Solution Structures and Backbone Dynamics of Escherichia coli Rhodanese PspE in Its Sulfur-Free and Persulfide-Intermediate Forms: Implications for the Catalytic Mechanism of Rhodanese^,

Abstract

Rhodanese catalyzes the sulfur-transfer reaction that transfers sulfur from thiosulfate to cyanide by a double-displacement mechanism, in which an active cysteine residue plays a central role. Previous studies indicated that the phage-shock protein E (PspE) from Escherichia coli is a rhodanese composed of a single active domain and is the only accessible rhodanese among the three single-domain rhodaneses in E. coli. To understand the catalytic mechanism of rhodanese at the molecular level, we determined the solution structures of the sulfur-free and persulfide-intermediate forms of PspE by nuclear magnetic resonance (NMR) spectroscopy and identified the active site by NMR titration experiments. To obtain further insights into the catalytic mechanism, we studied backbone dynamics by NMR relaxation experiments. Our results demonstrated that the overall structures in both sulfur-free and persulfide-intermediate forms are highly similar, suggesting that no significant conformational changes occurred during the catalytic reaction. However, the backbone dynamics revealed that the motional properties of PspE in its sulfur-free form are different from the persulfide-intermediate state. The conformational exchanges are largely enhanced in the persulfide-intermediate form of PspE, especially around the active site. The present structural and biochemical studies in combination with backbone dynamics provide further insights in understanding the catalytic mechanism of rhodanese.

Solution NMR Structure and Functional Analysis of the Integral Membrane Protein YgaP from Escherichia coli

“…N-labeled rhodanese-F were assigned using triple resonance three-dimensional constant time (ct)-TROSY 3 -HNCA, three-dimensional ct-TROSY-HNCACB (13), and three-dimensional ct-TROSY-HN(CO)CA (14) experiments. The side-chain 1 H resonances were assigned using three-dimensional ct-HCCH-total correlation spectroscopy (mixing time ( m ) ϭ 21.7 ms) (15) and three-dimensional H NOESY experiments ( m ϭ 60 ms) (17,18).…”

“…4C). Similar to GlpE and PspE (3,4), the active loop folds in a semicircular cradle-like conformation centered around the Cys-63 S ␥ atom defining the enzyme catalytic pocket. All backbone amide protons of residues Gln-64 -Thr-69 point toward the S ␥ atom of Cys-63, although their side chains point away from the catalytic pocket (Fig.…”

“…YgaP has a molecular mass of 18.6 kDa and is composed of 174 amino acid residues, of which 119 -174 are predicted by the membrane protein topology prediction method TMHMM (1) to form two transmembrane helices, whereas residues 1-118 are predicted to form a cytoplasmic rhodanese domain with sulfurtransferase activity (2,3). YgaP therefore belongs to the family of rhodaneses (thiosulfate-cyanide sulfurtransferase), which is a large superfamily of enzymes found in bacterial, archaeal, and eukaryotic cells.…”

“…YgaP is thereby the only membrane protein in E. coli with a rhodanese domain. These enzymes are supposed to have sulfurtransferase activity (3). Rhodanese domain-containing proteins provide both eukaryotic and prokaryotic organisms with labile reactive sulfides.…”

“…In the first step, the thiol group of the cysteine reacts with the thiosulfate anion (S 2 O 3 Ϫ ) to form an enzyme-persulfide intermediate, which reacts in a second step with the cyanide ion to produce thiocyanate (SCN Ϫ ) (7)(8)(9). Although the crystal structure of the sulfur-free and the persulfide form of one member of the rhodanese family (GlpE) as well as the solution state NMR structure of another member (PspE) have been determined (3,4,10), the molecular mechanism of the sulfurtransferase activity remains unclear (11), but the dynamics appear to be involved (3).…”