Catalytic Mechanism of Sep-tRNA:Cys-tRNA Synthase

Background: SepCysS catalyzes the conversion of tRNA-bound O-phosphoserine to Cys. Results: Three Cys residues are essential for SepCysS activity, and two of them form disulfide and persulfide intermediates. Conclusion: SepCysS transfers sulfur through generating disulfide and persulfide enzyme adducts. Significance: Elucidation of the sulfur transfer mechanism of SepCysS is crucial for understanding the tRNA-dependent Cys biosynthesis and sulfur metabolism in methanogens. Sep-tRNA:Cys-tRNA synthase (SepCysS) catalyzes the sulfhydrylation of tRNA-bound O-phosphoserine (Sep) to form cysteinyl-tRNACys (Cys-tRNACys) in methanogens that lack the canonical cysteinyl-tRNA synthetase (CysRS). A crystal structure of the Archaeoglobus fulgidus SepCysS apoenzyme provides information on the binding of the pyridoxal phosphate cofactor as well as on amino acid residues that may be involved in substrate binding. However, the mechanism of sulfur transfer to form cysteine was not known. Using an in vivo Escherichia coli complementation assay, we showed that all three highly conserved Cys residues in SepCysS (Cys64, Cys67, and Cys272 in the Methanocaldococcus jannaschii enzyme) are essential for the sulfhydrylation reaction in vivo. Biochemical and mass spectrometric analysis demonstrated that Cys64 and Cys67 form a disulfide linkage and carry a sulfane sulfur in a portion of the enzyme. These results suggest that a persulfide group (containing a sulfane sulfur) is the proximal sulfur donor for cysteine biosynthesis. The presence of Cys272 increased the amount of sulfane sulfur in SepCysS by 3-fold, suggesting that this Cys residue facilitates the generation of the persulfide group. Based upon these findings, we propose for SepCysS a sulfur relay mechanism that recruits both disulfide and persulfide intermediates.

Sep-tRNA:Cys-tRNA synthase (SepCysS) catalyzes the sulfhydrylation of tRNA-bound O-phosphoserine (Sep) to form cysteinyl-tRNA Cys (Cys-tRNA Cys ) in methanogens that lack the canonical cysteinyl-tRNA synthetase (CysRS). A crystal structure of the Archaeoglobus fulgidus SepCysS apoenzyme provides information on the binding of the pyridoxal phosphate cofactor as well as on amino acid residues that may be involved in substrate binding. However, the mechanism of sulfur transfer to form cysteine was not known. Using an in vivo Escherichia coli complementation assay, we showed that all three highly conserved Cys residues in SepCysS (Cys 64 , Cys 67 , and Cys 272 in the Methanocaldococcus jannaschii enzyme) are essential for the sulfhydrylation reaction in vivo. Biochemical and mass spectrometric analysis demonstrated that Cys 64 and Cys 67 form a disulfide linkage and carry a sulfane sulfur in a portion of the enzyme. These results suggest that a persulfide group (containing a sulfane sulfur) is the proximal sulfur donor for cysteine biosynthesis. The presence of Cys 272 increased the amount of sulfane sulfur in SepCysS by 3-fold, suggesting that this Cys residue facilitates the generation of the persulfide group. Based upon these findings, we propose for SepCysS a sulfur relay mechanism that recruits both disulfide and persulfide intermediates.
Aminoacyl-tRNAs are normally synthesized by direct attachment of specific amino acids to their cognate tRNAs. However, at least four aminoacyl-tRNAs (Asn-tRNA Asn , Gln-tRNA Gln , Cys-tRNA Cys , and Sec-tRNA Sec ) can be generated through tRNA-dependent modification reactions (1). For the biosynthesis of Cys-tRNA Cys in methanogenic archaea, tRNA Cys is initially aminoacylated with Sep 3 by SepRS; then Sep-tRNA Cys is converted to Cys-tRNA Cys by SepCysS (2). The SepRS/SepCysS pathway is the primary pathway for Cys biosynthesis in methanococci (3), which do not possess O-acetylserine sulfhydrylase, the bacterial cysteine biosynthesis enzyme (4). Furthermore, the canonical CysRS is either absent (5) or nonessential in these organisms (6). SepRS and SepCysS are conserved in most methanogenic archaea (except the Methanobrevibacter and Methanosphaera species) and the Archaeoglobus species, which are sulfur-reducing archaea and have many enzymes of the methanogenesis pathway (7). Phylogenetic analysis suggested that the SepRS/SepCysS pathway is perhaps ancient and was already present at the time of the last universal common ancestor (7).
