Reaction of Thiosulfate Dehydrogenase with a Substrate Mimic Induces Dissociation of the Cysteine Heme Ligand Giving Insights into the Mechanism of Oxidative Catalysis

Abstract

Thiosulfate dehydrogenases are bacterial cytochromes that contribute to the oxidation of inorganic sulfur. The active sites of these enzymes contain low-spin c-type heme with Cys^-/His axial ligation. However, the reduction potentials of these hemes are several hundred mV more negative than that of the thiosulfate/tetrathionate couple (E_m, +198 mV), making it difficult to rationalize the thiosulfate oxidizing capability. Here, we describe the reaction of Campylobacter jejuni thiosulfate dehydrogenase (TsdA) with sulfite, an analogue of thiosulfate. The reaction leads to stoichiometric conversion of the active site Cys to cysteinyl sulfonate (C_α-CH₂-S-SO₃^-) such that the protein exists in a form closely resembling a proposed intermediate in the pathway for thiosulfate oxidation that carries a cysteinyl thiosulfate (C_α-CH₂-S-SSO₃^-). The active site heme in the stable sulfonated protein displays an E_m approximately 200 mV more positive than the Cys^-/His-ligated state. This can explain the thiosulfate oxidizing activity of the enzyme and allows us to propose a catalytic mechanism for thiosulfate oxidation. Substrate-driven release of the Cys heme ligand allows that side chain to provide the site of substrate binding and redox transformation; the neighboring heme then simply provides a site for electron relay to an appropriate partner. This chemistry is distinct from that displayed by the Cys-ligated hemes found in gas-sensing hemoproteins and in enzymes such as the cytochromes P450. Thus, a further class of thiolate-ligated hemes is proposed, as exemplified by the TsdA centers that have evolved to catalyze the controlled redox transformations of inorganic oxo anions of sulfur.

Figure 1

Cys^–/His-ligated c-heme present in the active site of thiosulfate dehydrogenase, as illustrated for A. vinosum TsdA with Cys¹²³, c-heme, Arg¹⁰⁹, and Arg¹¹⁹ shown as sticks (PDB ID: 4WQ7).

Figure 2

LCMS and electronic absorbance of di-Fe(III) CjTsdA (black), after anaerobic incubation with 1.5 mM sulfite (red), then fully reoxidized with ferricyanide (blue). (A) Deconvoluted mass spectra after exposure to iodoacetate, see the text for details. Arrows indicate 37,202, 37,259, and 37,281 Da, the masses corresponding to CjTsdA with cysteinate¹³⁸ in unmodified, alkylated, and sulfonated forms, respectively, see the Supporting Information for details. (B) Electronic absorbance spectra. Samples (see Table S1 for the protein concentration) in anaerobic 50 mM HEPES, 50 mM NaCl, pH 7.

Figure 3

Reaction of di-Fe(III) CjTsdA with sulfite. For clarity, only the heme irons and active site Cys¹³⁸ are shown.

Figure 4

RT-MCD of di-Fe(III) CjTsdA (black), after anaerobic incubation with 1.5 mM sulfite (red) followed by sufficient ferricyanide to fully reoxidize (blue). The dashed traces are expanded ×10. Samples (see Table S1 for protein concentrations) in anaerobic 50 mM HEPES, 50 mM NaCl, pH 7.

Figure 5

Representative protein film cyclic voltammograms for CjTsdA before (A) and after (B) sulfite-conjugation of Cys¹³⁸. Baseline subtracted current (lines). Circles show the sum of contributions from two n = 1 centers with E_m −134 and +145 mV (A) and E_m, +75 and +154 mV (B) with individual contributions shown as dashed lines for the latter. These E_m values are also shown by the solid vertical lines for each heme. Dotted vertical lines show E_m values previously reported for CjTsdA. Scan rate is 10 mV s^–1 in anaerobic 50 mM HEPES, 50 mM NaCl, pH 7.

Figure 6

Proposed catalytic cycle for thiosulfate oxidation by TsdA enzymes where protonation of the active site cysteinate is critical to its dissociation from Heme 1. Two conserved Arg side chains in the substrate-binding pocket are indicated as positive charges.