenite concentrations lower than 100 mg · L-1, for which a shoulder at 11872.4 eV is observed on the XANES spectrum, an additional first-neighbor shell is necessary to fit the EXAFS data. Fitting this additional contribution using an As-O pair yields an unrealistic As-O distance value around 1.9 Å, whereas using an As-C pair yields an As-C distance of 1.97 ( 0.03 Å. However, this distance is longer than the As(V)-C distance measured by EXAFS in the As(V)-methylated molecule arsenobetaine (1.91 ( 0.03 Å, Table 2) or calculated from the crystal structure of tetramethylarsonium (1.90 ( 0.02 Å; 32). In contrast, this distance is consistent with that reported from crystal structure determination of As(III)-methylated compounds (28). Therefore, our EXAFS data suggest that a minor fraction of arsenic is present as an As(III)-methylated compound, exhibiting an absorption maximum at 11872.4 5344

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FIGURE 2. X-ray absorption near-edge structure (XANES) spectra at the As Kr-edge of E. gracilis cells (Eg-As10 to Eg-As200) and culture supernatants (Eg-S-As10 to Eg-S-As200) after a 10 day exposure to As(III) (As(III) concentrations ranging from 10 (As10) to 200 mg · L-1 (As200)), compared with the spectra of the standards As(III)-tris glutathione (As-(GS)3), arsenobetaine (AB), As(III) (1 wt%) adsorbed on ferrihydrite and As(V) (1 wt%) coprecipitated with ferrihydrite.

TABLE 2. Fitting Results for Arsenic K-edge EXAFS data of 1 Week-Old E. gracilis Cells Exposed to As(III) and of the As-tris-glutathione (As-(GS)3) and Arsenobetaine (AB) Model Compoundsa sample

AB As-(GS)3 Eg-50 ppm

N ((0.3)

3.5 3.2 2.1 0.8 Eg-100 ppm 2.5 0.2 Eg-200 ppm 2.5

bond As-C As-S As-S As-C As-S As-C As-S

R (Å) σ (Å) E0 (eV) ((0.03) ((0.02) ((3) 1.91 2.25 2.26 1.96 2.26 1.98 2.25

χ2

0.05 0.05 0.05

14 13 16

32.0 20.0 33.9

0.05

15

25.9

0.05

13

40.2

a

Coordination number (N), interatomic distance (R), Debye-Waller parameter (σ), and energy offset (E0). Fit quality was estimated by a reduced χ2 parameter (see text). Standard deviations indicated under bracket are estimated from the fit of the As-tris-glutathione model compound.

eV that falls within the range of values reported for As(III)methylatedcompoundsintheavailableliterature(11870.5-11873.8 eV setting the maximum absorption energy of inorganic As(V) at 11875 eV (31)).

FIGURE 3. Unfiltered k3-weighted EXAFS data of E. gracilis cells exposed to As(III) for 10 days (Eg-As50 to Eg-As200) and references As(III)-tris glutathione (As-(GS)3) and arsenobetaine (AB). (A) Unfiltered k3χ(k) data and (B) corresponding Fourier Transforms. Dashed lines show the data and solid lines the best fits on the k3χ(k) data (see Table 2).

a rapid disappearance of the As-methylated component with increasing concentration of arsenite in the culture medium, leading to a pure As(III)-thiol component in cells cultivated at 200 mg · L-1 As(III). Given the detection limit of the XANES spectroscopic method for identifying minor As-containing species (29), XANES spectra of the culture supernatants indicate that less than 5% extracellular arsenic could be in the form of As(GS)3, the major fraction being inorganic As(III), independent of the initial arsenite concentrations used in our cultures (Figure 2). We analyzed the arsenic content and speciation in the cultures performed in the presence of 0.5 mM BSO after 4 days (maximum effect of BSO on the cell growth, Figure 1) and after 7 days (Supporting Information Table S1 and Figure S2). Arsenic concentrations in the supernatants (Supporting Information Table S1) and XANES spectra (Supporting Information Figure S2) are similar to those obtained in the absence of BSO. These data indicate that As is mainly bound by sulfur ligands, even in the presence of the glutathione synthesis inhibitor. It is likely that BSO does not completely deplete the glutathione pool in the cells, given the low concentration of BSO (0.5 mM) used in our experiments (33). However, the effect of BSO on cell growth confirms that glutathione or phytochelatin play a major role in arsenic detoxification in E. gracilis.

Discussion

FIGURE 4. Proportions of As-S and As-C bonds in E. gracilis cells exposed to 10-200 mg · L-1 As(III) for 10 days. For the Eg-As10 sample, the proportions are calculated from the fit of the XANES spectrum using AB and As(III)-(GS)3 as standards (see Supporting Information Figure S1, χ2 ) 6.10-4). For other samples, the proportions are calculated with the EXAFS best fits presented in Table 2. All proportions are multiplied by the total As concentration in the dry cells (Table 1). These results highlight the relative increase of As-S bonds with increasing initial As(III) concentration in the culture medium. In all experiments, the proportion of As-C bonds is minor and decreases from 28% to undetectable levels with increasing arsenite concentration in the culture medium from 50 mg · L-1 to 200 mg · L-1 As(III) (Figure 4). The proportion of As-S bonds increases concomitantly from 72% to about 100% (Table 2). These results are in good agreement with the XANES results indicating (i) the presence of a mixture of As-methylated species with As(III)-thiol compounds in cells cultivated at the lowest As(III) concentrations (10 and 50 mg · L-1), and (ii)

