Towards Understanding the Pathway for Hydrogen Sulfide Metabolism

Abstract

Hydrogen sulfide (H2S) is the most recently identified member of a small family of labile biological signaling molecules, termed gasotransmitters, which includes nitric oxide and carbon monoxide. H2S is the only gasotransmitter that is enzymatically metabolized a process that occurs in the mitochondria. H2S needs to be tightly regulated because it is toxic at high concentrations and leads to physiological defects at low concentrations. For example, a genetic defect that affects the metabolic pathway of H2S is ethylmalonic encephalopathy, a fatal disorder that is characterized by extremely high levels of H2S. On the other hand, animal model studies provide compelling evidence for a functional association between abnormally low levels of H2S and cardiovascular disease. In light of H2S’s critical role, the goal of this thesis was to identify and characterize two human enzymes that are proposed to comprise part of the metabolic pathway of H2S in mammals: Sulfide:quinone oxidoreductase (SQOR) and thiosulfate:glutathione sulfurtransferase (TST). The present study postulates that human sulfide:quinone oxidoreductase (SQOR), a membrane-bound enzyme, catalyzes the first step in the mitochondrial metabolism of H2S. The reaction involves a two-electron oxidation of H2S to S0 (sulfane sulfur) and uses coenzyme Q as an electron acceptor. The fact that SQOR is a membrane-associated protein has made its expression and isolation challenging. We successfully purified and characterized human SQOR. Cyanide, sulfite, or sulfide can act as the sulfane sulfur acceptor in reactions that produce thiocyanate, thiosulfate, or a putative sulfur analog of hydrogen peroxide (H2S2), respectively. Thiosulfate is a known intermediate in the oxidation of H2S within animals and the major product formed in glutathione-depleted cells or mitochondria. Importantly, oxidation of H2S by SQOR with sulfite as the sulfane sulfur acceptor is rapid and highly efficient at physiological pH (kcat/Km,H2S = 2.9 × 107 M-1 s-1). We propose that this highly efficient oxidation of H2S by SQOR is the predominant source of the thiosulfate in mammalian tissues and that sulfite is the physiological acceptor of the sulfane sulfur. Our proposal opposes an alternative hypothesis that glutathione is an acceptor of the sulfane sulfur, which we have compelling evidence against. The discovery that sulfite was the physiological acceptor of the sulfane sulfur and SQOR produced thiosulfate, led us to postulate a role in H2S metabolism for a TST that transfers the sulfane sulfur of thiosulfate to glutathione producing GSS- and sulfite. We postulate that the TST links together the SQOR and sulfur dioxygenase (SDO) steps in the pathway because it consumes the thiosulfate from the SQOR reaction and produces glutathione persulfide (GSS-), a substrate required for SDO. Although an active TST enzyme had been found in yeast, attempts by other laboratories to isolate and characterize the mammalian enzyme have been unsuccessful. We also discovered genes that encode for human and yeast TST…