Enzymology of H2S biogenesis, decay and signaling

Abstract

Significance: Hydrogen sulfide (H2S), produced by the desulfuration of cysteine or homocysteine, functions as a signaling molecule in an array of physiological processes including regulation of vascular tone, the cellular stress response, apoptosis, and inflammation.

Recent advances: The low steady-state levels of H2S in mammalian cells have been recently shown to reflect a balance between its synthesis and its clearance. The subversion of enzymes in the cytoplasmic trans-sulfuration pathway for producing H2S from cysteine and/or homocysteine versus producing cysteine from homocysteine, presents an interesting regulatory problem.

Critical issues: It is not known under what conditions the enzymes operate in the canonical trans-sulfuration pathway and how their specificity is switched to catalyze the alternative H2S-producing reactions. Similarly, it is not known if and whether the mitochondrial enzymes, which oxidize sulfide and persulfide (or sulfane sulfur), are regulated to increase or decrease H2S or sulfane-sulfur pools.

Future directions: In this review, we focus on the enzymology of H2S homeostasis and discuss H2S-based signaling via persulfidation and thionitrous acid.

FIG. 1.

Pathways for H₂S production and oxidation. H₂S biogenesis catalyzed by the trans-sulfuration pathway enzymes, CBS and CSE occurs in the cytoplasm. The AAT/MST pathway exists in the cytoplasm and in the mitochondrion. Sulfide is oxidized in the mitochondrion by SQR to generate persulfide. In the second step, the persulfide is oxidized by ETHE1, a dioxygenase, to generate sulfite that can either be oxidized by rhodanese or by sulfite oxidase. Electrons released in the SQR reaction are captured by ubiquinone and transferred to the electron transport chain at the level of complex III. AAT, aspartate aminotransferase; CBS, cystathionine β-synthase; CSE, cystathionine γ-lyase (γ-cystathionase); ETHE1, sulfur or persulfide dioxygenase; SQR, sulfide quinone oxidoreductase; H₂S, hydrogen sulfide. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 2.

H₂S-generating reactions catalyzed by CBS, CSE, and AAT/MST. The trans-sulfuration enzymes, CBS and CSE catalyze multiple H₂S-generating reactions. The canonical reaction catalyzed by each enzyme in the trans-sulfuration pathway is shown at the top of the respective group. The AAT/MST pathway involves an initial transamination reaction followed by a sulfur transferase reaction. MST, mercaptopyruvate sulfurtransferases.

FIG. 3.

Structures of H₂S-producing enzymes. (a) The structure of full-length dimeric Drosophila CBS showing the lower catalytic domains containing PLP and heme and the upper regulatory subunits (PDB file 3PC4). The heme and PLP shown in stick representation, bind to the catalytic domains while SAM binds to the regulatory domain. (b) Structure of homotetrameric human CSE in which the subunits are shown in different shades (PDB file 2NMP). PLP is seen in three of the four subunits and is shown in ball representation. The two CXXC motifs are shown in sphere representation in one of the subunits. (c) Structure of human MST (PDB file 3OLH). The active site cysteine (Cys248) that is modified as a persulfide is shown in sphere representation. SAM, S-adenosylmethionine; PLP, pyridoxal 5′-phosphate. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 4.

Redox-dependent regulation of human CBS by gases. Ferric CBS can be reduced by NADPH in the presence of the diflavin methionine synthase reductase to give ferrous CBS. The latter can react with CO or NO to give ferrous-CO or ferrous-NO CBS, respectively, which upon air oxidation revert to ferric CBS. The NO- and CO-liganded forms of CBS are inactive.

FIG. 5.

Structures of enzymes in the sulfide oxidation pathway. Structures of (a) SQR from the hyperthermophilic bacterium Aquifex aeolicus (PDB file: 3HYW) and (b) Arabidopsis ETHE1 (PDB file:2GCU) showing the mononuclear nonheme iron (sphere) in the active site. The three subunits of SQR are shown in different shades and the FAD is shown in stick representation in (a). FAD, flavin adenine dinucleotide. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 6.

Minimal mechanism for the reaction catalyzed by human SQR. An active site disulfide is attacked by the incoming sulfide nucleophile and gives a persulfide and a free active site cysteine. The latter attacks the bound flavin forming a 4a adduct. Nucleophilic displacement by an acceptor (A) results in the transfer of the sulfane sulfur atom, reformation of the active site disulfide, and a two-electron reduction of the flavin.

FIG. 7.

Overall reaction catalyzed by ETHE1. The resting form of the enzyme has water molecules coordinated to the vacant ligand sites at the 3-His-1-Asp mononuclear ferrous iron site. Binding of the substrate persulfide displaces the water molecules. Iron-based oxidation chemistry results in the formation of the products, sulfite and thiol.

FIG. 8.

Proposed models for H₂S-based signaling. (A) Sulfane sulfur is reactive and needs to be sequestered under cellular conditions. Release of H₂S from persulfide is promoted under alkaline conditions and in the presence of a reductant such as glutathione (27). Alternatively, the sulfane sulfur can be transferred to an acceptor such as thioredoxin from where it is released as H₂S with the concomitant oxidation of thioredoxin. (B) Formation of persulfide requires oxidation of the target protein to sulfenic acid or disulfide followed by attack of sulfide (32). Alternatively, H₂S₂, the two-electron oxidation product of H₂S, can be attacked by a protein thiolate (59). (C) HSNO is formed by the reaction of H₂S and S-nitrosated protein. HSNO can diffuse across the cell membrane and serve as a source of H₂S or HNO depending on the whether the nucleophile attacks at the nitrogen or sulfur atom, respectively of HSNO (15). HSNO, thionitrous acid.