A Hybrid Mechanism for the Synechocystis Arsenate Reductase Revealed by Structural Snapshots during Arsenate Reduction*

Background: Arsenate reductases catalyze the reduction of arsenate to arsenite. Results: Structures of Synechocystis arsenate reductase in different reaction stages are determined and a hybrid catalytic mechanism is established. Conclusion: The mechanism involves an intramolecular thiol-disulfide cascade during arsenate reduction and subsequent enzyme reactivation by the glutathione-glutaredoxin pathway. Significance: The study provides insights into the evolution of arsenate reductases. Evolution of enzymes plays a crucial role in obtaining new biological functions for all life forms. Arsenate reductases (ArsC) are several families of arsenic detoxification enzymes that reduce arsenate to arsenite, which can subsequently be extruded from cells by specific transporters. Among these, the Synechocystis ArsC (SynArsC) is structurally homologous to the well characterized thioredoxin (Trx)-coupled ArsC family but requires the glutaredoxin (Grx) system for its reactivation, therefore classified as a unique Trx/Grx-hybrid family. The detailed catalytic mechanism of SynArsC is unclear and how the “hybrid” mechanism evolved remains enigmatic. Herein, we report the molecular mechanism of SynArsC by biochemical and structural studies. Our work demonstrates that arsenate reduction is carried out via an intramolecular thiol-disulfide cascade similar to the Trx-coupled family, whereas the enzyme reactivation step is diverted to the coupling of the glutathione-Grx pathway due to the local structural difference. The current results support the hypothesis that SynArsC is likely a molecular fossil representing an intermediate stage during the evolution of the Trx-coupled ArsC family from the low molecular weight protein phosphotyrosine phosphatase (LMW-PTPase) family.


Evolution of enzymes plays a crucial role in obtaining new
biological functions for all life forms. Arsenate reductases (ArsC) are several families of arsenic detoxification enzymes that reduce arsenate to arsenite, which can subsequently be extruded from cells by specific transporters. Among these, the Synechocystis ArsC (SynArsC) is structurally homologous to the well characterized thioredoxin (Trx)-coupled ArsC family but requires the glutaredoxin (Grx) system for its reactivation, therefore classified as a unique Trx/Grx-hybrid family. The detailed catalytic mechanism of SynArsC is unclear and how the "hybrid" mechanism evolved remains enigmatic. Herein, we report the molecular mechanism of SynArsC by biochemical and structural studies. Our work demonstrates that arsenate reduction is carried out via an intramolecular thiol-disulfide cascade similar to the Trx-coupled family, whereas the enzyme reactivation step is diverted to the coupling of the glutathione-Grx pathway due to the local structural difference. The current results support the hypothesis that SynArsC is likely a molecular fossil representing an intermediate stage during the evolution of the Trx-coupled ArsC family from the low molecular weight protein phosphotyrosine phosphatase (LMW-PTPase) family.
Arsenate reductase (ArsC) 4 comprises several families of enzymes that adopt different protein folds but have acquired a similar role in arsenic detoxification through convergent evolution (1). The enzyme catalyzes the reduction of arsenate (H 2 AsO 4 Ϫ /HAsO 4 2Ϫ ) to arsenite (H 3 AsO 3 ), which can be subsequently extruded from cells via specific transporters (1,2). After reducing one molecule of arsenate, the arsenate reductase itself becomes oxidized and inactivated. Upstream electron donors are thus essential for enzyme reactivation.
Among the well characterized arsenate reductases, a family that utilizes thioredoxin (Trx) as the upstream redox partner is represented by the Gram-positive bacteria Staphylococcus aureus plasmid pI258 ArsC and Bacillus subtilis chromosomal ArsC proteins, and termed the Trx-coupled ArsC family (3)(4)(5)(6)(7)(8)(9). Structural and biochemical evidences suggest that the family is an evolutionary descendent of a class of low molecular weight protein phosphotyrosine phosphatases (LMW-PTPases), and its arsenate reducing ability is drafted from the PTPase activity (5,8). On the other hand, two distinct ArsC families utilizing the glutathione (GSH)-glutaredoxin (Grx) redox pathway are represented by the Gram-negative bacteria Escherichia coli plasmid R773 ArsC and the yeast Saccharomyces cerevisiae ACR2p (or Leishmania major LmACR2) proteins (10 -14). These two families are structurally dissimilar, and are both unrelated to the Trx-coupled ArsC family (1,5). In contrast, a newly discovered arsenate reductase from cyanobacteria Synechocystis sp. strain PCC 6803 (abbreviated as SynArsC) shows high sequence and structural homology with both LMW-PT-Pase and Trx-coupled ArsC (15)(16)(17), whereas its requirement of the GSH-Grx system as the upstream electron donor instead of the Trx redox system stands out to be perplexing (15). Because the SynArsC protein shares sequence homology with neither the E. coli R773 ArsC (10 -12) nor yeast ACR2p (13)(14), no conclusions could be drawn concerning its detailed catalytic mechanism. The exact role of GSH/Grx in arsenate reduction by SynArsC, as well as the molecular mechanism of why SynArsC shows selectivity of Grx over Trx system, remains unclear.