SepCysS is a pyridoxal 5Ј-phosphate (PLP)-dependent enzyme converting the tRNA-bound Sep moiety to cysteine using an undefined sulfur donor (2). Sulfide, cysteine, and thiophosphate were sufficient sulfur donors in in vitro assays. However, all of them support only a very slow formation of cysteine (8). Therefore, the natural sulfur donor and the sulfur delivery mechanism of SepCysS remain unclear. A persulfide group is proposed to be a sulfur donor for cysteine biosynthesis, mainly based upon the fact that SepCysS is most closely related to cysteine desulfurase in terms of primary sequence (7) and overall structure (9). Cysteine desulfurase catalyzes sulfur transfer by mobilizing the thiol group of free cysteine, yielding a persulfide enzyme adduct and alanine (10). The persulfidic sulfur (R-S-SH), which is covalently linked to a conserved Cys residue of cysteine desulfurase, is then donated to other sulfur carrier proteins for the biosynthesis of Fe-S clusters, thionucleotides, or sulfur-containing vitamins (11). Analogous to cysteine desulfurase, SepCysS may mobilize sulfur from a sulfur source, form a persulfide group on a Cys residue, and then donate the sulfur to Sep-tRNA Cys to generate Cys-tRNA Cys .
Three Cys residues of SepCysS (Cys 39 , Cys 42 , and Cys 247 of the Archaeoglobus fulgidus enzyme) have been proposed to be candidates for persulfidation according to two observations (9). First, the three Cys residues are completely conserved in Sep-CysS, indicating that they are possibly essential for activity. Second, the crystal structure of A. fulgidus SepCysS showed that, similar to cysteine desulfurase, the enzyme forms a dimer, and the active cleft is located at the dimer interface. Cys 247 is located near the active site in the crystal structure. Cys 39 and Cys 42 , although located within a disordered region in the crystal structure, could be modeled proximal to the active site (9). However, whether the three Cys residues are involved in sulfur delivery awaits further investigation.
Here, we present that three Cys residues (Cys 64 , Cys 67 , and Cys 272 of Methanocaldococcus jannaschii SepCysS corresponding to Cys 39 , Cys 42 , and Cys 247 of the A. fulgidus enzyme) are essential for the sulfhydrylation reaction in vivo. More specifically, Cys 64 and Cys 67 are responsible for forming a disulfide linkage and carrying a sulfane sulfur. Therefore, we propose that through disulfide formation and disulfide-persulfide rearrangement, sulfur from a persulfide sulfur donor is transferred to SepCysS and in turn delivered to the tRNA-bound Sep for Cys-tRNA Cys formation.
Cloning, Expression, and Purification of Recombinant His 6 -SepCysS-The SepCysS gene from M. jannaschii (MJ1678) and A. fulgidus (AF0028) were cloned into pET15b vector as described (12). The mutations of MJ1678, including six single mutants (K234A, C64A, C67A, C113A, C209A, and C272A), three double mutants (C64A/C67A, C64A/C272A, and C67A/C272A), and one triple mutant (C64A/C67A/ C272A), were constructed using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA). The same set of mutants was also constructed for A. fulgidus SepCysS. The DNA oligonucleotides used for mutagenesis were designed using a web-based program primerX or a program provided by Stratagene. The manufacturer's recommended protocol was followed except that electrocompetent DH10␤ cells were used for transformation. All mutations were confirmed by plasmid DNA sequencing.
