Yann Desfougères

Jean-Michel Poitou

Henri Wròblewski

Laure Béven

An improved non-denaturing method for the purification of spiralin, the main membrane lipoprotein of the pathogenic bacteria Spiroplasma melliferum

Keywords: CE-HPLC, Detergents, Membrane lipoprotein, Spiralin, Spiroplasma melliferum, Mollicutes

Highlights

-An improved procedure for the purification of a bacterial lipoprotein is presented.

-The new procedure shortens the purification from 24 hours to 4 hours.

-Detergent screening revealed that spiralin is mostly insoluble in Sarkosyl and Triton X-100.

-Milligram quantities of highly pure spiralin can be obtained.

-The procedure is non-denaturing.

Introduction

Bacterial membrane lipoproteins are bound to the lipid bilayer by a di-or a tri-fattyacylated N-terminal cysteine [for a recent review: [START_REF] Nakayama | Lipoproteins in bacteria: structures and biosynthetic pathways[END_REF] ]. This covalent modification occurs in proteins belonging to different families acting as cell receptors, enzymes, transporters, or adhesins. Some of them, such as E. coli Braun's lipoprotein have also a structural function [START_REF] Dramsi | Covalent attachment of proteins to peptidoglycan[END_REF][START_REF] Yem | Physiological characterization of an Escherichia coli mutant altered in the structure of murein lipoprotein[END_REF][START_REF] Braun | Covalent lipoprotein from the outer membrane of Escherichia coli[END_REF]. In host-bacteria interactions, the lipidic moiety of lipoproteins modulate the activity of the vertebrate immune system through binding to host Toll-like receptors [START_REF] Kovacs-Simon | Lipoproteins of bacterial pathogens[END_REF]. Some lipoproteins further display antigen variability within their polypeptide chain enabling the pathogen to evade the host immune defenses [START_REF] Van Der Woude | Phase and antigenic variation in bacteria[END_REF]. In Mollicutes, murein-less bacteria including Mycoplasma spp. and Spiroplasma spp., lipoproteins are tightly anchored to the external leaflet of the plasma membrane and face the host environment. A large number of mycoplasmal lipoproteins have been associated to bacterial cytadherence [START_REF] Hopfe | Multifunctional cytoadherence factors[END_REF], to phase, size, and antigenic variation [START_REF] Citti | Phase and antigenic variation in mycoplasmas[END_REF], or to immunomodulatory effects [START_REF] Chambaud | Interactions between mycoplasma lipoproteins and the host immune system[END_REF][START_REF] Rottem | Interaction of mycoplasmas with host cells[END_REF][START_REF] Szczepanek | Host Immune Responses to Mycoplasmas[END_REF]. Spiralin is the major membrane lipoprotein of a certain number of Spiroplasma species infecting plants and arthropods. This lipoprotein was first discovered in Spiroplasma citri [START_REF] Wróblewski | Purification and characterization of spiralin, the main protein of the Spiroplasma citri membrane[END_REF] and later in S. melliferum [START_REF] Wróblewski | Amphiphilic properties of spiralin, the major surface antigen of spiroplasmas. A preliminary report[END_REF], S. kunkelii, and S. phoeniceum [START_REF] Foissac | Sequence analysis of Spiroplasma phoeniceum and Spiroplasma kunkelii spiralin genes and comparison with other spiralin genes[END_REF]. Spiralin is one of the most well-studied Mollicute lipoproteins as evidenced by the large range of structural, expression and functional data available [START_REF] Béven | Inhibition of spiralin processing by the lipopeptide antibiotic globomycin[END_REF][START_REF] Brenner | Spiralin, a mycoplasmal membrane lipoprotein, induces T-cell-independent B-cell blastogenesis and secretion of proinflammatory cytokines[END_REF][START_REF] Castano | Secondary structure of spiralin in solution, at the air/water interface, and in interaction with lipid monolayers[END_REF][START_REF] Duret | Spiralin is not essential for helicity, motility, or pathogenicity but is required for efficient transmission of Spiroplasma citri by its leafhopper vector Circulifer haematoceps[END_REF][START_REF] Killiny | Spiroplasma citri Spiralin Acts In Vitro as a Lectin Binding to Glycoproteins from Its Insect Vector Circulifer haematoceps[END_REF][START_REF] Wróblewski | Topology and acylation of spiralin[END_REF]. Spiralin purified from S. melliferum membrane possesses immunostimulating activity on human peripheral blood mononuclear cells and murine splenocytes [START_REF] Brenner | Spiralin, a mycoplasmal membrane lipoprotein, induces T-cell-independent B-cell blastogenesis and secretion of proinflammatory cytokines[END_REF]. In the phytopathogenic S. citri, spiralin is essential for an efficient transmission of the bacteria to the plant host by the insect vector [START_REF] Duret | Spiralin is not essential for helicity, motility, or pathogenicity but is required for efficient transmission of Spiroplasma citri by its leafhopper vector Circulifer haematoceps[END_REF], is involved in the invasion of the insect cells, and exhibits glycoconjugate binding properties [START_REF] Duret | Invasion of insect cells by Spiroplasma citri involves spiralin relocalization and lectin/glycoconjugate-type interactions[END_REF]. In addition, given the high abundance of the protein, which is located exclusively on the outer leaflet of the bacterial membrane, a mechanical function for spiralin has been proposed in addition to its putative role as a lectin. However, structural information that could help understanding the function of spiralin is still missing. For instance, no information on the tertiary structure of spiralin is available to date. Similarly, no thorough mass spectrometry analysis of the protein to elucidate whether spiralin is a target of unidentified posttranslational modifications as proposed before [START_REF] Foissac | Spiralin polymorphism in strains of Spiroplasma citri is not due to differences in posttranslational palmitoylation[END_REF] has been conducted.

Valuable information about the function of spiralin could thus be obtained from structural studies. However, the availability of efficient purification procedures is still nowadays the bottleneck of lipoprotein studies. The general strategy of recombinant technology consisting of overproducing the protein in a genetically modified host cell has allowed the purification of a large number of proteins. However, in the case of Mollicutes lipoproteins, many difficulties may be encountered when using the recombinant methodology.

The solubilization of the overproduced amphiphilic protein may prove difficult without loss of the native structure. The host cell may also fail to perform the covalent modifications of the naturally produced protein. Incorrect interpretation of the membrane targeting signals and/or toxicity due to the accumulation of the foreign protein in the cell membrane(s) may occur.

In the case of S. citri spiralin, a recombinant protein could be expressed in E. coli [START_REF] Mouches | Expression of the Spiroplasma citri spiralin gene in Escherichia coli. Use of the recombinant plasmid carrying this gene as a molecular probe[END_REF].

However, the recombinant product proved to be localized not only in the inner membrane but also in the outer membrane and cytoplasm of E. coli [START_REF] Blanchard | Localization of spiralin in Escherichia coli cells transformed with the recombinant plasmid pESI[END_REF]. In addition, the recombinant spiralin did not appear to be correctly post-translationally modified as three forms differing in their apparent molecular mass could be detected in E. coli, and not in S. citri [START_REF] Blanchard | Localization of spiralin in Escherichia coli cells transformed with the recombinant plasmid pESI[END_REF]. In the past, we developed different strategies to purify spiralin [START_REF] Wróblewski | Purification and characterization of spiralin, the main protein of the Spiroplasma citri membrane[END_REF][START_REF] Castano | Secondary structure of spiralin in solution, at the air/water interface, and in interaction with lipid monolayers[END_REF][START_REF] Wróblewski | Topology and acylation of spiralin[END_REF]. Starting from S. citri membranes, the purification process of the native spiralin entailed essentially three steps: [START_REF] Nakayama | Lipoproteins in bacteria: structures and biosynthetic pathways[END_REF] removal of extrinsic proteins using Tween 20 or Sarkosyl, (2) selective extraction of spiralin with 0.2 M sodium deoxycholate (DOC) or 100 mM CHAPS, and (3) fractionation of the DOC/CHAPS-soluble material by preparative agarose-suspension electrophoresis (ASE). However, this protocol is time-consuming and the amount of protein obtained is limited to 1 mg per separation. Moreover, the different steps were empirically designed. In this work, we aimed at optimizing the spiralin purification protocol by screening a variety of detergents for the initial extraction step, and establishing a cation exchange chromatography for the final purification. We could thereby shorten the total duration of the process and the final separation step offered a higher adaptability to amount of available starting material.

Materials and methods

Reagents

Acrylamide and N,N'-methylene bisacrylamide were from BDH (U. K.). Agarose (Indubiose A37) was from IBF (France). HECAMEG,

N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate (SB-12, lauryl sulfobetaine), N-tetradecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate (SB-14, tetradecyl sulfobetaine), 3-[(3-cholamidopropyl) dimethylammonio]-1-
propyl sulfonate (CHAPS), N-octyl-ß-D-glucopyranoside (OG), : nonaethyleneglycol octylphenol ether (Triton X-100) and sodium lauroyl-N-sarcosinate (Sarkosyl) were obtained from Sigma (U.S.A.). Sodium cholate and sodium deoxycholate (DOC) were from Calbiochem (La Jolla, California, U. S. A.). Sodium dodecylsulfate (SDS) was from Merck (Germany). N-dodecyl-N,N-dimethylammonio butyrate (DDMAB) and N-dodecyl-N,Ndimethylammonio undecanoate (DDMAU) were generous gifts from Y. Chevalier (CNRS UPR 9031, Vernaison, France). Cholylsarcosine was a generous gift from Diamalt (München, Germany). N-octanoyl-beta-D-glucosylamine (NOGA) was synthesized through the procedure described in Brenner-Henaff et al. (1993).

Protein concentration determination

Protein concentration was determined with the method of Lowry et al. [START_REF] Lowry | Protein measurement with the Folin phenol reagent[END_REF] modified by Markwell et al. [START_REF] Markwell | A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples[END_REF], using bovine serum albumin as standard. Titration of individual proteins was performed by scanning densitometry after SDS-PAGE and Coomassie brilliant blue R250 staining.