As a "hybrid" arsenate reductase, SynArsC probably represent an intermediate stage during evolution of arsenate reductases (1,15). Elucidation of its enzymatic mechanism would provide intriguing insights for understanding the evolutionary relationship between arsenate reductase and PTPase families. Herein we present a structure-based mechanism of SynArsC by using a combination of biochemical and biophysics methods, with four SynArsC structures representing different reaction stages during arsenate reduction determined. Our results clearly establish a hybrid mechanism that can be divided into two stages: the arsenate reduction stage and the enzyme reactivation stage. SynArsC alone is required in the arsenate reduction stage, utilizing a thiol-disulfide cascade mechanism essentially similar to the Trx-coupled ArsC family (5,6,8,9). Nevertheless, the oxidation of SynArsC results in a "loop to helix" transition at the active site, which is exactly the opposite of the "helix to loop" transition as observed in the Trx-coupled ArsC family (5,6,8,9). The different local conformation is incompatible with Trx interaction, and requires the GSH-Grx system as the upstream reductant. Our current results provide the structural insights for understanding the hybrid mechanism of SynArsC, and favor the hypothesis that SynArsC may represent an earlier stage during the enzyme evolution from LMW-PTPase to arsenate reductase.

Experimental Procedures
Protein Expression and Purification-The slr0946 gene encoding SynArsC was cloned into the pET-28a(ϩ) vector (Novagen) with or without the N-terminal His tag, and the resulting plasmid was used as templates in PCR amplification with primers designed to generate the C8S, C13S, C35S, C80S, C82S, C13S/C35S, C13S/C35S/C82S, and C8S/C13S/C35S mutants. Similar to as previously reported (18), the plasmids were transformed into the E. coli BL21(DE3) strain (Novagen) for expression. The culture was first grown in 1 liter of Luria-Bertani medium at 35°C with 50 mg/liter of kanamycin. When the A 600 reached 0.8, the cells were collected by centrifugation at 4,000 ϫ g and resuspended in 500 ml of M9 minimal medium (with kanamycin) with NH 4 Cl and glucose as the nitrogen and carbon sources. After shaking for 1 h at 35°C, protein expression was induced by adding isopropyl ␤-D-thiogalactoside to a final concentration of 0.4 mM. After 8 h induction, the cells were centrifuged at 7,000 ϫ g at 4°C, resuspended in 30 mM Tris buffer (pH 8.5), and frozen at Ϫ80°C. For preparation of 13 C/ 15 N-labeled or 15 N-labled proteins, 15 NH 4 Cl and [ 13 C 6 ]glucose or 15 NH 4 Cl only were used in the M9 media.
The proteins were initially purified either using anion-exchange chromatography (Q-Sepharose fast flow column, GE Healthcare) eluted with a 0 -0.5 M NaCl gradient for constructs without the N-terminal His tag, or nickel-affinity chromatography (nickel-nitrilotriacetic acid column, Qiagen) for constructs with the N-terminal His tag. The N-terminal His tag was removed by thrombin (Sigma) incubation overnight at room temperature. A subsequent gel-filtration chromatography (Superdex-75) using an ÄKTA FPLC system (GE Healthcare) was further used to obtain protein samples with Ͼ90% purity as judged by SDS-PAGE. The samples prepared with or without His tags exhibit similar catalytic activities as well as NMR spectral properties.
NMR Sample Preparations-The sample of the Cys 8 -Cys 80 disulfide-bridged SynArsC intermediate (int-SynArsC) was prepared using the C13S/C35S/C82S triple mutant. The protein was first purified in the presence of dithiothreitol (DTT) to ensure a fully reduced state. The DTT was subsequently removed by gel filtration, and the SynArsC C13S/C35S/C82S sample was mixed with arsenate at a molar ratio of 1:5 and incubated for 5 min at room temperature followed by a second gel filtration chromatography. Formation of the Cys 8 -Cys 80 disulfide bridge was verified by Ellman's test of free thiols using 5,5Ј-dithiobis-2-nitrobenzoic acid as the reagent (19), mass spectroscopy (20,21), and NMR spectroscopy (22).