The resulting plasmids were individually transformed into the Escherichia coli BL21-CodonPlus (DE3) strain (Stratagene) for expression of recombinant proteins. The transformed cells were grown in 1 liter of Luria-Bertani (LB) medium supplemented with 100 g/ml ampicillin at 37°C with shaking until they reached an absorbance at 600 nm of 0.6ϳ0.8. Isopropyl ␤-D-1-thiogalactopyranoside was added to a final concentra-tion of 1 mM to induce the production of recombinant proteins. After an additional 5-h incubation at 30°C or overnight incubation at 15°C, the cells were harvested by centrifugation at 5,000 ϫ g for 20 min at 4°C. For aerobic protein purification, cells were disrupted by sonication, and His-tagged SepCysS was purified with nickel-nitrilotriacetic acid resin (Qiagen) as described (2). For anaerobic purification of M. jannaschii Sep-CysS, the harvested E. coli cells were transferred into the anaerobic chamber (atmosphere of 95% N 2 and 5% H 2 ) and resuspended in 20 ml of buffer A (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4). The cells were disrupted by the addition of 2 ml of BugBuster (Novagen). RNA and DNA were digested with 10 l of Benzonase (Sigma) by incubation at room temperature for 30 min. E. coli host proteins were then denatured by heating at 75°C for 30 min and separated from soluble proteins by centrifugation at 100,000 ϫ g for 30 min at 4°C. For further purification of recombinant SepCysS, the supernatant was applied to 1 ml of TALON Metal Affinity Resins (Clontech) equilibrated with buffer A. Proteins bound to the column were eluted with 5 ml of buffer B (20 mM sodium phosphate, 0.5 M NaCl, 200 mM imidazole, pH 7.4). The elution fractions of the desired proteins were analyzed by SDS-PAGE, and the purified proteins were then dialyzed against buffer C (50 mM HEPES-NaOH, 20 mM MgCl 2 , pH 8.0), concentrated to a volume of 1 ml with a 30-kDa cutoff centrifugal filter (Millipore), and stored at Ϫ20°C until use. Protein concentrations were determined with the bicinchoninic acid assay (13).
Complementation of E. coli ⌬selA Mutant-The M. jannaschii wild-type (MJ1678) and mutant SepCysS genes (K234A, C64A, C67A, C272A, C209A, and C113A) were transformed into the E. coli ⌬selA strain JS1 together with the M. jannaschii phosphoseryl-tRNA Sec kinase gene (MJ1538). The complementation experiment was performed as described (14). Briefly, the transformed strains were plated on glucose-minimum medium agar plates and grown for 2 days at 30°C. The plates were then overlaid with agar containing 1 mg/ml benzyl viologen, 0.25 M sodium formate, and 25 mM KH 2 PO 4 , adjusted to pH 7.0. The appearance of blue/purple colonies is an indication of the production of active formate dehydrogenase H (15) and therefore a complementation with active SepCysS.
Spectroscopic Analysis of PLP-Absorption spectra of purified recombinant M. jannaschii SepCysS (20 M) were measured with the spectrophotometer system (Shimadzu, UV-2401PC) aerobically in cuvettes with a 1.0-cm light path at room temperature. The absorption spectra of the wild-type enzyme were compared with the mutant SepCysS (K234A) with and without potassium borohydride (20 mM) treatment.
Sulfur Transfer Assay with Radioactive Sulfur-M. jannaschii SepCysS was purified anaerobically as described above. The following procedure was carried out under anaerobic conditions. Purified maltose binding protein-tagged IscS  Fig. S1). The reaction was stopped by the addition of nonreducing SDS loading dye. The protein mixture was then separated by SDS-PAGE, and the radioactivity retained on the gel was followed by autoradiography.
Determination of Disulfide Content of SepCysS by Alkaline Sulfide/Cyanolysis Assay-The alkaline sulfide/cyanolysis assay (16) was used to determine disulfide linkages in SepCysS. The A. fulgidus wild-type SepCysS and five mutants including C247A, C39A/C42A, C39A/C247A, C42A/C247A, and C39A/ C42A/C247A were purified in the presence of 10 mM DTT to remove intrinsic persulfidic sulfur. The protein preparations were then dialyzed extensively against an anoxic buffer (50 mM HEPES-NaOH, 20 mM MgCl 2 , pH 8.0, degassed with N 2 for 1 h) to remove DTT and thus allow generation of disulfide linkages with trace amount of oxygen or other oxidants in the buffer. Circular dichroism spectra showed that no structural changes are introduced to these mutant proteins (data not shown). The wild-type and mutant SepCysS proteins (5 g/l) were then treated with sulfide (2 mM) at pH 9.0 anaerobically at room temperature for 3 h to allow disulfide linkages to react with sulfide and in turn form persulfide groups. Persulfide groups in the proteins were then reacted with cyanide (10 mM) at room temperature to produce thiocyanate (16). The amount of thiocyanate was measured by absorbance at 470 nm after reaction with the ferric reagent (17).
