Biosynthesis of a Central Intermediate in Hydrogen Sulfide Metabolism by a Novel Human Sulfurtransferase and Its Yeast Ortholog

Human sulfide:quinone oxidoreductase (SQOR) catalyzes the conversion of H₂S to thiosulfate, the first step in mammalian H₂S metabolism. SQOR’s inability to produce the glutathione persulfide (GSS^–) substrate for sulfur dioxygenase (SDO) suggested that a thiosulfate:glutathione sulfurtransferase (TST) was required to provide the missing link between the SQOR and SDO reactions. Although TST could be purified from yeast, attempts to isolate the mammalian enzyme were not successful. We used bioinformatic approaches to identify genes likely to encode human TST (<i>TSTD1</i>) and its yeast ortholog (<i>RDL1</i>). Recombinant TSTD1 and RDL1 catalyze a predicted thiosulfate-dependent conversion of glutathione to GSS^–. Both enzymes contain a rhodanese homology domain and a single catalytically essential cysteine, which is converted to cysteine persulfide upon reaction with thiosulfate. GSS^– is a potent inhibitor of TSTD1 and RDL1, as judged by initial rate accelerations and ≥25-fold lower <i>K</i>_m values for glutathione observed in the presence of SDO. The combined action of GSS^– and SDO is likely to regulate the biosynthesis of the reactive metabolite. SDO drives to completion <i>p</i>-toluenethiosulfonate:glutathione sulfurtransferase reactions catalyzed by TSTD1 and RDL1. The thermodynamic coupling of the irreversible SDO and reversible TST reactions provides a model for the physiologically relevant reaction with thiosulfate as the sulfane donor. The discovery of bacterial Rosetta Stone proteins that comprise fusions of SDO and TSTD1 provides phylogenetic evidence of the association of these enzymes. The presence of adjacent bacterial genes encoding SDO–TSTD1 fusion proteins and human-like SQORs suggests these prokaryotes and mammals exhibit strikingly similar pathways for H₂S metabolism

Human Sulfide:Quinone Oxidoreductase Catalyzes the First Step in Hydrogen Sulfide Metabolism and Produces a Sulfane Sulfur Metabolite

Sulfide:quinone oxidoreductase (SQOR) is a membrane-bound enzyme that catalyzes the first step in the mitochondrial metabolism of H₂S. Human SQOR is successfully expressed at low temperature in <i>Escherichia coli</i> by using an optimized synthetic gene and cold-adapted chaperonins. Recombinant SQOR contains noncovalently bound FAD and catalyzes the two-electron oxidation of H₂S to S⁰ (sulfane sulfur) using CoQ₁ as an electron acceptor. The prosthetic group is reduced upon anaerobic addition of H₂S in a reaction that proceeds via a long-wavelength-absorbing intermediate (λ_max = 673 nm). Cyanide, sulfite, or sulfide can act as the sulfane sulfur acceptor in reactions that (i) exhibit pH optima at 8.5, 7.5, or 7.0, respectively, and (ii) produce thiocyanate, thiosulfate, or a putative sulfur analogue of hydrogen peroxide (H₂S₂), respectively. Importantly, thiosulfate is a known intermediate in the oxidation of H₂S by intact animals and the major product formed in glutathione-depleted cells or mitochondria. Oxidation of H₂S by SQOR with sulfite as the sulfane sulfur acceptor is rapid and highly efficient at physiological pH (<i>k</i>_cat/<i>K</i>_m,H₂S = 2.9 × 10⁷ M^–1 s^–1). A similar efficiency is observed with cyanide, a clearly artificial acceptor, at pH 8.5, whereas a 100-fold lower value is seen with sulfide as the acceptor at pH 7.0. The latter reaction is unlikely to occur in healthy individuals but may become significant under certain pathological conditions. We propose that sulfite is the physiological acceptor of the sulfane sulfur and that the SQOR reaction is the predominant source of the thiosulfate produced during H₂S oxidation by mammalian tissues

Use of Tissue Metabolite Analysis and Enzyme Kinetics To Discriminate between Alternate Pathways for Hydrogen Sulfide Metabolism

Hydrogen sulfide (H₂S) is an endogenously synthesized signaling molecule that is enzymatically metabolized in mitochondria. The metabolism of H₂S maintains optimal concentrations of the gasotransmitter and produces sulfane sulfur (S⁰)-containing metabolites that may be functionally important in signaling. Sulfide:quinone oxidoreductase (SQOR) catalyzes the initial two-electron oxidation of H₂S to S⁰ using coenzyme Q as the electron acceptor in a reaction that requires a third substrate to act as the acceptor of S⁰. We discovered that sulfite is a highly efficient acceptor and proposed that sulfite is the physiological acceptor in a reaction that produces thiosulfate, a known metabolic intermediate. This model has been challenged by others who assume that the intracellular concentration of sulfite is very low, a scenario postulated to favor reaction of SQOR with a considerably poorer acceptor, glutathione. In this study, we measured the intracellular concentration of sulfite and other metabolites in mammalian tissues. The values observed for sulfite in rat liver (9.2 μM) and heart (38 μM) are orders of magnitude higher than previously assumed. We discovered that the apparent kinetics of oxidation of H₂S by SQOR with glutathione as the S⁰ acceptor reflect contributions from other SQOR-catalyzed reactions, including a novel glutathione:CoQ reductase reaction. We used observed metabolite levels and steady-state kinetic parameters to simulate rates of oxidation of H₂S by SQOR at physiological concentrations of different S⁰ acceptors. The results show that the reaction with sulfite as the S⁰ acceptor is a major pathway in liver and heart and provide insight into the potential dynamics of H₂S metabolism