Thiosulfate dehydrogenase (TsdA) from Allochromatium vinosum: structural and functional insights into thiosulfate oxidation

Abstract

Although the oxidative condensation of two thiosulfate anions to tetrathionate constitutes a well documented and significant part of the natural sulfur cycle, little is known about the enzymes catalyzing this reaction. In the purple sulfur bacterium Allochromatium vinosum, the reaction is catalyzed by the periplasmic diheme c-type cytochrome thiosulfate dehydrogenase (TsdA). Here, we report the crystal structure of the "as isolated" form of A. vinosum TsdA to 1.98 Å resolution and those of several redox states of the enzyme to different resolutions. The protein contains two typical class I c-type cytochrome domains wrapped around two hemes axially coordinated by His(53)/Cys(96) and His(164)/Lys(208). These domains are very similar, suggesting a gene duplication event during evolution. A ligand switch from Lys(208) to Met(209) is observed upon reduction of the enzyme. Cys(96) is an essential residue for catalysis, with the specific activity of the enzyme being completely abolished in several TsdA-Cys(96) variants. TsdA-K208N, K208G, and M209G variants were catalytically active in thiosulfate oxidation as well as in tetrathionate reduction, pointing to heme 2 as the electron exit point. In this study, we provide spectroscopic and structural evidence that the TsdA reaction cycle involves the transient presence of heme 1 in the high-spin state caused by movement of the Sγ atom of Cys(96) out of the iron coordination sphere. Based on the presented data, we draw important conclusions about the enzyme and propose a possible reaction mechanism for TsdA.

Keywords: Allochromatium vinosum; Crystal Structure; Cytochrome c; Enzyme Kinetics; Enzyme Mechanism; Enzyme Mutation; Sulfur; Tetrathionate; Thiosulfate Dehydrogenase; TsdA.

FIGURE 1.

TsdA structure and comparison with SoxA. A, TsdA overall fold with N-terminal domain (residues 5–138) represented in blue and C-terminal domain (residues 139–237) in red; the heme prosthetic groups are colored by atom type (yellow for carbon, blue for nitrogen, red for oxygen, and dark red for iron; this color code will be used in all figures). B, TsdA domains (colored as in A), superposed with the heme groups displayed in the same colors as their corresponding domains (heme 1 in blue and heme 2 in red). C, structural superposition of A. vinosum TsdA with R. sulfidophilum SoxA, for which the N-terminal domain (residues 51–150) is shown in green (corresponding heme group in dark green), and the C-terminal domain (residues 151–250) is shown in yellow (corresponding heme group in orange). D, structural superposition of A. vinosum TsdA with R. sulfidophilum SoxA individual domains (color code as in B and C).

FIGURE 2.

Heme coordination of “as isolated” TsdA (PDB code 4QW7). A, heme 1 is coordinated by His⁵³ and Cys⁹⁶. B, heme 2 is coordinated by His¹⁶⁴ and Lys²⁰⁸. A schematic representation is shown in pale gray with heme moieties and coordinating amino acid residues shown in sticks (color code as in Fig. 1 with sulfur atoms in green).

FIGURE 3.

TsdA heme environments. A and C, TsdA electrostatic surface potential highlighting the exposure of hemes 1 and 2, respectively. B and D, enlarged view of hemes 1 and 2, respectively; the amino acid residues forming the cleft's entrance are labeled.

FIGURE 4.

Vicinity of heme 1 distal side and ligand switch at heme 2. 2mF_o − DF_c electron density map contoured at 1σ level around heme 1 in TsdA-K208N variant showing an alternate conformation for Cys⁹⁶ and a thiosulfate (PDB code 4QWC) (A), and heme 1 from a bisulfite-soaked crystal displaying a persulfurated cysteine, a sulfite, and water molecule (PDB code 4QWB) (B). C, heme 1 from a dithionite-soaked crystal showing a Cys⁹⁶ double conformation, one of which is in a sulfinate form, a thiosulfate and a sulfide ion coordinating the heme (PDB code 4QW9). D, heme 2 from a dithionite-soaked crystal revealing a ligand switch upon reduction; Lys²⁰⁸ moves toward the interior of the protein, whereas Met²⁰⁹ coordinates heme 2 (PDB code 4QW9).

FIGURE 5.

Heme coordination of TsdA variants. 2mF_o − DF_c electron density map contoured at 1.5σ level around heme 2 in TsdA-K208N (PDB code 4WQC) (A), heme 2 in TsdA-K208G (PDB code 4WQE) (B), and heme 1 in TsdA-K208G (PDB code 4WQE) (C) with a thiosulfate, sulfite, and water molecule. D, same representation as in C with a modeled tetrathionate ion, represented in thinner sticks, based on the spatial arrangement of the other observed ions.