Determination of Thiol Content of SepCysS by DTNB Titration-The anaerobically purified M. jannaschii wild-type SepCysS and three mutants including C64A, C67A, and C272A were subjected to DTNB titrations under denaturing condition to determine the presence of disulfide bond in the resting state of the enzyme. The enzyme sample (0.5-2 nmol) in 200 l of denaturing buffer containing 0.1 M sodium phosphate, pH 7.3, 1 mM disodium EDTA, and 6.4 M guanidinium chloride was mixed with 200 l of 10 mM DTNB. The mixture (400 l) was incubated for 30 min at room temperature, and the production of 2-nitro-5-thiobenzoate anion (TNB 2Ϫ ) was quantified by absorbance at 412 nm using an extinction coefficient of 13,700 M Ϫ1 cm Ϫ1 (18). The mixture without addition of protein was used as a blank control. All reactions were performed in triplicate.
Determination of Persulfide Content of SepCysS by Fluorescent Labeling-For detection of persulfide groups, the anaerobically purified M. jannaschii SepCysS (ϳ2 nmol) in 100 l of buffer C (50 mM HEPES-NaOH, 20 mM MgCl 2 , pH 8.0) was incubated anaerobically with 20 nmol of the fluorescent dye 1,5-I-AEDANS at 37°C for 30 min to derivatize thiol groups in the protein (19). The reaction mixture was then applied to a Microcon YM-10 Centrifugal Filter unit (Millipore) and centrifuged at 14,000 ϫ g for 15 min. The protein in the retentate was washed four times with 350 l of buffer C to remove unreacted fluorescent dye. The protein sample was then reduced to a volume of 50 l by centrifugal filtration, supplemented with 50 l of 10 mM DTT, and incubated at 37°C for 20 min. In this step, persulfide groups, if reduced by DTT, would release the fluorescent dye into the solution. Finally, the reduced protein sample was applied to a Microcon YM-10 Centrifugal Filter and centrifuged at 14,000 ϫ g to dryness. The free fluorescent dye collected in the filtrate was quantified using the QuantaMaster Fluorescence Spectrofluorometer (Photon Technology International) with ex 337 nm and em 498 nm. The standard curve was generated with 0.01-0.5 nmol of 1,5-I-AEDANS in 100 l of buffer C with 5 mM DTT. As a negative control, the M. jannaschii SepCysS protein (ϳ2 nmol) in 100 l of buffer C was incubated with 50 nmol of DTT for 30 min at 37°C to reduce persulfide groups before reaction with the fluorescent dye.

Identification of Disulfide Linkage and Sulfane Sulfur in Sep-CysS by Mass Spectrometry-
The buffer of the anaerobically purified recombinant M. jannaschii SepCysS (MJ1678) was exchanged with 0.1 M ammonium bicarbonate-formic acid buffer, pH 7.2, by passage through a 0.5-ml Zeba Desalt Spin Column (Thermo Scientific). The protein (200 g) was digested with 2 g of trypsin (Promega) in a volume of 100 l at 37°C overnight, and then the samples were dried in vacuum. For the control experiment, the protein was incubated with 10 mM DTT at 60°C for 1 h to remove persulfide group before trypsin digestion.