Preparation of S. melliferum membranes

S. melliferum (strain BC-3 T , ATCC 33219) [START_REF] Clark | Spiroplasma-Melliferum, a New Species From the Honeybee (Apis-Mellifera)[END_REF] was grown under microaerobic conditions as previously described [START_REF] Béven | Effect of natural amphipathic peptides on viability, membrane potential, cell shape and motility of mollicutes[END_REF]. Cells were harvested by centrifugation at 10,000g for They were finally centrifuged at 38,000g and dispersed in 10 mM HEPES buffer (pH 7.4). All the operations were performed at 0-4°C and 5 ml membrane suspensions containing 10 mg protein ml -1 were stored at -80°C until use.

Membrane protein extraction with detergents

Proteins were extracted with detergents by mixing one volume of membrane suspension (10 mg of protein per ml of 100 M Na phosphate buffer, pH 7.5) with one volume of detergent solubilized in water. After 1 h with intermittent shaking, the mixture was centrifuged at 260,000g for 15 min (Beckman TL-100 ultracentrifuge, TLA 100.1 rotor) to separate solubilized proteins from insoluble material. Operations were performed at 18°C when SDS was used and at 4°C in the case of other detergents.

Preparation of antibodies

Antibodies against S. melliferum membrane proteins were elicited in three rabbits by subcutaneous inoculation, twice a month, of isolated membranes. Each inoculum was composed of 1 mg of membrane protein in 0.5 ml of 150 mM NaCl and emulsified with 0.7 ml of Freund adjuvant. The latter was used complete only for the first inoculation. After 3 months of immunization, the rabbits were bled and the three sera were pooled. The same procedure was used to obtain the antibodies which can recognize spiralin specifically. In that case, the immunogen was composed of 10 µg of pure spiralin detergent-free micelles per inoculum.

Chromatographic techniques

Preparative protein separations were performed by cation exchange chromatography in a column of methacrylate co-polymer resin beads covalently bonded with propylsulfonic acid functionalities (Waters Protein Pack SP8HR: resin particle diameter, 8 µm; pore size, 0.1 µm; column internal diameter, 1 cm; column length, 10 cm) and a Waters 625 HPLC system. The samples contained membrane proteins solubilized with 100 mM CHAPS in 20 mM sodium citrate buffer (pH 4.5) containing 4 mM DTT. Elution of the proteins adsorbed on the column was performed with a salt concentration gradient using the following buffers: 20 mM sodium citrate buffer (pH 4.5) containing 16 mM CHAPS and 2 mM DTT (Buffer A) and buffer A containing 1 M NaCl (Buffer B). Elution conditions (1 ml min -1 ): 0 to 5 min, 100% buffer A; 5 to 40 min, linear gradient to 30% buffer B; 40 to 50 min, 30% buffer B; 50 to 60 min, linear gradient up to 100% buffer B; 60 to 75 min, 100% buffer B, and 75 to 80 min, linear gradient down to 0% buffer B. The eluates were monitored by light absorbance at 280 nm and fractions were analyzed by SDS-PAGE.

The homogeneity of spiralin detergent-free micelles was analyzed by size-exclusion HPLC (SEC) in a Superdex 200 HR column (diameter, 1 cm; height, 30 cm) (Pharmacia, Uppsala, Sweden). The buffer used for column equilibration and protein elution (0.4 ml min -1 ) was 50 mM sodium phosphate buffer pH 7.0 containing 150 mM NaCl. Spiralin and reference proteins (thyroglobulin, ferritin, catalase, apotransferrin, hemoglobin and cytochrome c) were detected by light absorption at 280 nm.

Preparation of spiralin detergent-depleted micelles

Detergent was removed using the Lipoprep ® (Dianorm) flow-through dialyzer [START_REF] Zumbuehl | Liposomes of controllable size in the range of 40 to 180 nm by defined dialysis of lipid/detergent mixed micelles[END_REF]. The central compartment of the Teflon cell was loaded with 8 ml of a solution of spiralin (2 mg protein per ml of 10 mM sodium phosphate buffer) containing 16 mM CHAPS. Dialysis was performed for 36 h at 6°C through two cellulose membranes (diameter, 5 cm; cut-off, 10 kDa). The solution was stirred with a magnet spinning at 60 rev. min -1 and the dialysis buffer (10 mM sodium phosphate buffer, pH 7.2) was pumped through the two side compartments at a flow rate of 5 ml min -1 .

SDS-PAGE

Protein extracts were diluted 1:10 in loading buffer (62.5 mM Tris-HCl pH 6.8, 20 mM DTT, 2% SDS, 40% glycerol, and 0.01% bromophenol blue). The proteins were separated in 120 x 120 x 1 mm polyacrylamide gels in the presence of 0.1% SDS with the method of Laemmli [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF]. Compositions of the stacking and separating gels were T = 4.8%, C = 2.6% and T = 10%, C = 2.6%, respectively, with %T being the total monomer (acrylamide + bisacrylamide) concentration and %C the percentage of bisacrylamide relative to total monomer. Proteins were silver stained according to the method of Tunón and Johansson [START_REF] Tunón | Yet Another Improved Silver Staining Method for the Detection of Proteins in Polyacrylamide Gels[END_REF].

Immunoelectrophoresis

Crossed immunoelectrophoresis [START_REF] Clarke | A quantitative immuno-electrophoresis method (Laurell electrophoresis)[END_REF] was performed as described previously [START_REF] Wróblewski | Comparison of the amino acid compositions and antigenic properties of spiralins purified from the plasma membranes of different spiroplasmas[END_REF], in the presence of 10 mM SB12 to avoid the precipitation of hydrophobic proteins. Antibodies against S. melliferum membranes were prepared as described above and the serum was used at a 1/20 dilution. The directions of migration are indicated on Figure 7. After electrophoresis, the gels were washed and dried, and the immunoprecipitates were stained with Coomassie brilliant blue R-250. Fused-rocket immunoelectrophoresis was performed as described previously [START_REF] Wróblewski | Purification and characterization of spiralin, the main protein of the Spiroplasma citri membrane[END_REF], using antibodies directed against S. melliferum membrane similarly to the crossed immunoelectrophoresis.

Circular dichroism spectroscopy

Circular dichroism spectra of spiralin were recorded from 190 to 260 nm at 20°C on a Jasco 810 (Jasco, Bouguenais, France) spectropolarimeter equipped with a thermostatically controlled quartz cell with a path length of 2 mm. Spectra were required at a step resolution of 0.1 nm and at a 50 nm min -1 speed (bandwidth: 1 nm). The sample was prepared in 10 mM sodium phosphate buffer pH 7.4 at a final protein concentration of 3.8 µM. When needed, 50 mM CHAPS or 35 mM SDS was included in the samples. For each analysis three scans were performed and subsequently averaged. Corrections were made for buffer contribution.

Spectrum deconvolution was performed using the CDPro package. The ContinLL method was chosen to estimate secondary structure of spiralin. Wavelengths from 190 to 240 nm were submitted for analysis and the SMP56 reference set was chosen [START_REF] Sreerama | On the analysis of membrane protein circular dichroism spectra[END_REF].

Transmission electron microscopy

Suspensions of detergent-free spiralin micelles (0.02 mg ml -1 of 50 mM sodium phosphate buffer pH 7.4) were deposited on glow-discharged carbon-coated 200-mesh copper grids. After one min of contact, the adsorbed micelles were washed three times with distilled water and negatively contrasted with 2% uranyl acetate. Micrographs were taken with a Philips CM12 microscope operating at 120 kV and at a magnification of x 45,000.

Results and Discussion

Selective extraction of spiralin from the spiroplasma membrane

Selective extraction of membrane proteins by detergents has proven to be a very powerful tool to purify proteins [START_REF] Arachea | Detergent selection for enhanced extraction of membrane proteins[END_REF][START_REF] Garavito | Detergents as tools in membrane biochemistry[END_REF]. Spiralin can be extracted from spiroplasma membranes by sequential action of detergents [START_REF] Wróblewski | Purification and characterization of spiralin, the main protein of the Spiroplasma citri membrane[END_REF]. In the original protocol describing the purification of spiralin, Tween 20 was used to deplete the whole membranes from proteins leaving most of the spiralin in the insoluble fraction. Then, spiralin was solubilized by DOC, which selectively extracted this protein. Later, Sarkosyl was used in place of Tween 20 to reduce the time required for the extraction, and DOC was replaced by CHAPS [START_REF] Wróblewski | Topology and acylation of spiralin[END_REF]. There was no rationale behind the choice of these detergents except that they are non-denaturing detergents. We then decided to screen for other detergents which would have low selectivity for spiralin and could be used to deplete non-spiralin proteins from the membranes.

Fifteen non-denaturing detergents were compared for their ability to extract spiralin from the plasma membrane of S. melliferum. In each case, the amount of spiralin vs. total protein extracted was determined as a function of detergent concentration. Fig. 1 illustrates the results obtained with cholylsarcosine, CHAPS, and Sarkosyl, and Table 1 recapitulates the data obtained with the whole series of detergents. Most of the detergents extracted spiralin efficiently and selectively except for Triton X-100 and Sarkosyl. Triton X-100 could extract only 20% of proteins from the membrane and provide low amounts of spiralin. On the other hand, Sarkosyl could extract more proteins and had less selectivity for spiralin than Triton X-100. Sarkosyl at concentrations ≥ 50 mM extracted 55% of total protein but only 15% of spiralin (Fig. 1A &1B). Among the alkylated ionic detergents, Sarkosyl had the unique property of being unable to efficiently extract spiralin from the plasma membrane even at a concentration ten times higher than its critical micellar concentration of 14 mM (Fig. 1B). On the opposite, bile salt derivatives and alkyl glycosides proved to be very potent detergents to extract selectively spiralin from plasma membranes. In particular, CHAPS and DOC solubilized spiralin with a high enrichment factor (2.0 and 1.8, respectively), and a low optimal concentration (60 and 50 mM, respectively). Detergent concentration has to be kept as low as possible during the purification procedure because it has to be removed in the last step of the purification. This is the reason why we chose not to use detergents that gave higher enrichment factor at higher optimal concentrations such as cholylsarcosine, for example.

