Conformational Analysis and Chemical Reactivity of the Multidomain Sulfurtransferase, <i>Staphylococcus aureus</i> CstA

The <i>cst</i> operon of the major human pathogen <i>Staphylococcus aureus</i> (<i>S. aureus</i>) 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 <i>cst</i> operon, <i>cstA</i> and <i>cstB</i>, protect <i>S. aureus</i> 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 <i>S. aureus</i> against sulfide toxicity <i>in vivo</i>. The N-terminal domain of CstA exhibits thiosulfate sulfurtransferase (TST; rhodanese) activity, and a Cys66 ³⁴S-persulfide is formed as a catalytic intermediate in both the presence and absence of the adjacent TusA-like domain using ³⁴S-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 <i>in situ</i> persulfurated SufS. C66A substitution in CstA^Rhod‑TusA abolishes C128 <i>S</i>-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 (<i>R</i>_g ≈ 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