The peptide samples obtained from trypsin digestion were resuspended in 30 l of deionized H 2 O containing 3% acetonitrile and 0.1% formic acid, and 8 l of sample was analyzed on an Agilent 1100 Capillary LC (Palo Alto, CA) interfaced directly to a linear ion trap (LTQ) mass spectrometer (Thermo Electron, San Jose, CA). Mobile phases A and B were H 2 O-0.1% formic acid and acetonitrile-0.1% formic acid, respectively. Peptides eluted from the C18 column were injected into the mass spectrometer during a 60-min linear gradient from 5 to 60% mobile phase B at a flow rate of 4 l/min. The instrument was set to acquire MS/MS spectra on the nine most abundant precursor ions from each MS scan with a repeat count of 3 and repeat duration of 5 s. Dynamic exclusion was enabled for 60 s. Generated raw MS/MS were converted into the mzXML format and then into the PKL format using ReAdW followed by mzMXL2Other (20). The peak lists were then searched using the Mascot 2.2 software (Matrix Science, Boston, MA) against the sequence of MJ1678 with the following parameters: full tryptic enzymatic cleavage with two possible missed cleavages, peptide tolerance of 1000 parts per million, fragment ion tolerance of 0.6 Da, and variable modifications with oxidation of methionyl residues (ϩ16 Da), disulfide linkage of Cys residues (Ϫ2 Da), and persulfide modification of Cys residues (ϩ32 Da).

Three Conserved Cys Residues Are Essential for SepCysS
Activity-SepCysS can utilize Sep-tRNA Sec as a substrate and generate Cys-tRNA Sec in E. coli (14). In the E. coli ⌬selA strain JS1, active formate dehydrogenase H cannot be produced due to the lack of selenocysteine incorporation (14). SepCysS in combination with phosphoseryl-tRNA Sec kinase complements the ⌬selA strain and converts Ser-tRNA Sec 3 Cys-tRNA Sec to produce the cysteine homolog of formate dehydrogenase H, which has reduced activity compared with the native selenocysteine enzyme (14). Therefore, the in vivo activity of SepCysS can be tested by measuring formate dehydrogenase H activity, which reduces benzyl viologen with formate as the electron donor and results in blue/purple colonies (14). Using this method, the activities of six M. jannaschii SepCysS mutant enzymes were examined. The K234A mutant, which contains an Ala substitution of Lys 234 (corresponding to Lys 209 in A. fulgidus SepCysS), was inactive in the benzyl viologen assay ( Fig. 1). This result is in agreement with the absorption spectra characterization (Fig. 2) and the crystal structure data (9) showing that Lys 234 forms a Schiff base with PLP and therefore is functionally essential.
To identify the Cys residues involved in sulfur delivery, five cysteines in M. jannaschii SepCysS were individually altered to Ala. The three Cys3 Ala mutants of the conserved Cys residues (Cys 64 , Cys 67 , and Cys 272 corresponding to Cys 39 , Cys 42 , and Cys 272 in A. fulgidus SepCysS) lost their ability to complement the ⌬selA strain (Fig. 1), indicating that these three cysteines are necessary for in vivo SepCysS activity. Interestingly, they are probably located near the SepCysS active site based upon the crystal structure and have been proposed as candidates for persulfidation (9). On the other hand, the other two Cys mutants (C113A and C209A) exhibited activity similar to that of wildtype SepCysS (Fig. 1), indicating that these two Cys residues are dispensable.
SepCysS Can Receive Sulfur from IscS-The conversion of Sep-tRNA Sec 3 Cys-tRNA Sec by SepCysS requires a sulfur donor in E. coli; therefore, we tested whether SepCysS can receive sulfur from another sulfur-carrying protein. The cysteine desulfurase IscS, which generates a persulfide enzyme adduct at the expense of free cysteine, is a central physiological sulfur donor for the biosynthesis of sulfur-containing cofactors and tRNAs in bacteria (10,21,22 Fig. 3A), indicating that 35 S was able to transfer from Cys directly to IscS.
When SepCysS was incubated with IscS and [ 35 S]Cys, radioactivity was detected in two bands that corresponded to IscS and SepCysS, respectively (lane 3 of Fig. 3A). Addition of the reducing reagent ␤-mercaptoethanol (1%, v/v) to the protein mixture removed the radioactive labeling (Fig. 3B); this suggests that 35 S was transferred in the sulfane sulfur form. Therefore, these results indicate that SepCysS is able to accept IscS-associated sulfane sulfur.