FIGURE 6.

Comparison of electronic absorbance spectra for A. vinosum TsdA Cys⁹⁶ variants. Complete spectra of recombinant TsdA-C96H (100 μg ml⁻¹; 3.6 μm) (A), TsdA-C96M (120 μg ml⁻¹; 4.3 μm) (B), and TsdA-C96G (125 μg ml⁻¹; 4.5 μm) (C) were recorded for the “as isolated” (thin line), oxidized (dotted line), and reduced states (thick line). The region from 550 to 750 nm is enlarged for the oxidized (D) and reduced (E) state of recombinant TsdAwt (250 μg ml⁻¹; 8.9 μm; thick line), TsdA-C96G (dashed line), TsdA-C96H (dotted line), and TsdA-C96M (thin line). Oxidation was achieved by the addition of 3.5 μm ferricyanide, and reduction was achieved by the addition of 10 mm sodium dithionite. All spectra were recorded in 50 mm BisTris-HCl buffer, pH 6.5, and set to the same 280 and 750 nm values.

FIGURE 7.

Comparison of electronic absorbance spectra for A. vinosum TsdA wild type and Lys²⁰⁸/Met²⁰⁹ variants. Complete spectra of recombinant TsdA wild type (160 μg ml⁻¹; 5.7 μm) (A), TsdA-K208G (224.6 μg ml⁻¹; 8.3 μm) (B), TsdA-K208N (200 μg ml⁻¹; 7.4 μm) (C), and TsdA-M209G (218 μg ml⁻¹; 8.1 μm) (D) were recorded for the “as isolated” (thin line), oxidized (dotted line), and reduced states (thick line). Oxidation was achieved by the addition of 7 μm ferricyanide, and reduction was achieved by the addition of 10 mm sodium dithionite. Spectra from 550 to 750 nm were recorded for highly concentrated protein solutions: TsdA wild type protein (1150 μg ml⁻¹; 41.1 μm) (E), TsdA-K208G (100 μg ml⁻¹; 37 μm) (F), TsdA-K208N (1123 μg ml⁻¹; 41.6 μm) (G), and TsdA-M209G (873 μg ml⁻¹; 32.3 μm) (H). All spectra were taken in 50 mm BisTris-HCl buffer, pH 6.5, and set to the same 280 and 750 nm values.

FIGURE 8.

Comparison of electronic absorption spectra for the A. vinosum TsdA wild type protein at different pH values. Spectra of recombinant TsdA wild type protein (160 μg ml⁻¹; 5.9 μm) at pH 5.0 (thin line), pH 6.5 (dotted line), and pH 8 (thick line) in the oxidized (A) and in the reduced state (B) were recorded in 100 mm ammonium acetate buffer (pH 5.0), 50 mm BisTris-HCl buffer (pH 6.5), or 50 mm Tris-HCl buffer (pH 8.0), respectively. Oxidation was achieved by the addition of 7 μm ferricyanide, and reduction was achieved by the addition of 10 mm sodium dithionite. The insets in A and B show details of the 580–750 nm region (640 μg ml⁻¹; 23.7 μm). C, comparison of electronic absorption spectra (550–750 nm) for A. vinosum wild type TsdA (160 μg ml⁻¹; 5.9 μm) in the oxidized (thin line), thiosulfate-reduced (dotted line), partially reduced (dashed line), and completely reduced (thick line) state. Partial reduction was achieved by the addition of 1 mm sodium dithionite, and complete reduction was achieved by the addition of 10 mm sodium dithionite. For the thiosulfate-reduced state, 2 mm thiosulfate were added. All spectra were recorded in 50 mm BisTris-HCl buffer, pH 6.5.

FIGURE 9.

Proposed mechanism of TsdA activity. A reaction scheme for the TsdA-catalyzed oxidation of two thiosulfate molecules to tetrathionate is shown. Two thiosulfate molecules enter the active site, inducing the Sγ atom of Cys⁹⁶ to tilt away from the iron coordination sphere. This is probably accompanied by protonation of Sγ. The two thiosulfates react directly with each other, liberating two electrons that reduce heme 1 and heme 2. Upon reduction, heme 2 undergoes a shift from His/Lys to His/Met ligation. Tetrathionate is released, and the hemes are oxidized. HiPIP (high-potential iron-sulfur protein) is a likely in vivo electron acceptor for A. vinosum TsdA (64). Cys⁹⁶ is deprotonated, and Sγ is re-established as the ligand to heme 1 iron.