Structural and biochemical analyses indicate that a bacterial persulfide dioxygenase-rhodanese fusion protein functions in sulfur assimilation

Abstract

Hydrogen sulfide (H₂S) is a signaling molecule that is toxic at elevated concentrations. In eukaryotes, it is cleared via a mitochondrial sulfide oxidation pathway, which comprises sulfide quinone oxidoreductase, persulfide dioxygenase (PDO), rhodanese, and sulfite oxidase and converts H₂S to thiosulfate and sulfate. Natural fusions between the non-heme iron containing PDO and rhodanese, a thiol sulfurtransferase, exist in some bacteria. However, little is known about the role of the PDO-rhodanese fusion (PRF) proteins in sulfur metabolism. Herein, we report the kinetic properties and the crystal structure of a PRF from the Gram-negative endophytic bacterium Burkholderia phytofirmans The crystal structures of wild-type PRF and a sulfurtransferase-inactivated C314S mutant with and without glutathione were determined at 1.8, 2.4, and 2.7 Å resolution, respectively. We found that the two active sites are distant and do not show evidence of direct communication. The B. phytofirmans PRF exhibited robust PDO activity and preferentially catalyzed sulfur transfer in the direction of thiosulfate to sulfite and glutathione persulfide; sulfur transfer in the reverse direction was detectable only under limited turnover conditions. Together with the kinetic data, our bioinformatics analysis reveals that B. phytofirmans PRF is poised to metabolize thiosulfate to sulfite in a sulfur assimilation pathway rather than in sulfide stress response as seen, for example, with the Staphylococcus aureus PRF or sulfide oxidation and disposal as observed with the homologous mammalian proteins.

Keywords: X-ray crystallography; enzyme kinetics; hydrogen sulfide; iron; sulfur.

Figure 1.

Organization of BpPRF and limited sequence comparison. A, domain organization of PRFs. PRFs form S. aureus (SaPRF) and A. acidocaldarius (AaPRF) have an N-terminal PDO domain, a non-catalytic rhodanese (RHOD) domain, and a catalytic rhodanese domain. BpPRF displays an N-terminal PDO domain and C-terminal catalytic rhodanese (Rhod) domain. B, sequence alignment of rhodanese active-site sequences from human (hTST) and bovine (RhoBov) rhodanese, human mercaptopyruvate sulfurtransferase (MST), E. coli single domain rhodanese (GlpE), human mitochondrial single-domain sulfurtransferase (TSTD1), S. aureus PRF (SaPRF or CstB), B. phytofirmans PRF (BpPRF), and A. acidocaldarius PRF (AaPRF). The conserved active-site cysteine is highlighted in yellow. C, comparison of the genomic contexts of CstB (top) and BpPRF (bottom). The operon encoding CstB (pink) includes a multidomain rhodanese (CstA, red) and an SQR homolog (light green). The adjacent locus includes the polysulfide-sensing repressor CsTR (yellow) and TauE (purple), a putative sulfite permease/transmembrane sulfur compound exporter. The loci adjacent to BpPRF (pink) include a putative peroxidase (cyan) and pyridoxal 4-dehydrogenase (blue), a putative transmembrane (TMS)-type transporter (dark green), and a putative LysR-type regulator (orange).

Figure 2.

Crystal structure of BpPRF. The X-ray crystal structure of ΔCBpPRF was solved at 1.79 Å resolution by molecular replacement using the AaPRF structure (PDB code 3TP9) as a template. A, structure of the BpPRF monomer consists of an N-terminal PDO domain (blue), a 15-residue linker region (orange), and a C-terminal rhodanese domain (red). The linker region lacked density for eight residues (dashed lines) suggesting a flexible, disordered state. The iron (orange sphere) ligands in the PDO domain and the active-site cysteine, Cys-314, in the rhodanese domain are shown in stick representation. The rhodanese domain β-hairpin extension is highlighted by the red arrow. B, close-up of the PDO-active site with representative electron density (3.0σ F_o − F_c omit density; gray mesh) for side chains of His-58, His-114, Asp-133, and three water molecules (shown as red spheres) coordinated to the iron center (orange sphere). C, close-up of the rhodanese domain active site with representative electron density (3.0σ F_o − F_c omit density; gray mesh) for the Cys-314 side chain displaying the additional density for the persulfide modification.