SepCysS Carries Sulfane Sulfur-Because sulfur transfer from IscS to SepCysS occurred in vitro, we tested whether Sep-CysS expressed in E. coli carries sulfane sulfur. Recombinant M. jannaschii SepCysS was purified anaerobically in the absence of reducing reagents; then the free sulfhydryl groups of the purified protein were labeled with I-AEDANS, a fluorescent derivative of iodoacetamide (19). After removal of unreacted fluorescent dye by centrifugal filtration, the protein was treated with 100-fold excess of DTT. If persulfide (R-S-SH) or another sulfane sulfur containing group (e.g. polysulfide, RSS n SH) is present in the protein, the fluorophore would be released from the protein into solution upon reduction with DTT. In three independent treatments of 2 nmol of wild-type protein, 30 Ϯ 10 pmol of the fluorophore were released; this suggests that ϳ1.5% of the purified protein contained sulfane sulfur. For either of the C64A or the C67A mutant, no detectable amount of fluorophore (Ͻ5 pmol) was released after reduction with DTT, suggesting that Cys 64 and Cys 67 are essential for the formation of the sulfane sulfur-containing group. For the C272A mutant, only 10 Ϯ 5 pmol of the fluorophore (ϳ0.5%) was released from 2 nmol of protein, which accounted for about one third of the wild-type level. The lower amount of sulfane sulfur in the   FEBRUARY 17, 2012 • VOLUME 287 • NUMBER 8

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C272A mutant suggests that Cys 272 stimulates the formation or stabilizes the sulfane sulfur-containing group.
SepCysS Forms Disulfide Linkage-Intramolecular disulfide bonds are often formed between the sulfane sulfur-carrying Cys and a second catalytic Cys as a result of sulfur donation by sulfur transfer enzymes, such as ThiI (23,24) and MnmA (25,26). Two methods were used to determine whether SepCysS forms disulfide bonds.
To test whether a disulfide bond can be formed by SepCysS in vitro, the A. fulgidus wild-type and mutant proteins were incubated with Na 2 S under alkaline conditions (16). In this process, disulfide bonds of the protein would be cleaved by sulfide to produce persulfide groups, which can then be quantified by the cyanolysis assay (16). As a control experiment, the protein was subjected to cyanolysis without alkaline sulfide treatment, and no significant amount of persulfide was detected. This control suggests that intrinsic persulfide was removed during the purification with DTT and would not interfere with the following assay. After removal of DTT by extensive dialysis with a nonreducing anoxic buffer, we tested whether disulfide linkages can be generated with trace amount of oxygen or other oxidants in the buffer. As a result, 1.2 Ϯ 0.1 disulfide linkages were observed in the wild-type SepCysS after dialysis (Fig. 3). The background level was determined with the triple mutant, which has all three conserved cysteines (Cys 39 , Cys 42 , and Cys 247 ) mutated to Ala. When either Cys 39 or Cys 42 (corresponding to Cys 64 or Cys 67 of M. jannaschii SepCysS, respectively) was substituted with Ala, the absorbance decreased to the background level (Fig. 4A), indicating that both cysteines are involved in disulfide bond formation. On the other hand, when Cys 247 (corresponding to Cys 272 of M. jannaschii SepCysS) was substituted with Ala, the absorbance was similar to that of the wild-type enzyme (Fig. 4A), suggesting that Cys 247 is not involved in forming intramolecular disulfide.
To test whether SepCysS contains disulfide bonds in the resting state, the M. jannaschii wild-type and mutant proteins were purified anaerobically in the absence of reducing reagents and then reacted anaerobically with DTNB. Under denaturing conditions, 6.5 Ϯ 0.5 free thiol groups were observed in the wildtype SepCysS (Fig. 4B), which contains 9 Cys residues according to the protein sequence. When Cys 64 and Cys 67 were individually substituted with Ala, the number of free thiol groups increased to 7.7 Ϯ 0.7 and 7.6 Ϯ 0.7 (Fig. 4B), respectively, suggesting that these two cysteines were inaccessible to DTNB in the wild-type protein and potentially formed a disulfide bond. The C272A mutation decreased the number of free thiol groups to 5.7 Ϯ 0.4 in the protein (Fig. 4B), suggesting that Cys 272 is not involved in forming disulfide. These observations were consistent with those obtained from the alkaline sulfide/ cyanolysis assay, which suggests that Cys 64 and Cys 67 naturally form a disulfide bond.
