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...

Background: H 2 S levels can be regulated by oxidation via sulfide quinone oxidoreductase (SQR). Results: Human SQR uses glutathione as an acceptor forming glutathione persulfide (GSSH), which is preferentially converted to thiosulfate by human rhodanese. Conclusion: At physiologically relevant concentrations, sulfide oxidation proceeds via GSSH to sulfite and thiosulfate. Significance: Our combined experimental and simulation studies reveal the organizational logic of the sulfide oxidation pathway.

The enzymes of the transsulfuration pathway, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), are important for the endogenous production of hydrogen sulfide (H(2)S), a gaseous signaling molecule. The relative contributions of CBS and CSE to H(2)S generation in different tissues are not known. In this study, we report quantification of CBS and CSE in murine liver and kidney and their contribution to H(2)S generation in these tissues and in brain at saturating substrate concentrations. We show that CBS protein levels are significantly lower than those of CSE; 60-fold and 20-fold in liver and kidney, respectively. Each enzyme is more abundant in liver compared with kidney, twofold and sixfold for CBS and CSE, respectively. At high substrate concentrations (20 mM each cysteine and homocysteine), the capacity for liver H(2)S production is approximately equal for CBS and CSE, whereas in kidney and brain, CBS constitutes the major source of H(2)S, accounting for ∼80% and ∼95%, respectively, of the total output. At physiologically relevant concentrations of substrate, and adjusting for the differences in CBS versus CSE levels, we estimate that CBS accounts for only 3% of H(2)S production by the transsulfuration pathway enzymes in liver.

Background: RBCs produce H 2 S but, lacking mitochondria, are devoid of the canonical sulfide oxidation pathway. Results: RBCs utilize methemoglobin to catalyze H 2 S oxidation producing thiosulfate and polysulfide. Conclusion: In the presence of NADPH and a reductase, ferric sulfide hemoglobin is converted to oxyhemoglobin, completing the sulfide oxidation cycle. Significance: We describe a novel mechanism for H 2 S oxidation that may be pertinent to other hemeproteins.

The transsulfuration pathway converts homocysteine to cysteine and represents the metabolic link between antioxidant and methylation metabolism. The first and committing step in this pathway is catalyzed by cystathionine ␤-synthase (CBS), which is subject to complex regulation, including allosteric activation by the methyl donor, S-adenosylmethionine (AdoMet). In this study, we demonstrate that methionine restriction leads to a >10-fold decrease in CBS protein levels, and pulse proteolysis studies reveal that binding of AdoMet stabilizes the protein against degradation by Ϸ12 kcal͞mol. These observations predict that under pathological conditions where AdoMet levels are diminished, CBS, and therefore glutathione levels, will be reduced. Indeed, we demonstrate this to be the case in a mouse model for spontaneous steatohepatitis in which the gene for the MAT1A isoenzyme encoding AdoMet synthetase has been disrupted, and in human hepatocellular carcinoma, where MAT1A is silenced. Furthermore, diminished CBS levels are associated with reduced cell viability in hepatoma cells challenged with tert-butyl hydroperoxide. This study uncovers a mechanism by which CBS is allosterically activated by AdoMet under normal conditions but is destabilized under pathological conditions, for redirecting the metabolic flux toward methionine conservation. A mechanistic basis for the coordinate changes in redox and methylation metabolism that are a hallmark of several complex diseases is explained by these observations. glutathione ͉ liver disease C ellular methylation and antioxidant metabolism are linked by the transsulfuration pathway, which converts the methionine cycle intermediate, homocysteine, to cysteine, the limiting reagent in glutathione synthesis. The balance between conserving methionine via transmethylation under conditions of methionine restriction and committing it to transsulfuration under conditions of plenty is regulated at two key control points, methionine adenosyltransferase (MAT) and cystathionine ␤-synthase (CBS) (Fig. 1). Aberrations in methylation and redox homeostasis are common to a number of chronic diseases including pathologies of the liver. In alcoholic liver disease and in hepatocellular carcinoma an increase in markers of oxidative stress is observed (1, 2). Furthermore, there is a switch in the expression of MAT genes from MAT1A to MAT2A in liver cancer, which correlates with lower S-adenosylmethionine (AdoMet) levels (3).Under normal conditions, coordinate regulation of methylation and antioxidant metabolism is achieved by the allosteric activation of CBS by AdoMet (Fig. 1). AdoMet is a V-type allosteric effector that increases CBS activity 2-to 3-fold (4, 5). Under conditions of plenty, methionine is directed toward cysteine synthesis via the transsulfuration pathway for use in glutathione and other cellular functions or directed toward catabolism. Cysteine is the limiting reagent in glutathione synthesis and in liver; Ϸ50% of the cysteine in glutathione is derived from methionine via the transsulf...