Consequently, we chose a sequential detergent extraction procedure as a first step for the purification of spiralin before chromatographic separation. S. melliferum membranes were first treated with 20 mM Sarkosyl which solubilized about 40% of proteins including very little amount of spiralin (ca. 8%) and the fraction insoluble in these conditions was subsequently treated with 50 mM CHAPS which permitted to obtain a solution which contains spiralin as a major protein (Fig. 2, lane1, 2, and 3). Using Sarkosyl allowed not only to shorten the step of removing extrinsic proteins, but also to operate at more moderate pH (pH 7.4 instead of 9.5) as reported before [START_REF] Wróblewski | Topology and acylation of spiralin[END_REF][START_REF] Wróblewski | Comparison of the amino acid compositions and antigenic properties of spiralins purified from the plasma membranes of different spiroplasmas[END_REF].

Purification of spiralin by cation-exchange HPLC (CE-HPLC)

The amount of spiralin obtained by preparative ASE is limited to 1 mg per separation.

Indeed, an increase of the column diameter aiming at increasing the loading capacity would result in an excessive temperature transverse gradient and a poorer resolution. We aimed at optimizing the spiralin purification protocol by using a final separation step offering a higher adaptability to large amounts of available starting material. Our choice fell on the technique of ion exchange HPLC.

The theoretical titration curve of S. melliferum spiralin was used to define the best conditions for purification by ion exchange HPLC. Since this protein has a pI of 8.4, a cation-exchange method would be more appropriate than the anion-exchange option. Furthermore, a pH of 4.5 appeared to be optimal since in these conditions, the protein would have a net charge ca. +15, in principle a good compromise for adsorption/desorption when using a strong cation exchanger.

The elution profile of CE-HPLC showed that the major single peak was eluted at 0.2 M NaCl as expected (Fig. 3). The isolated major peak was analyzed by SDS-PAGE and provided a single band at the expected size for spiralin (28.7 kDa) (Fig. 2,lane4). It should be noted that the amount of spiralin in the HPLC peak is larger than suggested by its height because spiralin has a low molar extinction coefficient ( ≈ 6520 M -1 cm -1 at 280 nm) due to the lack of tryptophan in its amino acid sequence [START_REF] Wróblewski | Purification and characterization of spiralin, the main protein of the Spiroplasma citri membrane[END_REF].

The time of purification was shortened by choosing the chromatographic procedure instead of the agarose-suspension electrophoresis separation: 12h for the ASE vs. 90 min for the CE-HPLC. Moreover, the capacity of the CE-HPLC column allows recovering up to 6 mg of purified spiralin per chromatography starting from 50 mg of total spiroplasma membranes.

The efficiency of the overall purification procedure was estimated to be around 60%. We also successfully used a low-pressure cation exchange chromatography system to purify even larger amounts of spiralin. This permitted to collect 14 mg of purified spiralin per purification starting from 50 mg of total spiroplasma membranes (data not shown).

Spiralin detergent-free micelles

The final step of the purification procedure consisted in removing both NaCl and CHAPS from the mixed micelle solution obtained by CE-HPLC. This was achieved by controlled dialysis using the Lipoprep equipment. During detergent removal, spiralin molecules aggregated under the form of globular particles as shown by transmission electron microscopy (Fig. 4). The size distribution of these particles revealed that the supra-molecular structures had a mean diameter of 19 nm (n=1359, sd = 4.7 nm). Further analysis by SEC confirmed that the mean diameter was in the same range of magnitude, i.e. 17.0 nm (Fig. 5A).

Spiralin was eluted together with thyroglobulin, which has a molecular mass of 669 kDa and a Stokes radius of 8.5 nm. The average mass of spiralin micelles ca. 700 kDa, was similar to the spiralin detergent-depleted micelles obtained when spiralin was purified by preparative agarose suspension electrophoresis and subsequent dialysis [START_REF] Castano | Secondary structure of spiralin in solution, at the air/water interface, and in interaction with lipid monolayers[END_REF]. The elution fractions of SEC were analyzed by fused-rocket immunoelectrophoresis (Fig. 5B). This result proved that the purified protein was spiralin. This, as explained below, suggests that the protein kept its native structure during the SEC step. Consequently, what was measured here is indeed the size of spiralin micelles rather than aggregates of unfolded protein.

Evidence for the native structure of purified spiralin

Far-UV circular dichroism spectra of spiralin detergent-depleted micelles and of spiralin solubilized with SDS are displayed in Fig. 6A. The spectrum of spiralin micelles is characteristic of a highly organized protein containing a high proportion of periodic secondary structures. Deconvolution of the spectra was realized using the CDPro package (Fig. 6B).

Results obtained with the ContinLL method indicate that spiralin has about 32% of ß-sheet structures and 18% of -helices consistently with previous observations [START_REF] Castano | Secondary structure of spiralin in solution, at the air/water interface, and in interaction with lipid monolayers[END_REF]. Moreover spiralin has 22% of turns. The addition of the mild detergent CHAPS did not have any effect on the content of secondary structure in spiralin, supporting the idea that the improved purification procedures could keep the spiralin structure similar to its native structure.

Strikingly, the spectrum of SDS-treated spiralin shows that the denatured protein has a high proportion of -helical structures (36%) and a low ß-sheet content (9%), while the proportion of turns and other structures (including disordered structures) is the same in the native and the denatured protein. Helix formation in the presence of SDS can be attributed to the presence of amphiphilic helices in spiralin [START_REF] Parker | Protein structures in SDS micelle-protein complexes[END_REF][START_REF] Welker | Hsp12 is an intrinsically unstructured stress protein that folds upon membrane association and modulates membrane function[END_REF][START_REF] Singarapu | Structural characterization of Hsp12, the heat shock protein from Saccharomyces cerevisiae, in aqueous solution where it is intrinsically disordered and in detergent micelles where it is locally α-helical[END_REF]. Primary structure analysis suggested that spiralin might contain amphiphilic -helices that could be non-structured in the absence of a hydrophobic environment. This was confirmed experimentally for one -helix [START_REF] Brenner | Conformation, pore-forming activity, and antigenicity of synthetic peptide analogues of a spiralin putative amphipathic alpha helix[END_REF][START_REF] Bondon | Solution structure determination by NMR spectroscopy of a synthetic peptide corresponding to a putative amphipathic alpha-helix of spiralin: resonance assignment, distance geometry and simulated annealing[END_REF]. Our preferred hypothesis is that spiralin possesses one or more amphipathic -helices that have no structure in solution but become ordered in the presence of detergent or another hydrophobic environment. The impact of SDS on the secondary structures of spiralin is thus consistent with the previous results [START_REF] Parker | Protein structures in SDS micelle-protein complexes[END_REF]. That this could reflect a physiological function of spiralin still remains to be determined.

Antigenicity of the purified spiralin was tested by crossed immunoelectrophoresis (CIE). Proteins from whole S. melliferum membranes or purified spiralin were loaded on separate gels (Fig 7). The second migration was performed in agarose gels containing antibodies directed against total membrane protein extracts of S. melliferum. Fig. 7A shows that spiralin is by far the most abundant membrane protein in S. melliferum membranes and that it keeps its antigenic properties after extraction by DOC. The high purity of spiralin after CE-HPLC is illustrated in Fig. 7B. No other protein than spiralin could be detected confirming the results obtained by SDS-PAGE. Moreover, the positive signal obtained by CIE suggests that the purified spiralin retained some original structural conformations during the whole purification processes. Indeed, in-gel immunoprecipitation is possible only if at least three distinct epitopes are available to react with antibodies [42]. It is thought that a vast majority of the epitopes on proteins are conformational epitopes [START_REF] Todd | The Immunogenicity and Antigenicity of Proteins[END_REF][START_REF] Vinion-Dubiel | Antigenic diversity among Helicobacter pylori vacuolating toxins[END_REF]. Therefore, given the rather small size of spiralin, the existence of at least three different epitopes strongly suggests that the protein kept its native structure during the purification procedure.

Conclusion

Mollicutes include a high number of pathogens for humans, animals and plants [START_REF] Razin | 1. Mycoplasma taxonomy and ecology[END_REF].

Because many lipoproteins are involved in Mollicutes pathogenicity or virulence, it is essential to have these proteins in a purified native form for functional studies aiming at better understanding Mollicutes-hosts interactions. Currently, most functional studies are carried out using truncated forms of Mollicutes lipoproteins to evaluate their effect on host cells. For example, synthetic lipopeptides corresponding to the N-terminal moiety of native lipoproteins, such as MALP-2 and FSL-1 derived from lipoproteins produced in mycoplasmas infecting mammals, initiated the innate immune response in host cells [START_REF] Mühlradt | Isolation, structure elucidation, and synthesis of a macrophage stimulatory lipopeptide from Mycoplasma fermentans acting at picomolar concentration[END_REF][START_REF] Into | Synthesis and characterization of a dipalmitoylated lipopeptide derived from paralogous lipoproteins of Mycoplasma pneumoniae[END_REF]; the production of a recombinant protein corresponding to S. citri spiralin polypeptidic moiety (thus lacking the acylated part) allowed to uncover the role of this lipoprotein in recognition of host cell receptors [START_REF] Duret | Invasion of insect cells by Spiroplasma citri involves spiralin relocalization and lectin/glycoconjugate-type interactions[END_REF]. The use of whole detergent extracts containing a mixture of lipoproteins in functional studies proved to be also useful in demonstrating the putative functional role of lipoproteins in the host immune response, as illustrated by the work on the human mycoplasma Mycoplasma hominis described in Truchetet et al. [START_REF] Truchetet | Potential Role of Mycoplasma hominis in Interleukin (IL)-17-Producing CD4+ T-Cell Generation Via Induction of IL-23 Secretion by Human Dendritic Cells[END_REF]. Now the availability of purified, complete lipoproteins in their native form could help validating the putative function in Mollicutes pathogenesis observed when using truncated lipoproteins. This should also enable us to gain more insights into the function of spiralin at the molecular level. Notably, protein-lipid interactions can now be tested in vitro in order to evaluate the importance of such interactions in living bacteria and in the context of the host-pathogen interactions.