Identification of Disulfide Linkage and Sulfane Sulfur in Sep-CysS by Mass Spectrometry-To confirm the presence of disulfide linkage and sulfane sulfur in SepCysS, the protein was purified anaerobically in the absence of reducing reagents and digested with trypsin, and the resulting peptides were then analyzed by LC-MS/MS (Fig. 5). For the peptide 53 AVYEY-WDGYSVCDYCHGR 70 , the unmodified precursor ion (2187.4 Da) was not detected. However, the precursor ions can be observed with either Ϫ2 Da (mass of Ϫ2H) or ϩ30 Da (mass of Ϫ2H ϩ 1S) shift. When the Ϫ2 Da shift was considered as the loss of dihydrogen caused by the formation of an intrapeptide disulfide between Cys 64 and Cys 67 , analysis of the MS/MS spectra of the Ϫ2 Da shifted precursor ion confirmed the expected amino acid sequence (Fig. 5A). The fragmentation of the ϩ30 Da precursor ion identified ϩ32 Da shifts of the disulfide bondcontaining fragment ions, suggesting that a sulfane sulfur was added to the disulfide bond (Fig. 5B). This fragmentation pattern presumably resulted from an oxidation (loss of dihydrogen) between a persulfide group on either Cys 64 or Cys 67 and a free thiol group from the other Cys to form a trisulfide linkage, which occurred either in vivo or during the protein purification and the mass spectrometry processes. No persulfide modification or disulfide linkage with another Cys-containing peptide was observed for Cys 272 .

DISCUSSION
The data presented here demonstrate that all three conserved Cys residues of SepCysS (Cys 64 , Cys 67 , and Cys 272 in M. jannaschii SepCysS) are required for the conversion of the tRNA-bound Sep moiety to Cys. Furthermore, the formation of disulfide and trisulfide (presumably derived from an oxidation of a persulfide and a thiol group or from a reaction between two persulfide group to expel a bisulfide) linkages between Cys 64 and Cys 67 suggests that SepCysS employs both disulfide and persulfide intermediates for sulfur transfer. The requirement of multiple cysteines for sulfur transfer by SepCysS resembles the sulfur relay enzymes for tRNA thiolation, e.g. ThiI for 4-thiouridine biosynthesis (23,24) and MnmA for 2-thiouridine biosynthesis (25,26). For both ThiI and MnmA, two cysteines are required for catalysis: one cysteine receives a sulfane sulfur from cysteine desulfurase and forms a persulfide enzyme adduct, and the other cysteine attacks the bridging sulfur of the persulfide to liberate the terminal sulfur and consequently forms a disulfide with the first cysteine. Based upon our findings that a disulfide is present in the resting state   FEBRUARY 17, 2012 • VOLUME 287 • NUMBER 8 of SepCysS and both Cys 64 and Cys 67 are required for the formation of a persulfide, it is conceivable that a sulfur donor (equivalent of S 2Ϫ ) may attack one sulfur of the disulfide linkage to generate a persulfide on either Cys 64 or Cys 67 , leaving the other Cys as a free thiol (Fig. 6). Then, a thiolate derived from deprotonation of the free thiol can attack the bridging sulfur of the persulfide to liberate the terminal sulfur for cysteine biosynthesis and consequently regenerate the disulfide for the next catalytic cycle (Fig. 6). This reaction scheme can also explain the in vitro activity with sulfide, which may directly attack the disulfide of SepCysS or the double bond of the dehydroalanyl moiety (8).

