Organization of the human mitochondrial hydrogen sulfide oxidation pathway

Abstract

Sulfide oxidation is expected to play an important role in cellular switching between low steady-state intracellular hydrogen sulfide levels and the higher concentrations where the physiological effects are elicited. Yet despite its significance, fundamental questions regarding how the sulfide oxidation pathway is wired remain unanswered, and competing proposals exist that diverge at the very first step catalyzed by sulfide quinone oxidoreductase (SQR). We demonstrate that, in addition to sulfite, glutathione functions as a persulfide acceptor for human SQR and that rhodanese preferentially synthesizes rather than utilizes thiosulfate. The kinetic behavior of these enzymes provides compelling evidence for the flow of sulfide via SQR to glutathione persulfide, which is then partitioned to thiosulfate or sulfite. Kinetic simulations at physiologically relevant metabolite concentrations provide additional support for the organizational logic of the sulfide oxidation pathway in which glutathione persulfide is the first intermediate formed.

Keywords: Enzyme Kinetics; Flavoprotein; Hydrogen Sulfide; Oxidation-Reduction (Redox); Quinone; Redox.

FIGURE 1.

Overview of the enzymology of H₂S homeostasis. Scheme showing enzymes involved in the mitochondrial sulfide oxidation pathway. SDO denotes sulfur dioxygenase.

FIGURE 2.

Alternative wiring modes in the sulfide oxidation pathway. Path 1, GSH is the acceptor. Path 2, sulfite is the acceptor and possibly originates via the cysteine catabolic pathway in which cysteine sulfinic acid formed in the cytoplasm is converted to β-sulfinylpyruvate in the mitochondrion. Path 3, an unknown acceptor couples to SQR. Rhd, TST, and SO refer to rhodanese, a thiol sulfurtransferase, and sulfite oxidase, respectively.

FIGURE 3.

Kinetic scheme of reactions catalyzed by SQR. E₁-free (unbound) enzyme. E₂, E₃₁, E₃₂, E₃₃, E₃₄, and E₄ are enzyme complexes with different intermediates. HS⁻, hydrogen sulfide; GSH, reduced glutathione; Cys, cysteine; Hcy, homocysteine; SO₃²⁻-sulfite; CysSH, cysteine persulfide; HcySH, homocysteine persulfide; S₂O₃²⁻, thiosulfate. The rate constants for the individual steps are denoted by k.

FIGURE 4.

Characterization of recombinant human SQR and rhodanese. A, purity of recombinant human SQR (left panel) and rhodanese (right panel) as assessed by SDS-PAGE analysis. The molecular (M) weight markers are shown. B, absorption spectrum of purified SQR (0.55 mg/ml) in 100 mm Tris buffer, pH 8.0, containing 0.03% DHPC. C, SQR-catalyzed reduction of CoQ₁ in the presence of sulfide and GSH. The reaction was conducted in 100 mm Tris buffer, pH 8.0 containing 5 mm GSH, 150 μm CoQ₁, and 0.05% DHPC at 25 °C and initiated by addition of 7 nm SQR. Inset, spectral changes at 272 nm (circle) and 317 nm (triangle) in the presence of 50 mm GSH.

FIGURE 5.

Kinetic characterization of H₂S oxidation by SQR in the presence of varying concentrations of small molecule acceptors. The reaction mixtures contained 0–0.6 mm sulfite (A), 0–80 mm GSH (B), 0–0.9 mm sulfide (C), 0–70 mm l-homocysteine (D), and 0–75 mm l-cysteine (E) in 100 mm potassium phosphate buffer, pH 7.4, 0.03% DHPC, 60 μm CoQ₁, 0.1 mg ml⁻¹ BSA, 150 μm sulfide (except when sulfide was used as acceptor in the reaction), and 0.05 μg of SQR at 25 °C. The data are representative of three independent experiments.

FIGURE 6.

Kinetic analysis of rhodanese using thiosulfate as substrate. Dependence of the reaction velocity on thiosulfate concentration in the presence of saturating concentrations of GSH (A), cysteine (B), or homocysteine (C) and dependence of the reaction velocity on GSH (D), cysteine (E), or homocysteine (F) concentrations in the presence of saturating concentration of thiosulfate. A–C, the reaction mixture contained thiosulfate (0.1–20 mm), 50 mm GSH (A), 50 mm cysteine (B), or 50 mm homocysteine (C) and 1 or 10 μg of rhodanese, and 0.4 mm lead acetate in 100 mm HEPES buffer, pH 7.4. D–F, the reaction mixture contains 3 mm thiosulfate, GSH, cysteine, or homocysteine, 1 or 10 μg of rhodanese, and 0.4 mm lead acetate in 100 mm HEPES buffer, pH 7.4, as described under “Experimental Procedures.” The reaction of rhodanese with thiosulfate and GSH, cysteine, or homocysteine leads to H₂S formation, which is detected as lead sulfide at 390 nm. The data are representative of three independent experiments.

FIGURE 7.

Kinetic analysis of thiosulfate generation by rhodanese. Dependence of the reaction velocity on the concentration of GSSH (A) or sulfite (B). The reaction mixture contained sulfite, GSSH, and 1 μg of rhodanese, and thiosulfate formation was monitored by HPLC as described under “Experimental Procedures.” The data are representative of three independent experiments. C, HPLC profiles of a reaction mixture containing 150 μm sulfite, 2 mm GSSH, and 1 μg of rhodanese (black line) and of a control mixture lacking rhodanese (gray line). The peak with a retention time of ∼21 min represents thiosulfate. Sulfite (SO₃²⁻), H₂S (present in the GSSH substrate due to the conditions employed for its synthesis, see “Experimental Procedures”), and thiosulfate (S₂O₃²⁻) are well separated. The peak labeled with an asterisk is derived from monobromobimane. The peak area of thiosulfate formed nonenzymatically from H₂S and sulfite in the control sample was subtracted from an area in the reaction mixture, and the concentration was determined using a calibration curve generated using standards of known concentrations.

FIGURE 8.

Simulation of SQR reaction rates at physiologically relevant concentrations of sulfane sulfur acceptors. Dependence of the SQR reaction rate on the concentration of H₂S at physiologically relevant concentrations of acceptors ([GSH] = 7 mm, [Cys] = 100 μm, and [Hcy] = 4 μm) and 190 μm CoQ₁ and 0.1 μm sulfite (A) or 1 μm sulfite (B). C, fraction of the SQR reaction rate contributed by sulfite increases monotonically at increasing concentrations of sulfite. H₂S, cysteine, homocysteine, GSH, and CoQ₁ concentrations were fixed at 1, 100, and 4 μm, 7 mm, and 190 μm, respectively.