The sample of the Cys 80 -Cys 82 disulfide-bridged oxidized state (oxi-SynArsC) was prepared using the C8S/C13S/C35S triple mutant. After removal of DTT by gel filtration, the ArsC C8S/C13S/C35S protein was incubated with 0.03% H 2 O 2 at room temperature, followed by a second gel filtration chromatography. The protein was kept at a low concentration (Ͻ0.02 mM) during the incubation to avoid intermolecular disulfide formation. Formation of the Cys 80 -Cys 82 disulfide bridge was verified by Ellman's test (19), mass spectroscopy (20,21), and NMR spectroscopy (22).
The NMR sample of reduced SynArsC in the absence of ligand (red-SynArsC) was prepared in 50 mM Tris-HCl buffer (pH 7.0) with 50 mM NaCl and 40 mM DTT. The sample of phosphate-bound SynArsC (Pi-SynArsC) was prepared in 50 mM sodium phosphate buffer (pH 7.0) with 50 mM NaCl and 40 mM DTT. The int-SynArsC sample was dissolved in 50 mM Tris-HCl buffer (pH 7.0) with 50 mM NaCl, and the oxi-SynArsC sample was dissolved in 50 mM sodium phosphate buffer (pH 7.0) with 50 mM NaCl. In all NMR samples, 10% D 2 O was added for field lock, and 0.01% 2,2-dimethyl-2-silapentanesulfonic acid was added as the internal chemical shift reference.
Detection of Arsenic Redox Species-For detection of the arsenic species, high performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS) was used (23,24). Wild-type or mutant SynArsC (1 M) was incubated with an equal molar of arsenate in 50 mM Tris-HCl buffer (pH 7.0) with 50 mM NaCl for 30 min at room temperature before loading onto the HPLC column (Hamilton PRP X100) for separation of arsenic species by anion exchange chromatography. The eluents containing 20 mM K 2 HPO 4 -KH 2 PO 4 (pH 5.9) were pumped at a flow rate of 1 ml/min, and mixed with NaBH 4 solution to generate volatile arsine, which was subsequently purged by an argon flux to be detected by atomic fluorescence spectrometry. The experiments were performed using the AF-610D2 chromatography-atomic fluorescence spectrometry instrument system (Beijing Rayleigh Analytical Instrument Corp., China).
Mass Spectroscopy-Mass spectrometry analyses of the molecular weights of SynArsC-C13S/C35S/C82S and C8S/ C13S/C35S mutants in both reduced and oxidized states were performed using a high-resolution electrospray ionization-Fourier transform-ion cyclotron resonance (ESI-FT-ICR) mass spectrometer (APEX Qe, Bruker Daltonics) equipped with a 9.4 tesla actively shielded magnet. For a straightforward interpre-tation, the obtained positive ion mass spectra were charge-deconvoluted (20). Oxidation of the two mutants was achieved by incubation with arsenate and H 2 O 2 , respectively.
For determination of disulfide bonds, protein samples were subjected to digestion using a combination of multiple proteases trypsin, Asp-N and Glu-C (Promega). The resulting peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described previously (21). Briefly, nanoflow reversed-phase LC separation was carried out on an EASY-nLC 1000 System (Thermo Scientific). Peptides eluted from the capillary column were electrosprayed directly onto a linear ion trap mass spectrometer (LTQ Velos, Thermo Scientific) for MS and MS/MS analyses.
NMR Spectroscopy-NMR experiments were carried out at 25°C on Bruker Avance 500-, 600-, and 800-MHz spectrometers equipped with triple-resonance cryoprobes. Spectra for chemical shift assignments have been reported previously (18), and three-dimensional 15 N-and 13 C-edited nuclear Overhauser effect spectroscopy (NOESY)-heteronuclear single quantum coherence (HSQC) spectra (mixing time 100 ms) were collected to confirm the assignments and generate interproton distance constraints for structure calculation. The backbone amide residual dipolar couplings were also measured for the Pi-SynArsC sample by using the pentaethylene glycol dodecyl ether (C 12 E 5 ) and n-hexane liquid crystal as the alignment medium (25), and by calculating the splitting differences using the 1 H-15 N in-phase-anti-phase HSQC experiment isotropic and weakly aligned samples (26).