Sulfur Transfer Mechanism of SepCysS
Although a S 2Ϫ equivalent is likely a sulfur donor for the persulfidation of SepCysS (described above), the low in vitro sulfhydrylation activity of SepCysS with sodium sulfide suggested that bisulfide is unlikely to be the physiological sulfur donor (2,8). Because SepCysS was able to receive sulfur from the IscS persulfide, an unidentified protein-carrying persulfide possibly provides the sulfur for the persulfidation of SepCysS. The nature of the protein sulfur carrier is still under investigation. In analogy to the sulfur transfer from IscS to IscU, which forms disulfide-bridged IscS-IscU complex during iron-sulfur cluster assembly (27), if the persulfide of a sulfur carrier protein nucleophilically attacks the disulfide of SepCysS, then an intermolecular disulfide between SepCysS and the sulfur carrier protein would be expected as a result of persulfide group transfer. In this scenario, an exogenous reductant would be required to dissociate the sulfur carrier protein from SepCysS and regenerate free thiol groups.
Although a disulfide or sulfane sulfur was not observed on Cys 272 in our experiments, the reduced level of persulfide group in the C272A mutant suggested that Cys 272 may facilitate the formation of the persulfide enzyme adduct. Moreover, replacement of Cys 272 with Ser also abolished the activity of SepCysS (32), suggesting that a covalent modification on Cys 272 is likely required for in vivo sulfur transfer. Scenarios such as that Cys 272 serves as an initial nucleophilic group that attacks on a persulfide sulfur donor or as a general acid-base that deprotonates Cys 64 or Cys 67 can account for the observations, but further investigations are required to clarify its function.
This study provides a framework for understanding the sulfur transfer for the formation of Cys-tRNA Cys by SepCysS. Sep-CysS is a first example that recruits both persulfide and disulfide intermediates in methanogens. Given the obligately anaerobic lifestyle of methanogens, the mechanism of maintaining the relatively oxidized persulfide and disulfide species in the reducing cytoplasm is unclear. Because methanogens lack glutathione (28,29), other thiol-containing compounds (e.g. coenzyme M, coenzyme B, and the heterodisulfide of coenzyme M and coenzyme B) may function as the redox-buffering molecules. Furthermore, the enzyme that delivers sulfur to Sep-CysS remains an open question for future research. Because cysteine desulfurase is not encoded in M. jannaschii and many other methanogens (3) and cysteine should not be the ultimate sulfur donor for Cys-tRNA Cys biosynthesis, a new type of enzyme that can initiate the generation of persulfide from a noncysteine sulfur source would be responsible for the persulfidation of SepCysS. Identification of this enzyme and its inter-  (31). The electron movement through PLP has been omitted. E and F, proposed sulfur relay mechanism is based upon the disulfide and persulfide intermediates of SepCysS presented in this study. A, amino group of Sep-tRNA Cys attacks the Schiff base (internal aldimine) between Lys 234 and PLP to form an external aldimine. B, electron delocalization leads to ␤-elimination of phosphate and formation of dehydroalanyl-tRNA Cys . C, double bond of dehydroalanyl-tRNA Cys is attacked by a persulfide sulfur donor to form the Cys moiety. D, Lys 234 forms the Schiff base with PLP, leading to the release of Cys-tRNA Cys and regeneration of the active site of SepCysS. E, sulfur donor (S 2Ϫ equivalent) attacks the disulfide between Cys 64 and Cys 67 , and the sulfur (in red) is consequently transferred to either Cys 64 or Cys 67 (Cys 64 shown in the scheme here) to generate a persulfide enzyme adduct of SepCysS. F, thiolate formed by the other Cys (Cys 67 shown in the scheme here) attacks the bridging sulfur of the persulfide to liberate the terminal sulfur (in red) as a formal equivalent of S 2Ϫ for Cys moiety biosynthesis. The intramolecular disulfide is consequently regenerated. G, a proposal of in vivo sulfur transfer from a persulfide sulfur donor (R-S-S Ϫ ) to SepCysS is presented here with dashed arrows. The sulfur transfer results in an intermolecular disulfide between the sulfur donor and SepCysS and a persulfide on SepCysS. An exogenous reductant is required to resolve the intermolecular disulfide and release the sulfur donor (R-S Ϫ ).
mediates would more thoroughly elucidate the Cys-tRNA Cys biosynthetic system and may shed light on a general mechanism of sulfur metabolism in methanogenic archaea.