Moreover, the impact of spiralin on membrane shape and dynamics can be assessed using model systems such as liposomes. The data thus obtained should confirm or rule out the putative mechanical role of spiralin. We previously described a procedure for the purification of spiralin [START_REF] Rottem | Interaction of mycoplasmas with host cells[END_REF] [START_REF] Castano | Secondary structure of spiralin in solution, at the air/water interface, and in interaction with lipid monolayers[END_REF]. The previous purification process included the selective extraction of the protein by detergents from spiroplasma membranes, and a subsequent separation by agarosesuspension electrophoresis. This previous strategy was used to purify spiralin and show its immunomodulatory function [START_REF] Brenner | Spiralin, a mycoplasmal membrane lipoprotein, induces T-cell-independent B-cell blastogenesis and secretion of proinflammatory cytokines[END_REF]. In this work, we found that Sarkosyl has a unique property of extracting large amounts of membrane proteins but not spiralin. This property was not obtained with any other detergents and suggests that spiralin is mostly insoluble in Sarkosyl.

Moreover, replacing the ASE separation by a CE-HPLC step shortened drastically the time of the purification procedure. The amount of purified spiralin per round was increased from 1 mg (ASE) to > 6 mg (CE-HPLC).

This new purification procedure is based on the poor solubilization of spiralin by Sarkosyl, while this detergent could solubilize a large number of other membrane proteins.

This peculiar behavior of spiralin during the extraction step could be due to specific spiralinlipid interactions. This assumption is based on the existence of preferred interactions of diverse acylated proteins with membrane parts enriched in specific lipids, such as sterols, sphingolipids -enriched membrane domains (rafts) in other biological membrane systems [START_REF] Simons | Lipid rafts and signal transduction[END_REF]. Following our purification process, both circular dichroism spectroscopy and crossed immunoelectrophoresis strongly suggested that spiralin kept a structure very close to its native structure. The high amount of protein that can be obtained by this new method opens the possibility for structural studies that are usually highly demanding in terms of protein material. We think that the whole strategy, based on a detergent screening for the double extraction step, will also prove to be useful for the purification of other lipoproteins, involved in Mollicutes pathogenesis and for which specific interactions with membrane lipids are suspected. This should also open the way for the analysis of the three dimensional structure of lipoproteins, a procedure that usually requires high amounts of protein. Furthermore, this approach allows to obtain large quantities of proteins from the natural organism making possible the study of posttranslational modifications of surface proteins. The importance of such modifications, such as proteolysis, has been recently put forward and may play an important role in pathogenicity and increase the diversity of the cell surface proteome [START_REF] Tacchi | Post-translational processing targets functionally diverse proteins in Mycoplasma hyopneumoniae[END_REF]. (d) Spiralin enrichment factor (EF= % spiralin yield / % total protein yield). The fraction solubilized with a given detergent is enriched with spiralin if EF > 1. EF = 4 is the maximum valuable that can be attained since spiralin represents 25% (by mass) of the total membrane protein.

  15 min and washed once in 50 mM sodium phosphate buffer containing 150 mM NaCl and 550 mM D(-)sorbitol. The cells were then dispersed in 50 mM Tris-HCl buffer (pH 8.0) containing 1 mM of 4-(2-aminoethyl)-benzene sulfonyl fluoride (protease inhibitor, AEBSF) and disrupted by sonication (2 x 30 sec) with 500-Watt Ultrasonic Processor equipped with a titanium-tapered microtip. Plasma membranes were centrifuged three times 1 h at 38,000g in 50 mM Tris-HCl buffer pH 8.0 to remove cytoplasmic components. They were dispersed and stirred overnight in 5 mM Tris-HCl buffer (pH 8.0) containing 5 mM EDTA and 1 mM DTT.

Figure captions Fig. 1 .

 1 Figure captionsFig. 1. Examples of S. melliferum membrane protein extractions by detergents. Membranes were treated with cholylsarcosine (triangles), deoxycholate (circles), or Sarkosyl (squares) at various concentrations of detergents. Extracted proteins were separated from the insoluble material by centrifugation, and total proteins (A) and spiralin (B) were quantified in the solubilized fraction by densitometry analysis after SDS-PAGE.

Fig. 2 .

 2 Fig. 2. Purity control of spiralin by SDS-PAGE. Lane 1, S. melliferum BC3 membrane proteins (10 µg) solubilized with 1% SDS; lanes 2 and 3, membrane proteins (2.5 µg) selectively extracted with 0.1 M CHAPS from membranes treated with 20 mM Sarkosyl; lane 4, spiralin (2 µg) purified by cation-exchange HPLC; lane M, molecular weight markers. S indicates the migration of spiralin. All the samples contained 2 mM DTT. Protein bands were silver stained.

Fig. 3 .

 3 Fig. 3. Chromatographic separation of spiralin. Spiralin purification by cation-exchange HPLC. Sample: 5 mg of protein extracted with 0.1 M CHAPS in 4.2 ml of 20 mM sodium phosphate-citrate buffer (pH 4.5) containing 16 mM CHAPS and 2 mM DTT. Column: cation-exchange Protein Pack (Waters Millipore) SP8HR (, 1 cm ; length, 10 cm). Flow rate: 1 ml min -1 . Proteins were desorbed from the column using a 0 to 0.6 M NaCl linear gradient from fraction 20 to fraction 100. Elution of the was monitored by recording light absorption at 280 nm.

Fig. 4 .

 4 Fig. 4. Analysis of spiralin detergent-free micelles by electron microscopy. Spiralin micelles were visualized by transmission electron microscopy after negative staining. Representative pictures (left) and the size distribution of the particles (right) are shown. The scale bar represents 200 mm.

Fig. 5 .Fig. 6 .

 56 Fig. 5. Analysis of spiralin detergent-free micelles by size-exclusion chromatography (SEC).A. Spiralin micelles (dotted line) were eluted from a Superdex 200 HR 10/30 column with 50 mM sodium phosphate pH 7.0 buffer containing 150 mM NaCl as the mobile phase (flow = 0.4 ml min -1 ). Elution was followed by recording absorbance at 280 nm. Standard proteins (solid line) were: A, thyroglobulin; B, apoferritine; C, catalase; D, apotransferrin; E,

Fig. 7 .

 7 Fig. 7. Antigenicity of spiralin. Proteins solubilized with DOC were analyzed by crossed immunoelectrophoresis in veronal buffer pH 8.6 containing 13 mM DOC. The samples were composed of 25 µg of S. melliferum membrane proteins (A) and 5 µg of pure spiralin (B). The immunoprecipitates were stained with Coomassie blue R250. S indicates the spiralin immunoprecipitation peak.

Figure 1 Protein

 1 Figure 1

Figure 2 S

 2 Figure 2 S Should appear in black and white in the online and the print versions single-column fitting figure kDa

Figure 3 Fractions 5

 35 Figure 3

Figure 6 Should 7

 67 Figure 6

  (a) Lowest detergent concentration giving the highest yield of spiralin extraction. (b) Percentage of total membrane protein extracted. (c) Percentage of spiralin extracted.

Table 1 .

 1 Comparison of the efficacies of different detergents for the extraction of S.

	Tables
	melliferum membrane proteins.
	Detergents	Concopt (mM) a Protein yield (%) b Spiralin yield (%) c EF d
	Alkylated ionic detergents
	DDMAB	100	86	70	0.8
	DDMAU	25	90	65	0.7
	Sarkosyl	50	55	15	0.3
	SB-12	150	33	85	2.6
	SB-14	150	41	95	2.3
	Bile salts and derivatives
	CHAPS	60	50	100	2.0
	Cholic acid	100	50	90	1.8
	Cholylsarcosine	150	38	100	2.6
	DOC	50	55	100	1.8
	Taurocholic acid	100	55	80	1.5
	TauroDOC	100	55	85	1.6
	Alkylglycosides
	HECAMEG	60	35	100	2.8
	NOGA	100	45	100	2.2
	OG	75	73	100	1.4
	POE detergents
	Triton X-100	60	25	20	0.8

Acknowledgements

The authors thank Jean-Yves Cremet and Marie Guéguen for technical support and assistance.


Shaofeng Wang

Xu Ma

Guoqing Zhang

Yongfeng Jia
email: yongfeng.jia@iae.ac.cn

Keisuke Hatada

Prof Yonfeng Jia

New insight into the local structure of hydrous ferric arsenate using full-potential multiple scattering analysis, density functional theory calculations, and vibrational spectroscopy

Hydrous ferric arsenate (HFA) is an important arsenic-bearing precipitate in the mining-impacted environment and hydrometallurgical tailings.

However, there is no agreement on its local atomic structure. The local structure of HFA was re-probed by employing a full-potential multiple scattering (FPMS) analysis, density functional theory (DFT) calculations, and vibrational spectroscopy.

