Signaling by HS is proposed to occur via persulfidation, a posttranslational modification of cysteine residues (RSH) to persulfides (RSSH). Persulfidation provides a framework for understanding the physiological and pharmacological effects of HS. Due to the inherent instability of persulfides, their chemistry is understudied. In this review, we discuss the biologically relevant chemistry of HS and the enzymatic routes for its production and oxidation. We cover the chemical biology of persulfides and the chemical probes for detecting them. We conclude by discussing the roles ascribed to protein persulfidation in cell signaling pathways.

Vitamin B12 is a complex organometallic cofactor associated with three subfamilies of enzymes: the adenosylcobalamin-dependent isomerases, the methylcobalamin-dependent methyltransferases, and the dehalogenases. Different chemical aspects of the cofactor are exploited during catalysis by the isomerases and the methyltransferases. Thus, the cobalt-carbon bond ruptures homolytically in the isomerases, whereas it is cleaved heterolytically in the methyltransferases. The reaction mechanism of the dehalogenases, the most recently discovered class of B12 enzymes, is poorly understood. Over the past decade our understanding of the reaction mechanisms of B12 enzymes has been greatly enhanced by the availability of large amounts of enzyme that have afforded detailed structure-function studies, and these recent advances are the subject of this review.

H 2 S, the most recently discovered gasotransmitter, might in fact be the evolutionary matriarch of this family, being both ancient and highly reduced. Disruption of ␥-cystathionase in mice leads to cardiovascular dysfunction and marked hypertension, suggesting a key role for this enzyme in H 2 S production in the vasculature. However, patients with inherited deficiency in ␥-cystathionase apparently do not present vascular pathology. A mitochondrial pathway disposes sulfide and couples it to oxidative phosphorylation while also exposing cytochrome c oxidase to this metabolic poison. This report focuses on the biochemistry of H 2 S biogenesis and clearance, on the molecular mechanisms of its action, and on its varied biological effects.Sulfur cycles through several biologically relevant oxidation states ranging from Ϫ2 as in hydrogen and metal sulfides to ϩ6 in sulfate. H 2 S, a colorless gas with the odor of rotten eggs, is important in the biogeochemical sulfur cycle and is used as an energy source by microbes such as the purple and green sulfur bacteria. It is a weak acid with pK a1 and pK a2 of 6.9 and Ͼ12 (1) and an aqueous solubility of ϳ80 mM at 37°C. Hence, at the physiological pH of 7.4, the ratio of HS Ϫ :H 2 S is 3:1. For brevity, H 2 S is used to refer to the total free sulfide pool (i.e. H 2 S ϩ HS Ϫ ϩ S 2Ϫ ) in this report unless noted otherwise. The ready ionization of H 2 S at physiological pH suggests impeded permeation through the lipid bilayer when compared with other gases, viz. NO or CO. On the other hand, transport of the gas, H 2 S, across the membrane does not appear to be facilitated (2).The toxicity of H 2 S is thought to have influenced evolution. The presence of a metastable H 2 S-enriched oceanic stratum is postulated to have limited early metazoan colonization of the continental shelf (3), and an increase in H 2 S has been implicated in the Permian-Triassic extinction Ͼ250 million years ago (4). However, the reputation of H 2 S as a toxic gas is enjoying a facelift, with increasing numbers of reports that it modulates a range of biological processes. Despite the rising interest in H 2 S biochemistry, fundamental questions regarding regulation of its production, its mechanism of action, and its destruction remain. In addition, perhaps most critical to the field is the issue of what constitutes biologically relevant levels of H 2 S with reports varying over a 10 5 -fold concentration range. Using a gas chromatography-based chemiluminescent sulfur detection method, free H 2 S (H 2 S ϩ HS Ϫ ) in blood was estimated to be ϳ100 pM, and in tissues, it was estimated to be ϳ15 nM (5). These values are considerably lower than the ϳ30 -300 M concentrations reported in a spate of recent studies (reviewed in Ref. 6). The high values can be ascribed to technical artifacts introduced by long processing times and harsh (either acidic or alkaline) conditions used to shift the equilibrium toward H 2 S or S 2Ϫ , respectively. Under these conditions, sulfide leaches from iron-sulfur cluster-containing p...

