Conformational Analysis and Chemical Reactivity of the Multidomain Sulfurtransferase, Staphylococcus aureus CstA

Abstract

The cst operon of the major human pathogen Staphylococcus aureus (S. aureus) is under the transcriptional control of CsoR-like sulfurtransferase repressor (CstR). Expression of this operon is induced by hydrogen sulfide, and two components of the cst operon, cstA and cstB, protect S. aureus from sulfide toxicity. CstA is a three-domain protein, and each domain harbors a single cysteine that is proposed to function in vectorial persulfide shuttling. We show here that single cysteine substitution mutants of CstA fail to protect S. aureus against sulfide toxicity in vivo. The N-terminal domain of CstA exhibits thiosulfate sulfurtransferase (TST; rhodanese) activity, and a Cys66 (34)S-persulfide is formed as a catalytic intermediate in both the presence and absence of the adjacent TusA-like domain using (34)S-SO3(2-) as a substrate. Cysteine persulfides can be trapped on both C66 in CstA(Rhod) and on C66 and C128 in CstA(Rhod-TusA) when incubated with thiosulfate, sodium tetrasulfide (Na2S4), and in situ persulfurated SufS. C66A substitution in CstA(Rhod-TusA) abolishes C128 S-sulfhydration, consistent with directional persulfide shuttling in CstA. Fully reduced CstA(Rhod-TusA) is predominately monomeric, and high resolution tandem mass spectrometry reveals that Cys66 and Cys128 can form a C66-C128 disulfide bond using a number of oxidants, which leads to a significant change in conformation. A competing intermolecular C128-C128' disulfide bond is also formed. Small-angle X-ray scattering measurements and gel filtration chromatography of reduced CstA(Rhod-TusA) reveal an elongated molecule (Rg ≈ 30 Å, 21.6 kDa) where the two domains pack "side-by-side" that likely places Cys66 and Cys128 far apart. These studies are consistent with the low yield of C66-C128 cross-link as a mimic of a persulfide transfer intermediate in CstA, and small, but measurable persulfide transfer from Cys66 to Cys128 within the CstA(Rhod-TusA) with inorganic sulfur donors.

Sulfide-responsive transcriptional repressor SqrR functions as a master regulator of sulfide-dependent photosynthesis

“…These results reveal that 45% of all sulfide-responsive genes in the R. capsulatus genome are directly or indirectly regulated by SqrR. The identified sulfideresponsive genes include a number of genes known to function in sulfur metabolism or assimilation, including thiosulfate-sulfite oxidoreductases and thiosulfate sulfurtransferases or rhodanese homology domain proteins (33,34), whose expression is strongly repressed by SqrR. Although originally described as functioning in cyanide detoxification (35), sulfurtransferases and structurally unrelated single-Cys-containing TusA domains (36) are generally involved in persulfide shuttling, and are capable of accepting sulfur from a variety of RSS, including thiosulfate and lowmolecular-weight persulfides (37).…”

Hydrogen Sulfide and Reactive Sulfur Species Impact Proteome S-Sulfhydration and Global Virulence Regulation in Staphylococcus aureus

“…31,33 Strikingly, two uncharacterized thioredoxin-like proteins encoded by locus tags NWMN_0774 (of known structure; see Figure 5) and NWMN_0779 were found to be S -sulfhydrated only in sulfide-stressed cells and are therefore excellent candidates as protein “depersulfidases” (Figure 2D; Table S2), as described previously for the mammalian thioredoxin (Trx). 31,33 We find that NWMN_0774 and NWMN_0779 are both capable of reducing S -sulfhydrated CstA Rhod (Figure S3D), corresponding to the N-terminal rhodanese domain of the sulfurtransferase CstA 35 as a model substrate, in the presence of authentic thioredoxin reductase, TrxB, and a reductant (Figure 6A; Table S3). We therefore designate NWMN_0774 and NWMN_0779 as trxP and trxQ (for thioredoxin depersulfidases P and Q), respectively.…”

“…We therefore designate NWMN_0774 and NWMN_0779 as trxP and trxQ (for thioredoxin depersulfidases P and Q), respectively. In addition to CstA Rhod , we also tested the depersulfidase activity of TrxP and TrxQ on S -sulfhydrated Ldh2 which is modified at Cys71 nearly exclusively in sulfide-stressed cells and S -sulfhydrated to ≈70% upon reaction with Na 2 S 4 35 in vitro. S -Sulfhydrated Ldh2 is efficiently reduced by the TrxP–TrxB pair, concomitant with the production of H 2 S (Figure 6E,F, Table S4).…”

“…(A) Scheme of the strategy used to identify S -sulfhydrated peptides and proteins before (0 mM sulfide) or after addition of sulfide (0.2 mM) to cells. The method takes advantage of similar reactivities of thiols vs persulfides toward the iodoacetamides, 35 with both moieties capped with biotinylated iodoacetamide (b-IAM), enriched by neutravidin capture, which, following a washing protocol, is treated with the reductant TCEP, followed by capping eluted peptides with IAM, with carbamidomethylated (CAM) peptides identified by UPLC-ESI-MS/MS. Three biological replicates ( i = 1–3) of untreated (N i ) and sulfide-treated (S i ) were analyzed in this way (Figure S4), with the mean and standard deviation shown for the number of S -sulfhydrated peptides (B) and corresponding proteins (C) enriched by this method.…”

Staphylococcus aureus CstB Is a Novel Multidomain Persulfide Dioxygenase-Sulfurtransferase Involved in Hydrogen Sulfide Detoxification

“…16 SQR and CstB, on the other hand, are predicted to comprise a system analogous to the first two steps in human mitochondrial H 2 S oxidation. Human SQR (hSQR) initially oxidizes H 2 S to glutathione persulfide (GSSH); subsequently, ETHE1 oxidizes GSSH to sulfite in the presence of molecular oxygen, while regenerating reduced glutathione (GSH).…”