Structure Calculations-The structures of SynArsC in different states were calculated by CYANA and refined by AMBER (27)(28)(29). Inter-proton distance restraints were generated from nuclear Overhauser effect (NOE) in the NOESY-HSQC spectra. Dihedral angles and constraints were determined from backbone chemical shifts using TALOS (30). Hydrogen bond restraints were obtained from hydrogen-deuterium exchange experiments in combination with the NOE pattern and secondary structural information. For Pi-SynArsC, backbone amide residual dipolar coupling restraints were also used. The CAN-DID module of the CYANA program was used to generate the initial structures (28). The 20 structures with the lowest target functions were selected as models for SANE to extend the NOE assignments (31). 200 structures were calculated using CYANA interactively and 100 structures with the lowest target functions were selected for further refinement by AMBER. Finally, the 20 lowest-energy conformers were selected to represent the solution structure of SynArsC in different states.

Results
The Initial Stage of Arsenate Reduction-SynArsC contains a total of five cysteine residues, Cys 8 , Cys 13 , Cys 35 , Cys 80 , and Cys 82 , among which only the Cys 8 , Cys 80 , and Cys 82 residues were identified essential for arsenate reduction activity (15,17). Sequence alignment with the Trx-coupled S. aureus and B. subtilis ArsC shows that the first and second cysteines (Cys 8 /Cys 80 of SynArsC and Cys 10 /Cys 82 of S. aureus and B. subtilis ArsC) are conserved, whereas the third cysteine (Cys 82 of SynArsC and Cys 89 of S. aureus and B. subtilis ArsC) is positioned differently (Fig. 1A). As depicted in Fig. 1, B and C, one of the most significant differences between the Trx-and Grx-coupled arsenate reductases is that the Trx-coupled ArsC from either S. aureus or B. subtilis is able to complete the reduction of arsenate and release of arsenite all by itself (5,6,8,9), whereas the Grx-coupled arsenate reductases from either E. coli or yeast require two molecules of GSH to complete this reaction (11)(12)(13). To clarify to which catalytic mechanism SynArsC more closely resembles, it is therefore critical to evaluate its ability to catalyze one cycle of arsenate reduction on its own.
To directly monitor the arsenic redox state, we incubated equal molar of arsenate with wild-type SynArsC or mutants, which was subsequently subjected to HPLC-HG-AFS analysis (23,24). The results show that in the fully reduced state, wildtype SynArsC is capable of reducing arsenate in the absence of GSH. Among the six mutants tested (C8S, C13S, C35S, C80S, C82S, and C13S/C35S/C82S), only the serine substitutions of Cys 8 and Cys 80 led to inactive reactions ( Fig. 2A). In particular, the C13S/C35S/C82S triple mutant maintains the ability to reduce arsenate in the absence of GSH or other reducing components, indicating the critical roles of Cys 8 and Cys 80 during arsenate reduction. Furthermore, we verified by mass spectroscopy that after incubation of SynArsC-C13S/C35S/C82S mutant with arsenate, the molecular mass of the protein is reduced by 2 Da, corresponding to the loss of two hydrogen atoms (Fig. 2B). Further evidence from proteolysis and tandem mass spectrometry (MS/MS) confirmed the formation of a Cys 8 -Cys 80 disulfide bond (Fig. 2C). Therefore, the initial step of arsenate reduction by SynArsC involves only the active cysteines Cys 8 and Cys 80 , and the reaction results in the formation of a Cys 8 -Cys 80 disulfide-bridged intermediate.
Our results appear to contradict the previous report by Li and co-workers (15), in which an oxidized form of SynArsC with the Cys 80 -Cys 82 disulfide bond was observed and suggested to be active for arsenate reduction. However, the enzymatic activity assays were conducted by tracing the changes of NADPH absorbance, and the reaction systems contain an excess of GSH (0.5-10 mM in Refs. 15-17) compared with the SynArsC concentrations (ϳ 1-5 M in Refs. [15][16][17]. Therefore the Cys 80 -Cys 82 disulfide bond is readily reduced in the presence of excess GSH as confirmed by NMR spectroscopy (data not shown), and the kinetic parameters should actually correspond to fully reduced SynArsC. Furthermore, Synechocystis cells are buffered by GSH and GSSG with concentrations of ϳ3.0 and 0.23 mM (32), corresponding to a highly reducing environment with an estimated oxidation-reduction midpoint potential E m of Ϫ198 -220 mV depending on the pH value (pH 7.0 or 7.5). The SynArsC Cys 80 -Cys 82 disulfide bond has an E m value of Ϫ165 mV at pH 7.0 (17) and is therefore expected to be reduced in vivo, further supporting our proposed scenario with a fully reduced SynArsC at the initial catalytic stage.