The present laboratory study provides new insight into arsenic resistance mechanisms at the molecular level in E. gracilis. Our results show that E. gracilis is tolerant to elevated arsenite concentrations (up to 200 mg · L-1 As(III)), as high as those encountered by Euglenae, such as E. mutabilis, in heavily contaminated AMD (15, 16). Accordingly, the arsenic concentration (315 mg · kg-1 As in dry cells) measured in E. gracilis exposed to 200 mg · L-1 As(III) in our experiments, is comparable with the concentrations (336 ( 112 mg · kg-1 As) measured in E. mutabilis cells exposed to 50-250 mg · L-1 As(III) in the Carnoule`s AMD (16). In microorganisms, cellular uptake of arsenite is thought to proceed by passive diffusion of the electrically neutral As(OH)3 molecule, through aquaglyceroporins (9, 34). Once in the cell, various organic molecules may complex dissolved As(III) under physiological conditions. In particular, GSH (γ-Glu-Cys-Gly) is a primary peptide involved in heavy metal and metalloid binding in the cytoplasm and is the substrate for phytochelatins (PC, (γ-Glu-Cys)n-Gly) synthesis (ref 35 and references therein). For instance, in E. gracilis, the synthesis of these molecules is induced in response to Cd exposure (17). GSH and phytochelatins are especially involved in arsenic binding in green algae (36) and in plants (13, 37, 38). Moreover, glutathione has been shown to play a key role in arsenic detoxification since it constitutes a source of reducing potential for the reduction of As(V) to As(III) in microorganisms (9, 39). Our XAS results (Figure 2) indicate that low amounts of As(III) are preferentially bound in methylated compounds, while increasing As(III) levels results in binding to S-groups in E. gracilis exposed to arsenite. Such coordination is fully consistent with that in As(III)-(GS)3 and suggests that this molecule and/or PCs may participate in As(III) detoxification in E. gracilis. The role of the GSH pathway is confirmed by the lower growth rate of E. gracilis cultures exposed to As(III), in the presence of BSO, an inhibitor of GSH synthesis (Figure 1). Our results suggest that such molecules likely play a main role in complexing As (III) within the cells, even in the absence of arsenate reduction. Two different models account for the fate of As(III)glutathione or As(III)-phytochelatin complexes: arsenite VOL. 42, NO. 14, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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efflux by a membrane protein and/or As(GS)3 transport into vacuole. Both mechanisms have been identified in yeast (8). Export of arsenic-glutathione complexes is known in mammals (10), including humans (40). Two types of ATP-binding cassette transporters are responsible for the export of arsenicglutathione complexes: multidrug resistance associated protein (MRP) and P-glycoprotein (P-gp) (8, 40). Similar mechanisms have been described in algae and plants exposed to Cd. On the one hand, export of Cdglutathione or Cd-PC complexes by membrane transporters has been reported in diatoms (41) and has recently been described in algae (42). An MRP gene was shown to be related to Cd sensitivity in the green alga Chlamydomonas reinhardtii (42). Moreover, a P-gp like protein is overexpressed under Cd stress in E. gracilis (43). On the other hand, heavy metals bound by glutathione or PCs can also end up in a vacuole (44), in chloroplasts (17) or in mitochondriae (18). These two latter processes have been reported in E. gracilis exposed to Cd (17, 18). In contrast, our observations indicate that arsenic is not accumulated inside E. gracilis cells. Our data are thus consistent with exportation of As(III) outside the cells. Moreover, our XANES results suggest that arsenic might be exported in an inorganic form rather than as As-(GS)3 or as As-PCs. Further studies are needed to determine the fate of As-(GS)3 or PCs that we suggest are involved in the As(III) detoxification pathway in E. gracilis, and to identify proteins involved in the arsenic export mechanism.

Acknowledgments Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory, a national user facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences. We are indebted to the SSRL staff, especially John R. Bargar, Joe Rogers, and Samuel Webb, for their technical assistance during the XAFS experiments. This work was supported by the ECCO/ECODYN CNRS/INSU Program, by ACI/FNS grant no. 3033, by SESAME IdF grant no. 1775, and by NSF-EMSI Grant CHE-0431425 (Stanford Environmental Molecular Science Institute). This is IPGP contribution no. 2375.

Supporting Information Available Additionnal figures showing the linear decomposition of the XANES spectrum of 10 day old E. gracilis cells exposed to 10 mg · L-1 As(III) (Figure SI1) and the XANES spectra of the BSO-treated cells (Figure SI2) as well as an additional table providing the As concentrations measured in cultures performed in the presence of BSO (Table SI1). This material is available free of charge via the Internet at http:// pubs.acs.org.

Literature Cited (1) Smedley, P. L.; Kinniburgh, D. G. A review of the source, behaviour and distribution of arsenic in natural waters. Appl. Geochem. 2002, 17, 517–568. (2) Morin, G.; Calas, G. Arsenic in soils, mines tailings and former industrial sites. Elements 2006, 2, 97–101. (3) O’Day, P. Chemistry and mineralogy of arsenic. Elements 2006, 2, 77–83. (4) Cervantes, C.; Ji, G.; Ramirez, J. L.; Siver, S. Resistance to arsenic compounds in microorganisms. FEMS Microbiol. Rev. 1994, 15 (4), 355–67. (5) Butcher, B. G.; Deane, S. M.; Rawlings, D. E. The chromosomal arsenic resistance genes of Thiobacillus ferroxidans have an unusual arrangement and confer increased arsenic and antimony resistance to Escherichia coli. Appl. Environ. Microbiol. 2000, 66 (5), 1826–33. (6) Rosen, B. P. Transport and detoxification systems for transition metals, heavy metals and metalloids in eukaryotic and prokaryotic microbes. Comp. Biochem. Physiol., Part A: Mol. Integr. Physiol. 2002, 133, 689–693. 5346

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