The FPMS simulations indicated that the coordination number of the As-Fe and/or Fe-As in HFA was approximately two. The DFT calculations constructed a structure of HFA with the formula of Fe(HAsO 4 ) x (H 2 AsO 4 ) 1-x (OH) y •zH 2 O. The presence of protonated arsenate in HFA was also evidenced by vibrational spectroscopy. The As and Fe K-edge XANES spectra of HFA were accurately reproduced by FPMS simulations using the chain structure, which was also a reasonable model for EXAFS fitting. The FPMS refinements indicated that the interatomic Fe-Fe distance was approximately 5.2 Å, consistent with that obtained by Mikutta et al. (ES&T, 2013) using wavelet analysis. All of the results suggested that HFA was more likely to occur as a chain with AsO 4 tetrahedra and FeO 6 octahedra connecting alternately in an isolated bidentate-type fashion. This finding is of significance for understanding the fate of arsenic and the formation of ferric arsenate minerals in an acidic environment.

INTRODUCTION

Hydrous ferric arsenate (HFA) is one of the important secondary arsenic precipitates in the mining-impacted environment and hydrometallurgical tailings, [START_REF] Krause | Solubilities and Stabilities of Ferric Arsenate Compounds[END_REF][2][START_REF] Jia | Infrared spectroscopic and X-ray diffraction characterization of the nature of adsorbed arsenate on ferrihydrite[END_REF][START_REF] Langmuir | Solubility products of amorphous ferric arsenate and crystalline scorodite (FeAsO 4 •2H 2 O) and their application to arsenic behavior in buried mine tailings[END_REF][START_REF] Nishimura | Crystalline phases in the system Fe(III)-As(V)-H 2 O at 25C[END_REF] such as acidic mining drainage [START_REF] Maillot | Structure and reactivity of As(III)-and As(V)-rich schwertmannites and amorphous ferric arsenate sulfate from the Carnoules acid mine drainage, France: Comparison with biotic and abiotic model compounds and implications for As remediation[END_REF] and gold [START_REF] Paktunc | Speciation and characterization of arsenic in gold ores and cyanidation tailings using X-ray absorption spectroscopy[END_REF] and uranium mine tailings. [START_REF] Langmuir | Solubility products of amorphous ferric arsenate and crystalline scorodite (FeAsO 4 •2H 2 O) and their application to arsenic behavior in buried mine tailings[END_REF][START_REF] Chen | Structural characterization of poorly-crystalline scorodite, iron(III)-arsenate co-precipitates and uranium mill neutralized raffinate solids using X-ray absorption fine structure spectroscopy[END_REF][START_REF] Essilfie-Dughan | Arsenic and iron speciation in uranium mine tailings using X-ray absorption spectroscopy[END_REF] In the Fe-As coprecipitation process, a widely employed method for the removal and fixation of arsenic from hydrometallurgical raffinates, HFA precipitates as a major arsenic-bearing phase at acidic pH values. [START_REF] Jia | In Preparation, characterization and solubilities of adsorbed and co-precipitated iron(III)-arsenate solids, Hydrometallurgy 2003[END_REF] The adsorbed arsenate on ferrihydrite was also observed to transform into HFA at pH values of 3-5. 2,[START_REF] Jia | Infrared spectroscopic and X-ray diffraction characterization of the nature of adsorbed arsenate on ferrihydrite[END_REF] HFA is thermodynamically metastable and can convert to scorodite during prolonged aging at ambient temperature [START_REF] Nishimura | Crystalline phases in the system Fe(III)-As(V)-H 2 O at 25C[END_REF] or elevated temperatures. [START_REF] Jia | Infrared spectroscopic and X-ray diffraction characterization of the nature of adsorbed arsenate on ferrihydrite[END_REF][START_REF] Le Berre | A study of the crystallization kinetics of scorodite via the transformation of poorly crystalline ferric arsenate in weakly acidic solution[END_REF] Therefore, precipitation of HFA followed by heat treatment was also employed for the synthesis of scorodite, [START_REF] Krause | Solubilities and Stabilities of Ferric Arsenate Compounds[END_REF][START_REF] Langmuir | Solubility products of amorphous ferric arsenate and crystalline scorodite (FeAsO 4 •2H 2 O) and their application to arsenic behavior in buried mine tailings[END_REF][START_REF] Dove | The Solubility and Stability of Scorodite, FeAsO 4 •2H 2 O[END_REF][START_REF] Rochette | Stability of arsenate minerals in soil under biotically generated reducing conditions[END_REF][START_REF] Harvey | Scorodite Dissolution Kinetics: Implications for Arsenic Release[END_REF] and this could provide an alternative method for the removal and fixation of arsenic in hydrometallurgical solutions with conventional autoclave [START_REF] Dutrizac | The Synthesis of Crystalline Scorodite, Feaso4.2h2o[END_REF] and ambient pressure scorodite precipitation technology. [START_REF] Filippou | Arsenic immobilization by controlled scorodite precipitation[END_REF][START_REF] Demopoulos | Precipitation of crystalline scorodite (FeAsO4 • 2H2O) from chloride solutions[END_REF][START_REF] Singhania | Temperature and seeding effects on the precipitation of scorodite from sulfate solutions under atmospheric-pressure conditions[END_REF] The local atomic structure of HFA controls its environmental behaviors and recrystallization process. Although various techniques, including X-ray diffraction (XRD), 2,[START_REF] Jia | Infrared spectroscopic and X-ray diffraction characterization of the nature of adsorbed arsenate on ferrihydrite[END_REF] Fourier transform infrared (FTIR) spectroscopy, [START_REF] Jia | Infrared spectroscopic and X-ray diffraction characterization of the nature of adsorbed arsenate on ferrihydrite[END_REF] total X-ray scattering (TXS), [START_REF] Mikutta | Total X-ray scattering, EXAFS, and Mössbauer spectroscopy analyses of amorphous ferric arsenate and amorphous ferric phosphate[END_REF] synchrotron-based extended X-ray absorption fine structure spectroscopy (EXAFS), [START_REF] Maillot | Structure and reactivity of As(III)-and As(V)-rich schwertmannites and amorphous ferric arsenate sulfate from the Carnoules acid mine drainage, France: Comparison with biotic and abiotic model compounds and implications for As remediation[END_REF][START_REF] Chen | Structural characterization of poorly-crystalline scorodite, iron(III)-arsenate co-precipitates and uranium mill neutralized raffinate solids using X-ray absorption fine structure spectroscopy[END_REF][START_REF] Paktunc | Synthesis and phase transformations involving scorodite, ferric arsenate and arsenical ferrihydrite: Implications for arsenic mobility[END_REF][START_REF] Paktunc | Characterization of ferric arsenate-sulfate compounds: Implications for arsenic control in refractory gold processing residues[END_REF][START_REF] Mikutta | New Clues to the Local Atomic Structure of Short-Range Ordered Ferric Arsenate from Extended X-ray Absorption Fine Structure Spectroscopy[END_REF] and wavelet analysis, [START_REF] Mikutta | New Clues to the Local Atomic Structure of Short-Range Ordered Ferric Arsenate from Extended X-ray Absorption Fine Structure Spectroscopy[END_REF] have been utilized to characterize the structure of HFA, there is still no agreement on its atomic structure. [START_REF] Chen | Structural characterization of poorly-crystalline scorodite, iron(III)-arsenate co-precipitates and uranium mill neutralized raffinate solids using X-ray absorption fine structure spectroscopy[END_REF][START_REF] Mikutta | Total X-ray scattering, EXAFS, and Mössbauer spectroscopy analyses of amorphous ferric arsenate and amorphous ferric phosphate[END_REF][START_REF] Paktunc | Synthesis and phase transformations involving scorodite, ferric arsenate and arsenical ferrihydrite: Implications for arsenic mobility[END_REF][START_REF] Paktunc | Characterization of ferric arsenate-sulfate compounds: Implications for arsenic control in refractory gold processing residues[END_REF][START_REF] Mikutta | New Clues to the Local Atomic Structure of Short-Range Ordered Ferric Arsenate from Extended X-ray Absorption Fine Structure Spectroscopy[END_REF][START_REF] Paktunc | Comment on "Structural characterization of poorly-crystalline scorodite, iron(III)-arsenate co-precipitates and uranium mill neutralized raffinate solids using X-ray absorption fine structure spectroscopy[END_REF][START_REF] Paktunc | Comment on "New Clues to the Local Atomic Structure of Short-Range Ordered Ferric Arsenate from Extended X-ray Absorption Fine Structure Spectroscopy[END_REF][START_REF] Jiang | Response to the comment on "Structural characterization of poorly-crystalline scorodite, iron(III)-arsenate co-precipitates and uranium mill neutralized raffinate solids using X-ray absorption fine structure spectroscopy[END_REF][START_REF] Mikutta | Response to Comment on "New Clues to the Local Atomic Structure of Short-Range Ordered Ferric Arsenate from Extended X-ray Absorption Fine Structure Spectroscopy[END_REF] Current views regarding the local atomic structure include the framework model, [START_REF] Chen | Structural characterization of poorly-crystalline scorodite, iron(III)-arsenate co-precipitates and uranium mill neutralized raffinate solids using X-ray absorption fine structure spectroscopy[END_REF][START_REF] Jiang | Response to the comment on "Structural characterization of poorly-crystalline scorodite, iron(III)-arsenate co-precipitates and uranium mill neutralized raffinate solids using X-ray absorption fine structure spectroscopy[END_REF] similar to the structure of scorodite, and the chain model, [START_REF] Paktunc | Synthesis and phase transformations involving scorodite, ferric arsenate and arsenical ferrihydrite: Implications for arsenic mobility[END_REF][START_REF] Paktunc | Characterization of ferric arsenate-sulfate compounds: Implications for arsenic control in refractory gold processing residues[END_REF][START_REF] Paktunc | Comment on "Structural characterization of poorly-crystalline scorodite, iron(III)-arsenate co-precipitates and uranium mill neutralized raffinate solids using X-ray absorption fine structure spectroscopy[END_REF][START_REF] Paktunc | Comment on "New Clues to the Local Atomic Structure of Short-Range Ordered Ferric Arsenate from Extended X-ray Absorption Fine Structure Spectroscopy[END_REF] similar to the structures of butlerite and/or fibroferrite. Both models were proposed mainly based on coordination numbers (CNs) and the interatomic distance of the As-Fe single scattering path derived from shell-fit analysis of As and Fe K-edge EXAFS spectra.

