Fast conformational exchange between the sulfur-free and persulfide-bound rhodanese domain of E. coli YgaP

Highlights

• Three dimensional solution NMR structure of YgaP rhodanese domain.

• Function validation of YgaP rhodanese domain to substrate Na₂S₂O₃.

• Fast exchange between the intact and persulfide-intermediate rhodanese domain.

Abstract

Rhodanese domains are abundant structural modules that catalyze the transfer of a sulfur atom from thiolsulfates to cyanide via formation of a covalent persulfide intermediate that is bound to an essential conserved cysteine residue. In this study, the three-dimensional structure of the rhodanese domain of YgaP from Escherichia coli was determined using solution NMR. A typical rhodanese domain fold was observed, as expected from the high homology with the catalytic domain of other sulfur transferases. The initial sulfur-transfer step and formation of the rhodanese persulfide intermediate were monitored by addition of sodium thiosulfate using two-dimensional ¹H–¹⁵N correlation spectroscopy. Discrete sharp signals were observed upon substrate addition, indicting fast exchange between sulfur-free and persulfide-intermediate forms. Residues exhibiting pronounced chemical shift changes were mapped to the structure, and included both substrate binding and surrounding residues.

Introduction

Rhodanese was first reported in 1933 as an enzyme capable of catalyzing the transfer of a sulfur atom from thiosulfate to the toxic cyanide, resulting in the non-toxic thiocyanate [1]. The cyanide detoxification activity of rhodanese has since been identified in all three major evolutionary phyla [2], and the enzyme has been given the full name thiosulfate:cyanide sulfurtransferase (TST, EC 2.8.1.1) [3]. Rhodanese domains are found as tandem repeats, single domain proteins or in combination with distinct protein domains [4].

The best-characterized are the mitochondrial bovine TST (Rhobov) [5], [6] and the Azotobacter vinelandii rhodanese (RhdA) enzymes [7], both of which contain two tandem rhodanese domains and an essential conserved Cys residue in the C-terminal rhodanese domain that is critical for catalysis. It has been reported that lack of the Cys residue in the N-terminal rhodanese domain also results in an inactive enzyme [5], [7]. Structurally, rhodanese domains are closely related to the human phosphatase Cdc25, both in tertiary structure and the location of the conserved Cys [8], [9]. In Escherichia coli, eight proteins have been identified as containing rhodanese domains [10], and three (GlpE, PspE, and YgaP) belong to the single-domain rhodanese homology family and contain the essential Cys [11]. Among them, YgaP is the only membrane-associated rhodanese with the predicted two transmembrane helices near the C-terminus [12].

Currently, two separate sulfur-transfer steps are believed to occur during catalysis. In the first step, the sulfhydryl (−SH) group of the conserved Cys residue attacks the thiosulfate (S₂O₃²⁻) anion, forming a covalent persulfide intermediate; in the second step, the persulfide intermediate is attacked by a cyanide (CN⁻) ion to release the thiocyanate (SCN⁻) product and regenerate the cysteine sulfhydryl group [3], [7]. The three-dimensional structure of PspE was determined using solution NMR. It was reported that backbone dynamics of the persulfide intermediate of PspE was less stable and more conformationally flexible than the ligand-free form, despite of similar conformations between the two forms [13].

In this work, we determined the NMR solution structure of the rhodanese domain of YgaP from E. coli. The structure showed a typical fold reminiscent of GlpE [11] and PspE [13]. During preparation of this communication, Eichmann et al. [14] reported the solution NMR structure of full-length YgaP in the presence of detergent micelles, which formed a homo-dimer with strong hydrophobic interactions between the two transmembrane helices of each subunit, although no direct interactions between the N-terminal rhodanese domains were visible. The YgaP rhodanese domain structure determined in the present study was highly similar to full length YgaP. Formation of the persulfide intermediate resulting from the first sulfur-transfer step was monitored using two dimensional NMR following addition of sodium thiosulfate. Interestingly, significant chemical shift perturbations were observed in both substrate binding and surrounding residues. Line shape analysis indicated that a fast exchange takes place between the ligand-free and persulfide-bound forms.