Catalytic mechanism of Sep-tRNA:Cys-tRNA synthase: sulfur transfer is mediated by disulfide and persulfide

Abstract

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.

FIGURE 1.

Complementation of the E. coli ΔselA mutant with phosphoseryl-tRNA^Sec kinase (PSTK) and SepCysS. The in vivo activities of M. jannaschii wild-type and mutant (K234A, C64A, C67A, C272A, C209A, and C113A) SepCysS converting Sep-tRNA^Sec to Cys-tRNA^Sec were tested by their ability to restore formate dehydrogenase H activity in the E. coli ΔselA mutant. The complementation was observed by formation of purple colonies resulted from reduction of benzyl viologen with formate. The complementation required co-expression of phosphoseryl-tRNA^Sec kinase, which converts Ser-tRNA^Sec to Sep-tRNA^Sec.

FIGURE 2.

Spectra of the M. jannaschii wild-type and K234A mutant SepCysS. The absorption between 400–430 nm was used as an indication of internal Schiff base formed between PLP and a Lys residue of the enzyme (30).

FIGURE 3.

Sulfur transfer from the cysteine desulfurase IscS to SepCysS. A, E. coli IscS (8 μm; lane 1), M. jannaschii SepCysS (20 μm; lane 2), and the two proteins together (lane 3) were incubated with 150 μm [³⁵S]Cys at 37 °C for 3 h, and then the incubation mixtures were analyzed by SDS-PAGE under nonreducing condition. The left and right panels show the Coomassie-stained gel and the PhosphorImager scan, respectively. The positions of maltose-binding protein-tagged IscS (∼90 kDa) and His-tagged SepCysS (∼45 kDa) are labeled. B, E. coli IscS (4 μm) and M. jannaschii SepCysS (20 μm) were incubated with 150 μm [³⁵S]Cys at 37 °C for 30 min, and then the incubation mixtures were analyzed by SDS-PAGE under nonreducing condition (lane 1) and reducing condition with 1% (v/v) β-mercaptoethanol (lane 2).

FIGURE 4.

Determination of disulfide linkages in SepCysS. A, disulfide content in A. fulgidus SepCysS was determined with the alkaline sulfide/cyanolysis assay. B, free thiol content in M. jannaschii SepCysS was determined with DTNB titration. + indicates that the Cys residue is intact; − indicates that the Cys residue is altered to Ala. Error bars represent S.D. from three independent assays.

FIGURE 5.

Identification of disulfide and sulfane sulfur modification of the M. jannaschii SepCysS by LC-MS/MS. Fragment b- and y-ions derived from the tryptic peptide ⁵³AVYEYWDGYSVCDYCHGR⁷⁰ (theoretical MH²⁺ = 1094.20) are labeled in green and blue, respectively. A, MS/MS fragmentation spectra of the −2 Da shifted precursor ion (MH²⁺ = 1093.53). Detected fragment b- and y-ions containing Cys⁶⁴ and Cys⁶⁷ with −2 Da shift are underlined. B, MS/MS fragmentation spectra of the +30 Da shifted precursor ion (MH²⁺ = 1109.56). Detected fragment b- and y-ions containing Cys⁶⁴ and Cys⁶⁷ with +30 Da shift are underlined.

FIGURE 6.

Proposed mechanism of sulfur delivery by SepCysS. A–D, proposed PLP-dependent Sep → Cys conversion is based upon the mechanism of Sep → Sec conversion catalyzed by SepSecS (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 Lys²³⁴ and PLP to form an external aldimine. B, electron delocalization leads to β-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, Lys²³⁴ 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 (S²⁻ equivalent) attacks the disulfide between Cys⁶⁴ and Cys⁶⁷, and the sulfur (in red) is consequently transferred to either Cys⁶⁴ or Cys⁶⁷ (Cys⁶⁴ shown in the scheme here) to generate a persulfide enzyme adduct of SepCysS. F, thiolate formed by the other Cys (Cys⁶⁷ shown in the scheme here) attacks the bridging sulfur of the persulfide to liberate the terminal sulfur (in red) as a formal equivalent of S²⁻ 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⁻) 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⁻).