However, the As-Fe CNs of HFA calculated from EXAFS spectra could vary significantly (1.7 -4), depending on the fitting strategies and initial model (See Table S1, Supporting Information), thus leading to different conclusions about the local structure of HFA. Chen et al. [START_REF] Chen | Structural characterization of poorly-crystalline scorodite, iron(III)-arsenate co-precipitates and uranium mill neutralized raffinate solids using X-ray absorption fine structure spectroscopy[END_REF] obtained CNs of 3.2 (As-Fe) and 3.8 (Fe-As) by fitting the EXAFS spectra of HFA and proposed the scorodite-like structure, i.e., arsenate tetrahedra connecting to four FeO 6 octahedra and vice versa. In comparison, Paktunc and coworkers obtained CNs of approximately 1.8 for both the As-Fe and Fe-As paths, hence suggesting that the local structure of HFA is better described by the chain model, i.e., single chains of corner-sharing FeO 6 octahedra bridged by AsO 4 tetrahedra. [START_REF] Paktunc | Synthesis and phase transformations involving scorodite, ferric arsenate and arsenical ferrihydrite: Implications for arsenic mobility[END_REF] Mikutta et al. obtained a Fe-Fe interatomic distance of approximately 5.3 Å according to the wavelet transform analysis and the total X-ray scattering results, which is in agreement with the crystallographic data of scorodite and significantly longer than that of the chain model (approximately 3.8 Å) as in butlerite or fibroferrite. [START_REF] Mikutta | Total X-ray scattering, EXAFS, and Mössbauer spectroscopy analyses of amorphous ferric arsenate and amorphous ferric phosphate[END_REF][START_REF] Mikutta | New Clues to the Local Atomic Structure of Short-Range Ordered Ferric Arsenate from Extended X-ray Absorption Fine Structure Spectroscopy[END_REF] They proposed that HFA was composed of isolated FeO 6 octahedra bridged by AsO 4 tetrahedra.

Compared to the EXAFS spectra, XANES has much stronger oscillation and is more sensitive to the structural details of the absorbing atoms. Based on full multiple scattering (FMS) calculations, XANES features have been widely adopted to probe the interatomic distance and angle of the local structure during the last two decades. [START_REF] Benfatto | MXAN: a new software procedure to perform geometrical fitting of experimental XANES spectra[END_REF][START_REF] Benfatto | Geometrical fitting of experimental XANES spectra by a full multiple-scattering procedure[END_REF][START_REF] Benfatto | The MXAN procedure: a new method for analysing the XANES spectra of metalloproteins to obtain structural quantitative information[END_REF][START_REF] Hayakawa | Progresses in the MXAN fitting procedure[END_REF][START_REF] Monesi | Local structure in LaMnO 3 and CaMnO 3 perovskites: A quantitative structural refinement of Mn K-edge XANES data[END_REF][START_REF] Demchenko | Full multiple scattering analysis of XANES at the Cd L-3 and O K edges in CdO films combined with a soft-x-ray emission investigation[END_REF][START_REF] Smolentsev | Full-potential XANES analysis for 3D local structure investigation of isocyanide Ni complex[END_REF] Hatada et al. developed the full-potential multiple scattering (FPMS) theory and successfully simulated the XANES spectra of tetrahedrally coordinated cations in GeCl 4 or quartz. [START_REF] Hatada | Full-potential multiple scattering for x-ray spectroscopies[END_REF][START_REF] Hatada | Full-potential multiple scattering for core electron spectroscopies[END_REF][START_REF] Hatada | Full-potential multiple scattering theory with space-filling cells for bound and continuum states[END_REF] In this study, we used FPMS analysis, in combination with density functional theory (DFT) calculations, EXAFS, and vibrational spectroscopy, to probe the local atomic structure of HFA. This would aid in the understanding of its formation and transformation processes in the environment and hydrometallurgical arsenic removal/fixation operations.

EXPERIMENTAL

Synthesis of Hydrous Ferric Arsenate and Scorodite. HFA was synthesized using sodium arsenate and ferric sulfate solutions according to a previously reported method. 2,[START_REF] Jia | Infrared spectroscopic and X-ray diffraction characterization of the nature of adsorbed arsenate on ferrihydrite[END_REF] In brief, 0.01 mol of As (from Na 2 HAsO 4 •7H 2 O) and 0.01 mol of Fe (from Fe 2 (SO 4 ) 3 •5H 2 O) were added to 500 mL of double distilled water, and the pH of the mixture was adjusted from 1.3 to 1.8 with a 1 M NaOH solution. Then, the mixture was maintained at that pH and room temperature for 3 hours. The resultant solid product was separated by filtration, washed with HCl solution (pH 1.8), and freeze-dried. Scorodite (FeAsO 4 •2H 2 O) was also prepared as a reference material by heating a mixture of 0.3 M Fe(III) and 0.3 M As(V) solutions in an autoclave at 175°C for 10 h followed by filtering, rinsing four times with HCl solution (pH 1.8) and freeze drying. [START_REF] Gomez | Vibrational spectroscopy study of hydrothermally produced scorodite (FeAsO 4 •2H 2 O), ferric arsenate sub-hydrate (FAsH[END_REF] Both synthesized materials were characterized using a Rigaku D/Max 2500 X-ray diffractometer with graphite monochromatized CuK α1 radiation.

The XRD patterns of synthetic HFA showed two broad diffraction peaks at 2θ values of approximately 28° and 58° (Figure S1 in the Supporting Information), which were consistent with previously reported results. [START_REF] Jia | Infrared spectroscopic and X-ray diffraction characterization of the nature of adsorbed arsenate on ferrihydrite[END_REF][START_REF] Paktunc | Synthesis and phase transformations involving scorodite, ferric arsenate and arsenical ferrihydrite: Implications for arsenic mobility[END_REF] After digestion with concentrated hydrochloric acid, the Fe/As molar ratio of HFA was determined to be 1.02±0.01 (n = 3), and the sulfate concentration was lower than 0.01%. The XRD pattern of synthetic scorodite matched well with the standard in the Joint Committee on Powder Diffraction Standards (JSPDS) database (Figure S1).

X-ray Absorption Spectroscopy (XAS) Measurements.

To collect the XAS spectra, approximately 0.1 g of the synthesized solid was pressed as a disc with a diameter of approximately 1 cm after grinding with an agate mortar and pestle. The As and Fe K-edge XAS spectra were obtained on the XAFS beamline at the Beijing Synchrotron Radiation Facility (BSRF). The storage ring was operated at 2.5 GeV and a ring current of 250 mA. A fixed double crystal monochromator with Si(111) crystals was used to monochromatize the white beam. The absolute position of the monochromator was calibrated to 7112 eV for Fe measurements at the first inflection point in the K-edge absorption spectrum of Fe foil and at 11919 eV for As measurements by setting the first inflection point in the L III -edge absorption spectrum of Au foil. The absorption spectra were collected in the energy range of 6910-7910 eV for the Fe K-edge spectra and in the range of 11660-12680 eV for the As K-edge spectra in the transmission mode at room temperature. The scan steps were set to 5 eV/step for the pre-edge, 0.5 eV/step for the XANES, and 3-5 eV/step for the EXAFS regions. Each individual sample was scanned three times, and the averaged results are reported.

ACS Paragon Plus Environment Environmental Science & Technology

The pre-processing of spectra was performed by using the Athena program in the Demeter package (version, 0.9.20). [START_REF] Ravel | HEPHAESTUS: data analysis for X-ray absorption spectroscopy using IFEFFIT[END_REF] The pre-edge of all spectra was fitted with a straight line in the range of 150 -50 eV before the absorption edge. The post-edge absorption background was subtracted by fitting a cubic polynomial spline to the data in the energy range of 0 -750 eV after the absorption edge. For Fourier transform, the background cutoff parameter (R bkg ) was set to 0.9 and 1.0 for As and Fe, respectively. EXAFS Analysis. EXAFS data analysis was performed by fitting the Fourier transformed experimental data with the theoretical amplitude and phase functions calculated by FEFF code. The crystallographic structure of scorodite [START_REF] Hawthorne | The hydrogen positions in scorodite[END_REF] was used as the FEFF input structure for the EXAFS data of scorodite, whereas the structure of HFA optimized by DFT calculations was used as the model structure of HFA. The least-squares fitting was performed in the k ranges of 3-14 Å -1 for As and 2-14 Å -1 for Fe K-edge spectra with Artemis (Demeter package, version 0.9.20) by using a Kaiser-Bessel window function. Based on the fitting results of scorodite, the amplitude reduction factor, S 0 2 , was set to 1.0 and 0.9 for the As and Fe K-edge EXAFS data, respectively. XANES Analysis. The X-ray absorption cross sections from the rising edge energy region to 150 eV for As and 200 eV for Fe K-edge XANES spectra after the absorption edge have been analyzed using the recently developed FPMXAN software. For the Fe XANES spectra analysis, the muffin-tin (MT) approximation was used for the shape of the potential. [START_REF] Benfatto | MXAN: a new software procedure to perform geometrical fitting of experimental XANES spectra[END_REF][START_REF] Benfatto | Geometrical fitting of experimental XANES spectra by a full multiple-scattering procedure[END_REF][START_REF] Hayakawa | Progresses in the MXAN fitting procedure[END_REF] For the As K-edge XANES spectra, a Wigner-Seitz cell was constructed by adding 10 empty spheres around the As atom (Figure S3) based on the FPMS theory. [START_REF] Hatada | Full-potential multiple scattering for x-ray spectroscopies[END_REF][START_REF] Hatada | Full-potential multiple scattering for core electron spectroscopies[END_REF][START_REF] Hatada | Full-potential multiple scattering theory with space-filling cells for bound and continuum states[END_REF] The real part of self energy was calculated by using the Hedin-Lunqvist (H-L) potential. Multiple scattering of hydrogen atoms was not considered during the calculations except for the hydrogen atom linked to the central As-O bond. The program used a Monte-Carlo search to alternatively optimize the structure and non-structural parameters (e.g., Fermi energy, energy shift, normalization factor, energy independent broadening, energy dependent broadening, overlapping factor, etc.) until the global minimum of the square residue was achieved. The fitting quality was evaluated using the goodness-of-fit parameter square residue function (S 2 ), which is defined by the following equation: [START_REF] Benfatto | MXAN: a new software procedure to perform geometrical fitting of experimental XANES spectra[END_REF][START_REF] Benfatto | Geometrical fitting of experimental XANES spectra by a full multiple-scattering procedure[END_REF]

( ) [ ] ∑ ∑ = = - - = m i i m i i i th i i w y y w n S 1 1 2 1 exp 2 ε (1)
where n is the number of independent parameters, m is the number of data points, y i th and y i exp are the theoretical and experimental values of the absorption, respectively, ε i is the error in each point of the experimental data set, and w i is a statistical weight. In this study, w i = 1 was assumed and the square residual function S 2 was equaled to the statistical χ 2 function.