Aims: Hydrogen sulfide (H 2 S) is a signaling molecule, which influences many physiological processes. While H 2 S is produced and degraded in many cell types, the kinetics of its turnover in different tissues has not been reported. In this study, we have assessed the rates of H 2 S production in murine liver, kidney, and brain homogenates at pH 7.4, 37°C, and at physiologically relevant cysteine concentrations. We have also studied the kinetics of H 2 S clearance by liver, kidney, and brain homogenates under aerobic and anaerobic conditions. Results: We find that the rate of H 2 S production by these tissue homogenates is considerably higher than background rates observed in the absence of exogenous substrates. An exponential decay of H 2 S with time is observed and, as expected, is significantly faster under aerobic conditions. The half-life for H 2 S under aerobic conditions is 2.0, 2.8, and 10.0 min with liver, kidney, and brain homogenate, respectively. Western-blot analysis of the sulfur dioxygenase, ETHE1, involved in H 2 S catabolism, demonstrates higher steady-state protein levels in liver and kidney versus brain. Innovation: By combining experimental and simulation approaches, we demonstrate high rates of tissue H 2 S turnover and provide estimates of steady-state H 2 S levels. Conclusion: Our study reveals that tissues maintain a high metabolic flux of sulfur through H 2 S, providing a rationale for how H 2 S levels can be rapidly regulated. Antioxid. Redox Signal. 17, 22-31.

Hydrogen sulfide (H2S) elicits pleiotropic physiological effects ranging from modulation of cardiovascular to CNS functions. A dominant method for transmission of sulfide-based signals is via posttranslational modification of reactive cysteine thiols to persulfides. However, the source of the persulfide donor and whether its relationship to H2S is as a product or precursor is controversial. The transsulfuration pathway enzymes can synthesize cysteine persulfide (Cys−SSH) from cystine and H2S from cysteine and/or homocysteine. Recently, Cys−SSH was proposed as the primary product of the transsulfuration pathway with H2S representing a decomposition product of Cys−SSH. Our detailed kinetic analyses demonstrate a robust capacity for Cys−SSH production by the human transsulfuration pathway enzymes, cystathionine beta-synthase and γ-cystathionase (CSE) and for homocysteine persulfide synthesis from homocystine by CSE only. However, in the reducing cytoplasmic milieu where the concentration of reduced thiols is significantly higher than of disulfides, substrate level regulation favors the synthesis of H2S over persulfides. Mathematical modeling at physiologically relevant hepatic substrate concentrations predicts that H2S rather than Cys−SSH is the primary product of the transsulfuration enzymes with CSE being the dominant producer. The half-life of the metastable Cys−SSH product is short and decomposition leads to a mixture of polysulfides (Cys−S−(S)n−S−Cys). These in vitro data, together with the intrinsic reactivity of Cys−SSH for cysteinyl versus sulfur transfer, are consistent with the absence of an observable increase in protein persulfidation in cells in response to exogenous cystine and evidence for the formation of polysulfides under these conditions.

Oxidative stress and diminished glutathione pools play critical roles in the pathogenesis of neurodegenerative diseases, including Alzheimer and Parkinson disease. Synthesis of glutathione, the most abundant mammalian antioxidant, is regulated at the substrate level by cysteine, which is synthesized from homocysteine via the transsulfuration pathway. Elevated homocysteine and diminished glutathione levels, seen in Alzheimer and Parkinson disease patients suggest impairments in the transsulfuration pathway that connects these metabolites. However, the very existence of this metabolic pathway in the brain is a subject of controversy. The product of the first of two enzymes in this pathway, cystathionine, is present at higher levels in brain as compared with other organs. This, together with the reported absence of the second enzyme, ␥-cystathionase, has led to the suggestion that the transsulfuration pathway is incomplete in the brain. In this study, we incubated mouse and human neurons and astrocytes and murine brain slices in medium with [35 S]methionine and detected radiolabel incorporation into glutathione. This label transfer was sensitive to inhibition of ␥-cystathionase. In adult brain slices, ϳ40% of the glutathione was depleted within 10 h following ␥-cystathionase inhibition. In cultured human astrocytes, flux through the transsulfuration pathway increased under oxidative stress conditions, and blockade of this pathway led to reduced cell viability under oxidizing conditions. This study establishes the presence of an intact transsulfuration pathway and demonstrates its contribution to glutathione-dependent redox-buffering capacity under ex vivo conditions in brain cells and slices.