In mammals, the two enzymes in the trans-sulfuration pathway, cystathionine ␤-synthase (CBS) and cystathionine ␥-lyase (CSE), are believed to be chiefly responsible for hydrogen sulfide (H 2 S) biogenesis. In this study, we report a detailed kinetic analysis of the human and yeast CBS-catalyzed reactions that result in H 2 S generation. CBS from both organisms shows a marked preference for H 2 S generation by ␤-replacement of cysteine by homocysteine. The alternative H 2 S-generating reactions, i.e. ␤-elimination of cysteine to generate serine or condensation of 2 mol of cysteine to generate lanthionine, are quantitatively less significant. The kinetic data were employed to simulate the turnover numbers of the various CBS-catalyzed reactions at physiologically relevant substrate concentrations. At equimolar concentrations of CBS and CSE, the simulations predict that H 2 S production by CBS would account for ϳ25-70% of the total H 2 S generated via the trans-sulfuration pathway depending on the extent of allosteric activation of CBS by S-adenosylmethionine. The relative contribution of CBS to H 2 S genesis is expected to decrease under hyperhomocysteinemic conditions. CBS is predicted to be virtually the sole source of lanthionine, and CSE, but not CBS, efficiently cleaves lanthionine. The insensitivity of the CBS-catalyzed H 2 S-generating reactions to the grade of hyperhomocysteinemia is in stark contrast to the responsiveness of CSE and suggests a previously unrecognized role for CSE in intracellular homocysteine management. Finally, our studies reveal that the profligacy of the trans-sulfuration pathway results not only in a multiplicity of H 2 S-yielding reactions but also yields novel thioether metabolites, thus increasing the complexity of the sulfur metabolome.

Although there is a growing recognition of the significance of hydrogen sulfide (H 2 S) as a biological signaling molecule involved in vascular and nervous system functions, its biogenesis and regulation are poorly understood. It is widely assumed that desulfhydration of cysteine is the major source of H 2 S in mammals and is catalyzed by the transsulfuration pathway enzymes, cystathionine ␤-synthase and cystathionine ␥-lyase (CSE). In this study, we demonstrate that the profligacy of human CSE results in a variety of reactions that generate H 2 S from cysteine and homocysteine. The ␥-replacement reaction, which condenses two molecules of homocysteine, yields H 2 S and a novel biomarker, homolanthionine, which has been reported in urine of homocystinuric patients, whereas a ␤-replacement reaction, which condenses two molecules of cysteine, generates lanthionine. Kinetic simulations at physiologically relevant concentrations of cysteine and homocysteine, reveal that the ␣,␤-elimination of cysteine accounts for ϳ70% of H 2 S generation. However, the relative importance of homocysteinederived H 2 S increases progressively with the grade of hyperhomocysteinemia, and under conditions of severely elevated homocysteine (200 M), the ␣,␥-elimination and ␥-replacement reactions of homocysteine together are predicted to account for ϳ90% of H 2 S generation by CSE. Excessive H 2 S production in hyperhomocysteinemia may contribute to the associated cardiovascular pathology.H 2 S is the newest member of a growing list of gaseous signaling molecules that modulate physiological functions (1-3). Concentrations of H 2 S ranging from 50 to 160 M have been reported in the brain (4), where it appears to function as a neuromodulator by potentiating the activity of the N-methyl-Daspartate receptor and by altering induction of long term potentiation in the hippocampus, important for memory and learning (5). H 2 S levels in human plasma are reported to be ϳ50 M, and in vitro studies suggest that it functions as a vasodilator by opening K ATP channels in vascular smooth muscle cells (6).A recent in vivo study has demonstrated the efficacy of H 2 S in attenuating myocardial ischemia-reperfusion injury by protecting mitochondrial function (7). The role of H 2 S in inflammation is suggested by several studies (8 -11); however, the underlying mechanism is unknown. Remarkably, H 2 S can also induce a state of suspended animation in mice by decreasing the metabolic rate and the core body temperature presumably by inhibiting cytochrome c oxidase in the respiratory chain (12).Endogenous H 2 S is presumed to be generated primarily by desulfhydration of cysteine catalyzed by the two pyridoxal phosphate (PLP) 3 -dependent enzymes in the transsulfuration pathway: cystathionine ␤-synthase (CBS) and cystathionine ␥-lyase (CSE) (13,14). In fact, it is widely assumed, based on the reported absences of CSE in the brain (15) and of H 2 S in the brain of CBS knock-out mice (16), that CBS is the primary source of H 2 S in this organ, whereas CSE plays the...