Molecular Orbital Theory Calculations. The structure of HFA cluster was optimized by performing DFT calculations using ORCA program (V3.0.3) in the gas phase. [START_REF] Neese | The ORCA program system[END_REF] All energy minimization calculations were carried out without symmetry and atomic constraints. After preliminary tests, hybrid functional B3LYP and def2-TZVP basis set [START_REF] Xu | Accuracy of Effective Core Potentials and Basis Sets for Density Functional Calculations, Including Relativistic Effects, As Illustrated by Calculations on Arsenic Compounds[END_REF] were utilized for geometric optimization, using the resolution of identity (RI) method. The atom-pairwise dispersion correction with the Becke-Johnson damping scheme (D3BJ) [START_REF] Grimme | Effect of the Damping Function in Dispersion Corrected Density Functional Theory[END_REF] was also applied for all calculations.

High-spin Fe 3+ , with each 3d electron occupying one of the five d-orbitals, was used for energy minimization. The spin multiplicity of the HFA cluster model (containing two Fe atoms) was set to 11 according to multiplicity = 2S + 1, where S is the spin of the unpaired electrons. [START_REF] Watts | Arsenic Adsorption onto Minerals: Connecting Experimental Observations with Density Functional Theory Calculations[END_REF] Harmonic vibrational frequencies of the geometrically optimized HFA was also calculated.

Raman Spectroscopy. The Raman spectra of synthetic HFA and scorodite were collected on a Raman microscope (Thermo Scientific™ DXR™ xi) equipped with a solid state laser diode operating at 785 nm. A 50X objective lens and 0.8 mW laser were applied. Approximately 50 mg of freeze-dried ground sample was mounted on a glass slide. All samples were measured from 60 to 3300 cm -1 in the line focus confocal mode with a detector exposure time of 8 s with the accumulation of 30 spectra. The wavenumber resolution was set at 4 cm -1 . All of the scans were measured at 10% of the laser output at the microscope exit to avoid radiation damage.

Before the measurements, the instrument was calibrated to 520 cm -1 by using a standard silicon sample.

Infrared Spectroscopy. FTIR spectra were scanned on a Nicolet 6700 Fourier transform infrared spectrometer (Thermo Fisher Scientific, USA). The spectra were collected in a range of 400-4000 cm -1 with a spectral resolution of 4 cm -1 and 200 co-added scans. Each sample of less than 0.050 mg was mixed with high-purity KBr and then pressed into a disk. Immediately, samples were recorded in the transmission mode. Peak fitting of infrared spectra was performed using a Lorentzian-Gaussian cross function with the minimum number of peaks. All infrared and Raman spectra were manipulated using the software OMNIC (v 8.2). The cluster radius used is 8 Å, containing 166 and 150 atoms for the As and Fe K-edge XANES spectra simulations, respectively. The results show that the experimental spectra of scorodite were accurately reproduced (S 2 = 7.7 for As and S 2 = 3.9 for Fe). The small discrepancy between the theoretical and experimental spectra in the first 20 eV from the edge is possibly due to the limited cluster size or an overestimation of the inelastic losses. [START_REF] Benfatto | Geometrical fitting of experimental XANES spectra by a full multiple-scattering procedure[END_REF] The controversy for the local atomic structure of HFA focused on the number and positions of FeO 6 octahedra/AsO 4 tetrahedra coordinating with the central atoms.

RESULTS AND DISCUSSION

XANES

Since HFA is the precursor of crystalline scorodite, [START_REF] Jia | Infrared spectroscopic and X-ray diffraction characterization of the nature of adsorbed arsenate on ferrihydrite[END_REF][START_REF] Nishimura | Crystalline phases in the system Fe(III)-As(V)-H 2 O at 25C[END_REF] it could be intuitively assumed that FeO 6 octahedra connect with AsO 4 tetrahedra in a similar structure to that of scorodite during the formation of the HFA. Therefore, we extracted the local structure of AsO 4 tetrahedra and FeO 6 octahedra from the crystallographic data of scorodite as the initial structure to distinguish which structure is proximal to the local structure of HFA (Figure 2). The interatomic distances and angles of the extracted local structure are summarized in Table S2 in the Supporting Information.

The theoretical As and Fe K-edge XANES spectra of different clusters of AsO 4 tetrahedra and FeO 6 octahedra were calculated. Only the non-structural parameters were optimized by fixing the atomic positions of each cluster. The comparisons between the theoretical spectra and experimental data are illustrated in Figure 2c and 2d. For As K-edge XANES spectra, it can be observed that the calculated spectrum of the configuration A1, representing one AsO 4 tetrahedron connecting with four FeO 6 octahedra, has significantly stronger oscillations than other clusters in the energy range of 50-80 eV, where it is strongly influenced by the backscattering of Fe atoms. As a result, the S 2 of 6.9 for configuration A1 is higher than those derived from clusters A5, A6, and A7 (one AsO 4 tetrahedron connects with two FeO 6 octahedra), indicating that the A1 configuration is not the best. Consistently, the Fe K-edge XANES spectra showed that the configuration F1 (one FeO 6 octahedron connects with four AsO 4 tetrahedra) did not give the smallest S 2 either, implying that the FeO 6 octahedron is less likely to connect with 4 AsO 4 tetrahedra in a manner similar to that of scorodite. These results suggested that in HFA, one AsO 4 tetrahedron/FeO 6 octahedron is more likely to connect with two FeO 6 octahedra/AsO 4 tetrahedra as a chain rather than a scorodite-like structure.

Furthermore, the XANES spectra are also very sensitive to the Fe-O-As angle between AsO 4 tetrahedra and FeO 6 octahedra. [START_REF] Benfatto | Geometrical fitting of experimental XANES spectra by a full multiple-scattering procedure[END_REF]44 It can be clearly observed from Å, with an average of 1.67±0.046 Å, which was comparable to the values derived from DFT calculations and EXAFS data (Table 1). The refined Fe-O bond lengths ranged from 1.84 to 2.15 Å, with an average of 1.99±0.11 Å, which was slightly shorter than those from DFT calculations and EXAFS data (Table 1). In addition, the FPMS optimization of the Fe K-edge XANES spectra gave an average interatomic Fe-Fe distance of 5.22 Å, which is highly consistent with the DFT result (~ 5.25 Å)

and the value of approximately 5.3 Å derived from the wavelet analysis reported by Mikutta et al. [START_REF] Paktunc | Speciation and characterization of arsenic in gold ores and cyanidation tailings using X-ray absorption spectroscopy[END_REF] These results further implied that our chain structure was reasonable for HFA. [START_REF] Paktunc | Synthesis and phase transformations involving scorodite, ferric arsenate and arsenical ferrihydrite: Implications for arsenic mobility[END_REF][START_REF] Paktunc | Comment on "Structural characterization of poorly-crystalline scorodite, iron(III)-arsenate co-precipitates and uranium mill neutralized raffinate solids using X-ray absorption fine structure spectroscopy[END_REF][START_REF] Paktunc | Comment on "New Clues to the Local Atomic Structure of Short-Range Ordered Ferric Arsenate from Extended X-ray Absorption Fine Structure Spectroscopy[END_REF] To verify this hypothesis, the structures of butlerite and fibroferrite were modified by replacing the S atoms with As atoms, and were optimized using DFT calculations. The optimized structures are shown in Figure S5 in the Supporting Information. The Fe-Fe interatomic distances in optimized butlerite-like and fibroferrite-like structures were 3.78 Å and 3.65 Å on average, respectively, which were significantly shorter than those from the DFT calculations (5.25 Å), the FPMS refinement (5.22 Å) and the wavelet analysis of the Fe K-edge EXAFS data (~5.3 Å). [START_REF] Paktunc | Characterization of ferric arsenate-sulfate compounds: Implications for arsenic control in refractory gold processing residues[END_REF] Furthermore, the theoretical As and Fe K-edge XANES spectra calculated using FPMS theory based on the optimized structures were compared with the experimental data. Significant differences were observed for both As (S 2 = 10.3 for butlerite and S 2 = 7.9 for fibroferrite) and Fe (S 2 = 2.2 for butlerite and S 2 = 4.2 for fibroferrite) (see Figure S6 in the Supporting Information). These results indicated that the chain structure of corner sharing FeO 6 was unlikely to be a reasonable model to describe the local atomic structure of HFA.