The Intramolecular Thiol-Disulfide Cascade-To obtain more details of the arsenate reduction mechanism by SynArsC, we used two-dimensional solution NMR spectroscopy to monitor the conformational changes of 15 N-labeled SynArsC upon incubation with arsenate. As expected, incubation of the SynArsC-C13S/C35S/C82S triple mutant (retaining Cys 8 and Cys 80 ) with arsenate results in a single set of peaks corresponding to the Cys 8 -Cys 80 disulfide-bridged state. However, incuba-tion of the SynArsC-C13S/C35S double mutant, which retains the three essential cysteine residues Cys 8 , Cys 80 , and Cys 82 , results in the appearance of two new sets of amide cross-peaks accompanied by the disappearance of the original peak set corresponding to the reduced state (Fig. 3A). This observation suggests the co-existence of two new conformational states after reaction with arsenate. Similar results could also be obtained by incubating wild-type SynArsC with arsenate.
Interestingly, the peak positions of the two conformations can be superimposed reasonably well with that of the oxidized samples of SynArsC-C13S/C35S/C82S (harboring a Cys 8 -Cys 80 disulfide bond) and SynArsC-C8S/C13S/C35S (harboring a Cys 80 -Cys 82 disulfide bond) mutants (Fig. 3B), strongly suggesting that arsenate incubation results in a mixture of Cys 8 -Cys 80 and Cys 80 -Cys 82 disulfide-bridged conformations. This speculation is further confirmed by MS/MS analysis of digested proteins in that both peptides containing Cys 8 -Cys 80 and Cys 80 -Cys 82 disulfide bonds were detected in the arsenate-incubated SynArsC-C13S/C35S sample (Fig. 2, D and E).
Because the Cys 8 -Cys 80 disulfide-bridged conformation has been demonstrated to be the product of the initial reaction step, the Cys 80 -Cys 82 disulfide-bridged state most probably result  (5,6,8,9). C, schematic presentation of the catalytic mechanisms of the Grx-coupled ArsC families (11)(12)(13). The catalytic active cysteine depicted in the scheme corresponds to Cys 12 in E. coli plasmid R773 ArsC and Cys 76 in yeast Acr2p. D, schematic presentation of the hybrid catalytic mechanism of SynArsC as proposed in the current study. from a subsequent intra-molecular thiol-disulfide transfer. However, the population of the Cys 8 -Cys 80 disulfide-bridged state appears larger than the Cys 80 -Cys 82 disulfide-bridged state as estimated from the relative intensities of the two peak sets. Neither extension of incubation time nor addition of excess arsenate succeeded in increasing the population of the later conformation. Moreover, overnight incubation with the prokaryotic disulfide isomerase DsbC from E. coli (33,34), with DsbC in the reduced state and SynArsC:DsbC molar ratios of 1:0.2, 1:0.5, and 1:1, also failed to change the relative populations of the two peak sets. These results suggest that the SynArsC protein may have reached equilibrium between the two redox states under our experimental condition and the thiol-disulfide transfer from Cys 8 -Cys 80 to Cys 80 -Cys 82 is incomplete in the absence of the upstream reductant GSH.
The above results show clear resemblance to the Trx-coupled ArsC family, whereas at the same time display significant different characteristics (4 -9). Both families utilize the first and second essential cysteines for arsenate reduction and form a disulfide-bridged intermediate state (Fig. 1, A, B, and D). An intra-molecular thiol-disulfide transfer reaction subsequently takes place to generate a state in which the second and third essential cysteines are covalently linked. In the Trx-coupled ArsC family, this intra-molecular thiol-disulfide transfer reaction occurs fast and efficiently as arsenate titration of wild-type B. subtilis ArsC quickly converts the protein spectra from the reduced state into the Cys 82 -Cys 89 disulfide-bridged oxidized state, without the observation of peaks corresponding to the Cys 10 -Cys 82 disulfide-bridged intermediate (9). In the case of SynArsC, however, the Cys 8 -Cys 80 disulfide-bridged intermediate could not only be trapped in the reaction using wild-type protein, but also constitutes more than half of the population, indicating the thiol-disulfide transfer is of low efficiency. This could be a contributing effect to the lower catalytic activity of SynArsC compared with the Trx-coupled family (V max ϭ 3.1 mol/min/mg for SynArsC and V max ϭ 14.5 mol/min/mg for S. aureus ArsC) (15,35).