Figure 3.

Close-up of the PDO-active site of BpPRF with GSH. A, stereo view of the C314S BpPRF PDO domain with GSH bound. The surrounding residues that may participate in substrate binding and/or stabilization are shown with electron density (3.0σ F_o − F_c omit density; gray mesh) for GSH. Tyr-176, Arg-142, and Lys-216 are hydrogen-bonded (black dashes) to GSH. Two waters (red spheres) are coordinated to the iron center (orange sphere) in addition to the sulfur atom of GSH. B, stereo view of C314S BpPRF PDO domain (blue) in complex with GSH overlaid on human PDO (15) (PDB code 4CHL) (gray). GSH (shown in green) is displayed in stick representation. Conserved residues predicted to be involved in substrate binding, stabilization, and/or positioning (15) are Tyr-176, Arg-193, Leu-212, Arg-142, and Pro-215 (BpPRF numbering). Lys-216 is not conserved in human PDO.

Figure 4.

Comparison of the BpPRF rhodanese domain with rhodanese. A, structural overlay of the BpPRF rhodanese domain (red) with the catalytic domain of bovine rhodanese (PDB code 1RHD, gray (34)). The active-site cysteine residues, Cys-247 (bovine) and Cys-314 (BpPRF), are shown in stick representation Structural differences are shown with arrows: a β-hairpin extension is seen only in BpPRF (red), and two loop extensions are seen only in bovine rhodanese (black). B, structural overlay of the rhodanese active-site loops from BpPRF (red) and bovine rhodanese (gray).

Figure 5.

Kinetics of PDO activity of BpPRF. Dependence of PDO activity on GSSH concentration for wild-type BpPRF (solid circles) and C314S BpPRF (open circles). Oxygen consumption by BpPRF in 100 mm sodium phosphate buffer, pH 7.4, was monitored at 22 °C in the presence of varying concentration of GSSH. The data are representative of three independent experiments.

Figure 6.

Kinetics of BpPRF-catalyzed sulfur transfer reactions. The sulfurtransferase activity associated with the rhodanese domain in wild-type BpPRF (A, C, and E) or with the isolated rhodanese domain (Rhod domain) (B, D, and F) was determined in the presence of a constant thiosulfate concentration (60 mm) and varying concentrations of cyanide (A and B), GSH (C and D), or cysteine (E and F). The data are representative of 3–4 independent experiments. The data were fitted with the Michaelis-Menten, sigmoidal, or Hill equation as described under “Experimental procedures.”

Figure 7.

Kinetics of BpPRF-catalyzed sulfur transfer reactions. Dependence of sulfurtransferase activity on varying thiosulfate concentrations in the presence of 60 mm cyanide (A and B), 30 mm GSH (C and D), and 50 mm cysteine (E and F) for the wild-type BpPRF and the isolated rhodanese domain (Rhod Domain). The reactions were performed as described under “Experimental procedures.” The data are representative of 3–4 independent experiments. Data were fitted with either Michaelis-Menten or Hill equation.

Figure 8.

Product analysis and reaction stoichiometry of the BpPRF-The reaction stoichiometry of BpPRF under single-turnover conditions was analyzed in the presence (A) and absence (B) of oxygen. The reactions were performed in the presence of 250 μm GSH, 250 μm thiosulfate and 250 μm enzyme. C, stoichiometry of BpPRF reactions under multiple turnover conditions was analyzed in the presence of 250 μm GSH, 250 μm thiosulfate and decreasing enzyme concentrations as described under “Experimental procedures.” Data are representative of 4–5 independent experiments.

Figure 9.

Modeling of potential BpPRF domain interaction interface. A, overlay of BpPRF PDO domain (blue) and the BpPRF rhodanese domain (red) with the PDO domain and catalytic rhodanese domain of AaPRF (PDB code 3TP9) (shown in gray). The active-site loop of AaPRF (yellow) with Cys-202 faces the iron center (orange sphere) in the PDO domain. B, electrostatic surface potential of the BpPRF domains in the same orientation as in A but rotated by 35°. The arrows highlight locations of the PDO- and rhodanese (Rhod)-active sites. Positive and negative electrical potential are shown in blue and red, respectively, and represent a range of −5 to +5 kT/e.