EXAFS Analysis. Fourier transforms of As and Fe K-edge EXAFS data of synthetic scorodite and HFA are compared in Figure 5. Consistent with previous reports, [START_REF] Chen | Structural characterization of poorly-crystalline scorodite, iron(III)-arsenate co-precipitates and uranium mill neutralized raffinate solids using X-ray absorption fine structure spectroscopy[END_REF][START_REF] Mikutta | New Clues to the Local Atomic Structure of Short-Range Ordered Ferric Arsenate from Extended X-ray Absorption Fine Structure Spectroscopy[END_REF] the magnitudes of the Fourier transform peaks of HFA at R + ∆R ~ 2.9 Å for the As K-edge EXAFS spectra and R + ∆R ~ 3.0 Å for the Fe K-edge EXAFS spectra, with both attributed to the As-Fe oscillations, are less than half of those of crystalline scorodite. This was thought to be the result of lower CNs of the nearest Fe/As atoms around the absorbers or large variation in the Fe-As interatomic distance. 22 The As and Fe K-edge EXAFS spectra were fitted by using the DFT optimized cluster structure of HFA as a model structure. The detailed least-square fitting results are summarized in Table S3 in the Supporting Information. The satisfactory goodness-of-fit parameters, R-factor (0.008 for As and 0.006 for Fe) and reduced χ 2 (110 for As and 28 for Fe), were obtained for both the As and Fe K-edge EXAFS data. As described above, comparisons in Table 1 show that the mean interatomic distances of As-O (1.68 Å, CN = 4), Fe-O (2.00 Å, CN = 6), and As-Fe (3.30 Å, CN = 1.5) are comparable to those derived from DFT calculations and FPMS analysis. These data further demonstrated that the chain structure of HFA proposed in this study is reliable.

Infrared and Raman Spectroscopy. Figure 6, Figure S7 and Figure S8 illustrate the infrared and Raman spectra of synthetic HFA and scorodite. The assignments of infrared and Raman bands for scorodite and HFA are listed in Table S4 in the Supporting Information.

The deconvoluted bands of infrared (400 -1200 cm -1 ) and Raman spectra (60 -1200 cm -1 ) for HFA are consistent (Figure 6). Due to the poorly crystalline nature, the bands on infrared and Raman spectra of HFA are poorly resolved and may represent composite of different vibrations. The shape and positions of the infrared and Raman bands of HFA are different from those of scorodite indicating that the protonation and/or metal complexation of AsO 4 tetrahedra in HFA was different from in scorodite. [START_REF] Xu | Accuracy of Effective Core Potentials and Basis Sets for Density Functional Calculations, Including Relativistic Effects, As Illustrated by Calculations on Arsenic Compounds[END_REF][START_REF] Watts | Arsenic Adsorption onto Minerals: Connecting Experimental Observations with Density Functional Theory Calculations[END_REF]45 According to the DFT calculated frequencies and previously reported infrared and Raman spectra of (hydrogen) arsenate group-bearing solids (Figure S9 and Table S4), [45][46][47][48][49][START_REF] Gomez | Molecular and structural investigation of yukonite (synthetic & natural) and its relation to arseniosiderite[END_REF][START_REF] Chtioui | Crystal structure, thermal analysis and IR spectroscopic investigation of (C 6 H 9 N 2 )H 2 XO 4 (X = As, P)[END_REF] the intense HFA infrared bands at 827 and the Raman bands at 825 may be attributed to the composite of ν 1 AsO 3 symmetric stretching vibration from HAsO 4 2-groups and As-OFe stretching vibrations from H 2 AsO 4 groups. [START_REF] Yang | Complexation of arsenate with ferric ion in aqueous solutions[END_REF] The infrared band at 877 cm -1 and Raman band at 889 cm -1 could be assigned to the ν 3 AsO 3 antisymmetric stretching vibration from HAsO 4 2-groups. [47][48][49] The infrared band at 951 cm -1 is possibly attributed to the ν 3 AsO 2 antisymmetric stretching vibration from H 2 AsO 4 -group. [START_REF] Chtioui | Crystal structure, thermal analysis and IR spectroscopic investigation of (C 6 H 9 N 2 )H 2 XO 4 (X = As, P)[END_REF] The Raman bands at 985 cm -1 and weak infrared bands at 1048 and 1108 cm -1 may be attributed to the ν 1 symmetric stretching vibration of SO 4 2-impurity. [START_REF] Gomez | Molecular and structural investigation of yukonite (synthetic & natural) and its relation to arseniosiderite[END_REF][START_REF] Zhang | Incorporation of arsenic into gypsum: Relevant to arsenic removal and immobilization process in hydrometallurgical industry[END_REF] The shoulder bands at 748 cm -1 (infrared)

and 766 cm -1 (Raman), locating in the range of As-OH stretching vibrations (707-772 cm -1 ) [45][46][47][48][START_REF] Gomez | Molecular and structural investigation of yukonite (synthetic & natural) and its relation to arseniosiderite[END_REF] , could be assigned to the composite of As-OH stretching vibrations from H 2 AsO 4 -groups and As-OFe stretching vibrations from HAsO 4 2-groups. Other observed bands were assigned to the following vibrations: 213 and 271 cm -1 in the Raman spectrum to the lattice vibrations; 424 cm -1 of the infrared spectrum and 360 and 412 cm -1 in the Raman spectrum to the composite of the ν 2 HAsO S5.

  Analysis. The normalized As and Fe K-edge XANES spectra of scorodite and HFA are compared in Figure1aand 1b. The figures show obvious differences at 11876-11888 eV and 11934-11980 eV for As as well as 7126-7135 eV and 7183-7300 eV for Fe K-edge XANES spectra, indicating that the structure of HFA is different from that of scorodite. The As and Fe K-edge XANES spectra of scorodite were simulated using FPMS calculations (Figure1c and 1d). The crystallographic data of scorodite with the space group Pcab (a = 8.937 Å, b = 10.278 Å, c = 9.996 Å, α = β = γ = 90°) were used as the starting simulation model.[START_REF] Hawthorne | The hydrogen positions in scorodite[END_REF]

Figures 2c and 2d 8 - 1

 81 Figures 2c and 2d that the clusters of AsO 4 tetrahedra and FeO 6 octahedra with

Figure 1 .Figure 2 .

 12 Figure 1. Arsenic (a) and iron (b) K-edge XANES spectra of HFA (black lines) and

Figure 4 .

 4 Figure 4. Comparison of the experimental and theoretical data of non-structural

34 Figure 5 .

 345 Figure 5. Arsenic and iron K-edge k 3 -weighted EXAFS spectra (left) and the

Figure 6 .

 6 Figure 6. Deconvoluted infrared (400 -1200 cm -1 ) (b) and Raman (60 -1200 cm -1 ) (c)

  Paktunc and coworkers argued that the local structure of HFA is similar to those of the ferric sulfates butlerite (FeSO 4 (OH)•2H 2 O) and fibroferrite (FeSO 4 (OH)•5H 2 O), which consist of corner-sharing FeO 6 octahedra single chains bridged by SO 4 tetrahedra.

Table 1 .

 1 AsO 4 ) 3 •5.5H 2 O) at 25°C depending on the pH values. 5 It would be easier for the HFA with our model structure to convert to ferric hydrogen arsenate minerals (FeH 3 (AsO 4 ) 2 •10H 2 O, and Fe(H 2 AsO 4 ) 3 •5.5H 2 O) than the scorodite model which consists of isolated AsO 4 tetrahedron connecting to four FeO 6 octahedra, and vice versa. It is also easier for the structure we proposed to transform to scorodite than the butlerite and/or fibroferrite-like structure consisting of single chains of corner-sharing FeO 6 octahedra bridged by AsO 4 tetrahedra which needs breakage of Fe-O-Fe bonds. The findings of the present work could also explain the reason why HFA showed Comparison of interatomic distance (Å) (coordination number) of scorodite and HFA among the results derived from crystallographic data (XRD), total X-ray scattering (TXS), As and Fe K-edge EXAFS spectra analysis, DFT calculations, and full-potential multiple scattering analysis (FPMS) of As and Fe K-edge XANES spectra

	4	2-/H 2 AsO 4	-

ACS Paragon Plus Environment Environmental Science & Technology

ACS Paragon Plus Environment Environmental Science & Technology

ACKNOWLEDGEMENTS

We thank the staff of the XAFS beamline at the Beijing Synchrotron Radiation Facility for their help with the As and Fe K-edge XAS spectral measurements. This ACS Paragon Plus Environment Environmental Science & Technology work was financially supported by the National Natural Science Foundation of China (No. 41530643), the Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDB14020203) and the National Natural Science Foundation of China (Nos. 41303088 and 41473111). We also gratefully acknowledge the European FP7 MSNano network under Grant Agreement (No. PIRSES-GA-2012-317554), COST Action MP1306 EUSpec, and the Marie Curie Intra-European Fellowship MS-BEEM (No. PIEF-GA-2013-625388). ACS Paragon Plus Environment Environmental Science & Technology

significantly higher solubility than scorodite [START_REF] Langmuir | Solubility products of amorphous ferric arsenate and crystalline scorodite (FeAsO 4 •2H 2 O) and their application to arsenic behavior in buried mine tailings[END_REF] which needs to break four covalent bonds to dissolve.

ASSOCIATED CONTENT

Supporting Information. Additional information contains the XRD patterns, the SEM images, the Wigner-Seitz cell, the DFT-optimized butlerite-like and fibroferrite-like chain structures and their the theoretical As and Fe K-edge XANES spectra, infrared and Raman spectra, previously reported As-Fe CNs, interatomic distance and bond angle in the local scorodite structure, the EXAFS fitting parameters, and frequency assignments.

AUTHOR INFROMATION

Corresponding Author

*Telephone: Tel: +86 (24) 83970503. Email: yongfeng.jia@iae.ac.cn.

Present Address

No.72, Wenhua Road, Shenyang, China, 110016.

Notes

The authors declare no competing financial interest. This supporting information contains 9 figures, 4 tables, and references.