Structures of SynArsC in Different Reaction
Stages-To obtain a structural basis for SynArsC arsenate reducing mechanism, we prepared four SynArsC samples representing different reaction stages during one reaction cycle of arsenate reduction, including: 1) the fully reduced form in the absence of substrate or substrate analogue, representing the initial stage of SynArsC in the reaction and designated as red-SynArsC; 2) the substrate analog phosphate-bound form, mimicking the stage in which arsenate binds to the active site and designated as Pi-SynArsC; 3) the Cys 8 -Cys 80 disulfide-bridged form trapped by incubating the C13S/C35S/C82S mutant with arsenate, corresponding to the reaction intermediate after arsenate is reduced to arsenite and designated as int-SynArsC; 4) the Cys 80 -Cys 82 disulfide-bridged form prepared using the C8S/ C13S/C35S mutant, mimicking the oxidized state and designated as oxi-SynArsC. The redox states of all samples were verified by Ellman's test of free thiols (19) as presented in Table  1. The formations of Cys 8 -Cys 80 and Cys 80 -Cys 82 disulfide bonds in int-SynArsC and oxi-SynArsC samples were confirmed by MS/MS analyses (the result for int-SynArsC is shown in Fig. 2C as mentioned above, and the result for oxi-SynArsC is similar to Fig. 2E and not shown herein). For 15 N/ 13 C-labeled protein samples, the redox states of the thiols were further supported by the NMR chemical shift assignment of cysteine C ␤ atoms (22) as shown in Table 1. Solution NMR structures for all four stages were determined and shown in Fig. 4 and the structural statistics are summarized in Table 2.
The Active Site Conformation-The active site of SynArsC is formed by the P-loop (Cys 8 -Arg 14 ) harboring the essential CX 5 R motif (36), together with the Cys 80 -Cys 82 segment and the Asp 103 residue from the loop connecting ␤4 and ␣3 (Fig.  5A). The side chain of the essential catalytic Cys 8 is positioned in the center of the active site cavity, and is surrounded by the main chain amide dipoles of the P-loop residues and the positive charged side chains of Arg 10 and Arg 14 . These together with the macrodipole from helix ␣1 pointing toward the active site may contribute to the stabilization of the nucleophilic thiolate of Cys 8 , which is essentially similar to the Trx-coupled ArsC family (5).
Phosphate, an arsenate analog, binds to the active site P-loop of SynArsC and stabilizes the local structure, resulting in a significant decrease of the P-loop backbone root mean square deviations in the structure ensemble of Pi-SynArsC compared with red-SynArsC (Fig. 4E). In red-SynArsC, a total of six backbone amide cross-peaks (Lys 9 -Arg 14 ) in the P-loop are missing in the 1 H-15 N HSQC spectra, suggesting conformational exchanges that lead to signal broadening. The P-loop in Pi-SynArsC becomes more rigid as more resonances were detectable, with all non-proline residues assigned except for Lys 9 and Arg 14 .
The local conformation of the 80s loop is also affected by phosphate binding. The positions of the Cys 80 residue are generally similar in either the absence or presence of phosphate. However, the tri-residue Gly 81 -Cys 82 -Gly 83 segment is flipped out in the red-SynArsC structures, generating a relative open environment for substrate entry to the active site, whereas it moves closer to the active site in Pi-SynArsC and forms a more compact local conformation (Fig. 5B). Comparison of the two structure ensembles reveals an average of 6.1 Ϯ 1.2 Å movement of the Cys 82 residue upon phosphate binding.
Accessing a Buried Disulfide Bond-Upon arsenate reduction to arsenite, a Cys 8 -Cys 80 disulfide-bridged intermediate (int-SynArsC) is formed. The Cys 8 -Cys 80 disulfide bond is mostly buried (Fig. 5C), with limited accessibility to upstream electron donor. The average solvent accessible areas for the two sulfur atoms of Cys 8 and Cys 80 are 0.7 and 3.3 Å 2 , respectively, and correspond to ϳ1 and 5% of the total solvent accessible areas of these atoms in the context of an isolated residue (calculated The tests were repeated three times per sample. b The C ␤ chemical shifts of cysteines lower than 32 ppm and larger than 35 ppm usually indicate reduced and oxidized states, respectively (22). from 20 representative conformers). A subsequent intramolecular thiol transfer event releases the thiol of Cys 8 and transfers the disulfide bond to Cys 80 -Cys 82 , which is located on the protein surface and gains increased solvent accessibility (Fig. 5D). The two sulfur atoms of Cys 80 and Cys 82 are estimated to have average solvent accessible areas of 4.1 and 8.1 Å 2 , respectively, corresponding to 6 and 12% of the total solvent accessible areas in the context of an isolated residue (calculated from 20 representative conformers). The 3-fold increase of solvent accessi-bility could considerably facilitate the following reaction with the Grx-GSH system. Fig. 6, A-C, shows the structure comparisons of SynArsC with S. aureus ArsC in different states (6). The int-SynArsC structure is highly similar to S. aureus ArsC in the Cys 10 -Cys 82 disulfide-bridged intermediate state, with the disulfide bonds occupying near identical positions. The largest difference is the location and local conformation of the third cysteine residues (mutated to Leu in S. aureus ArsC and Ser in int-SynArsC as  shown in Fig. 6B). Notably, int-SynArsC shows significantly elevated conformational heterogeneity compared with other states. The backbone root mean square deviations of the int-SynArsC structure ensemble are almost twice as large as in other states (Fig. 4E). A total of 16 backbone amide signals are missing in the NMR spectra, including the whole Val 7 -Met 17 segment in the P-loop, the active residues Cys 80 , and the nearby Gly 81 and Val 84 residues, suggesting intermediate conformational exchanges on the NMR time scales. Moreover, the int-SynArsC sample is more prone to degradation. These properties are consistent with the notion that int-SynArsC represents a reaction intermediate, and are also similarly observed in the intermediate state of S. aureus ArsC (6). Similar to the Trxcoupled ArsC family, the transition from int-SynArsC to oxi-SynArsC not only exposes the buried disulfide bond onto protein surface, but also brings the protein back to a structurally stabilized state. A Loop to Helix Conformational Transition-During the transition from int-SynArsC to oxi-SynArsC, both residues Cys 80 and Cys 82 undergo a 6 -7 Å movement to form a disulfide bond (Fig. 5D). The highly flexible Gly 81 forms a small bulge in oxi-SynArsC, allowing disulfide bond formation between the closely positioned cysteines Cys 80 and Cys 82 . The covalent bond generates conformational constraint on the Gly 81 -Leu 86 segment and pulls it toward the structure core. The Gly 83 -Val 84 -Asn 85 segment in oxi-SynArsC therefore forms a small helix, whereas this segment adopts a relatively extended conformation in both red-SynArsC and Pi-SynArsC. In the structures of int-SynArsC, however, the segment shows an intermediate local conformation that is less extended compared with red-SynArsC and Pi-SynArsC, whereas the one-turn helix is not readily formed (Fig. 5E). Therefore, upon arsenate reduction and self-oxidization, the local conformation of the 80s segment in SynArsC undergoes a loop to helix transition.
This conformational change is exactly the opposite of the helix to loop transition observed in the Trx-coupled ArsC family (5,6,8,9). In both S. aureus and B. subtilis ArsC structures, the Cys 82 -Cys 89 segment forms a small helix in the reduced state, whereas it becomes disrupted upon Cys 82 -Cys 89 disulfide bond formation and adopts a "looped out" conformation. Therefore, the oxidized form of SynArsC shows considerable conformational differences with the Trx-coupled ArsC family in the 80s segment (Fig. 6C). The oxidized S. aureus and B. subtilis ArsC proteins show a relatively flat molecular surface around the 80s segment, which has been demonstrated to form the interaction surface of ArsC and the upstream reductant Trx (37). In SynArsC, the formation of a protruding helix in the corresponding region results in steric hindrance for interaction with Trx, and the disulfide bond is exposed to an opposite direction and would be incompatible for Trx interaction (Fig. 6D).
In contrast, the small GSH molecule is free to access the disulfide bond and act as an upstream reductant. Because Cys 82 is more solvent-exposed compared with Cys 80 , it is more likely to be attacked and covalently linked to the GSH peptide. Inspection of the surface electrostatic potential of SynArsC shows a patch of negative charges near the Cys 82 residue (Fig.  6E), which is complementary to the electropositive area near the active Cys 18 residue of Synechocystis GrxA (17). Therefore, upon covalent linkage with GSH, the nearby electronegative area of SynArsC, together with the negative charges of GSH peptide itself, may act in concert to facilitate interaction with GrxA.
A Hybrid Mechanism of SynArsC-Based on the above results, we can establish a structure-based functional mechanism of SynArsC as shown in Fig. 1D. It has a hybrid feature, with an intra-molecular thiol-disulfide cascade mechanism for reducing arsenate similar to the Trx-coupled ArsC family (4 -9), and a GSH/Grx-dependent regeneration mechanism similar to the Grx-coupled ArsC families (10 -14). A brief description of the catalytic cycle is summarized as follows.
In the free reduced form (red-SynArsC), an empty and dynamic substrate binding pocket facilitates interaction with the substrate. In the substrate analog-bound form (Pi-Sy-nArsC), the active site becomes stabilized. The local environment at the active site pocket enables the nucleophilic attack on arsenate by Cys 8 to form the covalent Cys 8 ⅐HAsO 3 Ϫ complex accompanied by the release of a water molecule. The second catalytic cysteine, Cys 80 is positioned nearby, with a 5.4 -8.9 Å distance from Cys 8 (calculated for the sulfur atoms in Pi-SynArsC structure), ready to attack the Cys 8 -HAsO 3 Ϫ bond and form the Cys 8 -Cys 80 disulfide bond while releasing one molecule of arsenite. The disulfide bond is buried in this intermediate state (int-SynArsC) and inaccessible to upstream reductant. Subsequent attack by the third catalytic cysteine, Cys 82 , could be facilitated by the significantly elevated conformational dynamics of int-SynArsC. This reaction transfers the disulfide to the protein surface, forming the oxidized state with a Cys 80 -Cys 82 disulfide bond (oxi-SynArsC). The 80s segment of oxi-SynArsC forms a short helix, which is distinct from the Trx-coupled ArsC structures. This local protrusion blocks Trx interactions while allowing the disulfide bond to be accessed by the small molecule GSH. Upon reduction by the GSH/Grx system, SynArsC returns to the fully reduced active form and is able to participate in the next cycle of arsenate reduction.

Discussion
The molecular mechanism of the SynArsC function derived from our current study is different from the previous hypothesis (15). The major discrepancy lies in the starting state of SynArsC in the reaction cycle. In the previous report, an oxidized form with the Cys 80 -Cys 82 disulfide was observed in a E. coli expressed sample and the protein was found to be dimeric (15). A hypothesized reaction scheme was proposed assuming an oxidized starting state, and one molecule of GSH is suggested necessary for arsenate reduction (15). In our study, we observed that the sample redox and oligomeric states are dependent on the protein purification procedure. By using anion exchange followed by gel filtration chromatography, we obtained SynArsC samples with the majority (Ͼ90%) in the fully reduced monomeric state without the use of reductant during the purification procedure. Varying extents of protein oxidation and oligomerization occur when the purification procedure was extended or when a lengthy dialysis step was added, suggesting that air exposure may be the cause of Cys 80 -Cys 82 disulfide bond formation and perhaps nonspecific inter-molecular disulfide bridging. Further evidences from the redox potentials of Cys 80 -Cys 82 disulfide bond (17) and the in vivo GSH/GSSG redox couple (32) helped confirm that the catalytic active form of SynArsC corresponds to the fully reduced state. The mechanism proposed in this study provides a thorough explanation of both the structural and activity data available.
From an evolutionary point of view, both SynArsC and Trxcoupled ArsC are closely related to the LMW-PTPase, and the arsenate reducing activity was suggested to be drafted from the PTPase activity (5,8,15). An intriguing question is whether SynArsC evolves from the Trx-coupled ArsC family and gradually diverts to the GSH/Grx redox pathway, or it represents a molecular fossil of an intermediate stage during the evolution of LMW-PTPase to Trx-coupled ArsC? Herein, we demonstrate that SynArsC utilizes an essentially identical thiol-disulfide cascade mechanism as the Trx-coupled ArsC family, whereas its last step of thiol transfer has much lower efficiency. This phenomenon is consistent with the lower arsenate reduction activity of SynArsC, and may be attributed to the distinct position of the third catalytic cysteine (Cys 82 of SynArsC is solvent exposed, whereas Cys 89 of S. aureus/B. subtilis ArsC locates in a relatively hydrophobic structural environment), as well as the differences in local conformation and amino acid composition. In addition, phylogenetic analysis suggested that SynArsC is sequentially closer to LWM-PTPase, and the PTPase activity of SynArsC appears slightly better than the Trx-coupled ArsC (V max of SynArsC toward the general substrate para-nitrophenyl phosphate is about 2-fold of S. aureus/B. subtilis ArsC) (15). Taken together, a more favorable scenario is that SynArsC represents an intermediate evolutionary stage between LWM-PTPase and Trxcoupled ArsC. It has acquired arsenate reduction activity but exhibits lower catalytic efficiency compared with the later evolved S. aureus/B. subtilis ArsC, and shows an oxidized conformation readily reduced by the low molecular weight GSH but not preferable for interaction with Trx. Further evolution refines the local conformation of the 80s segment for optimization of the arsenate reducing activity. This process may work in concert with other evolutionary pressures (i.e. the fact that some Gram-positive bacteria such as B. subtilis lack GSH) to gradually shift the position of the third active cysteine and modify the local conformation, generating a novel ArsC family that has higher arsenate reduction activity and utilize the Trx system as the upstream reductant.