Hydrogen sulfide (H2S) Hydrogen sulfide (H2S) is the most recent addition to the gasotransmitter family,

including nitric oxide (NO) and carbon monoxide (CO). These gasotransmitters are small molecules of gas
that has the remarkable ability to freely diffuse through a cell membrane to induce an array of
intracellular signaling responses (17-19). Their production and metabolism are enzymatically regulated,
and chemical donors can duplicate the effects of the gasotransmitters. H2S is regarded to have
physiological importance in the cardiovascular, neuronal, immune, renal, respiratory, gastrointestinal,
reproductive, and endocrine systems. H2S carries out its physiological function by targeting membrane
ion channels, proteins, enzymes, and transcription factors (17; 18; 20).

The key mechanism by which H2S may carry out these effects is through the S-sulfhydration of proteins
by converting cysteine-SH groups to –SSH (20). Indeed, H2S can S-sulfhydrate about 10-25% of liver
proteins, including actin, tubulin, and glyceraldehyde-3- phosphate dehydrogenase, under physiological
conditions (20). This suggests that posttranslational modification by H2S may be an important signaling
mechanism in the cardiovascular system (18). The pursuit of determining the endogenous level of H2S is
by far one of the most controversial issues in the H2S field. In the blood or plasma, H2S level has been
reported to be above 35 µM in several species, such as rat, mouse, or human (21-23). In brain tissue, H2S
was detected in the range of 50–160 μM. These measurements of detecting H2S are based upon the
methylene blue method; this is most common assay used that relies on trapping H2S with zinc followed
by acidification, which is then measured spectrophotometrically. However, this method of H2S detection
involves harsh conditions that release bound sulfide from proteins and amino acids; hence, caution
should be used when interpreting these results. Thankfully, advancements have been made where more
sensitive methods (gas chromatography and polarographic H2S sensor) can accurately measure free H2S
concentrations (as low as 14 nM), without releasing stored sulfide (24-26). Despite this, there is still
uncertainty about the exact level of endogenous H2S level in mammalian biological tissues. [Project
copy]

For example, erythrocytes, supplemented with glucose and sulfur and containing electron carriers such
as NADH, NADPH and reduced glutathione (GSH), spontaneously react with sulfur to produce H2S
(Searcy and Lee 1998). Iron-sulfur cluster containing proteins carrying Fe2S2, Fe3S4 or Fe4S4 clusters are
another source of non-enzymatically generated H2S. These include proteins such as ferredoxins and
‘Rieske’ proteins among others (Beinert et al. 1997). In the presence of reducing agents such as
glutathione, H2S can also be released from bound sulfur in, for example, persulfides in neurons and
astrocytes (Ishigami et al. 2009).

Although non-enzymatic routes for H2S generation have been identified, it seems likely that the bulk, if
not all, of the H2S which is generated for the purpose of carrying out biological functions in the body is
derived enzymatically. This no doubt explains the huge emphasis in recent years in understanding as
much as possible about the biochemistry, molecular biology and physiological relevance of the H2S
synthesizing enzymes. The list of reported biological effects of both endogenous and exogenously
administered H2S is far reaching and expanding on an almost weekly basis. Numerous molecular targets
for this gas have also been identified.

The biological effects of H2S are outside the scope of this present review and are dealt with elsewhere
in this volume. Rather, we will provide here an overview of the H2S synthesizing enzymes. This will
encompass a discussion of the way in which H2S synthesizing enzymes are regulated in health and in
disease, as well as a consideration of the limitations of the currently available H2S synthesizing enzyme
inhibitors which have been widely used to probe the biological roles of this gas.

Three pathways of H2S degradation exist: (1) desulfurization to thiosulfate by mitochondrial

oxidation, then to sulfite or sulfate; (2) cytosolic methylation to dimethylsulfide; and (3)
sulfhemoglobin formation by the binding to hemoglobin.
A previous investigation shows that after H2S is synthesized, it is quickly absorbed or stored as a
form of bound or acid-labile sulfur, while free H2S is maintained at a baseline level. Bound sulfur
is stored intracellularly in neurons and astrocytes of rats and releases H2S in the presence of
physiologic concentrations of the endogenous reducing substances glutathione and cysteine.
Acid-labile sulfur is another form of stored H2S. It localizes in the mitochondria and the release
of H2S occurs at a pH below 5.4

Hughes MN, Centelles MN, Moore KP. Making and working with hydrogen sulfide: the
chemistry and generation of hydrogen sulfide in vitroand its measurement in vivo: a review.
Free Radic Biol Med 2009; 47: 1346–53.
In mammalian tissues, H2S is produced endogenously from enzymic desulfydration of cysteine catalyzed
by cystathionine-γ-lyase (also termed γ -cystathionase CSE), cystathionine-β-synthase (CBS),

these enzymes, CBS and CSE are pyridoxal -5- phosphate dependent pathways being predominantly
responsible for the synthesis of H2S in vivo [4,6].

Thus, L-cysteine may be hydrolyzed by CBS to produce H2S, with L-serine as the by-product, or
hydrolyzed by CSE to produce H2S, pyruvate and ammonia.

In an alternative pathway, CSE may catalyze the conversion of cystine (Cys-S-S-Cys) to thiocysteine,
which is then hydrolyzed by CSE to cysteine and H2S.

These two key cytosolic enzymes are also involved in the trans-sulfuration pathway of methionine and
homocysteine metabolism, with CBS catalyzing the condensation of homocysteine and serine to form
cystathionine in an irreversible reaction. Cystathionine may then be hydrolyzed by CSE to cysteine with
α-ketobutyrate and ammonia as by-products.

In addition, several other pathways for H2S synthesis have been described. Thus, cysteine may react with
a ketoacid to form 3- mercaptopyruvate, which undergoes desulfuration by 3-mercapto pyruvate
sulfurtransferase (MPST) to form H2S and pyruvate.

Furthermore, CBS can catalyze the reaction on the thiol of cysteine to form S-thiolate (Cys-S-R) with the
concomitant release of H2S. Chen et al. [10]

reported also that CBS could catalyze the condensation of cysteine and homocysteine to form
cystathionine and H2S. However, most studies suggest that H2S is synthesized in mammalian tissues
predominantly through the enzymatic action of either CBS or CSE on cysteine. All the other pathways
involving additional enzymes and substrates are likely to play only a minor role in the biosynthesis of
H2S.

However, CBS and CSE have different tissue distributions; cystathionine β-synthase is primarily
responsible for production of H2S in the central nervous system, while cystathionine γ-lyase is primarily
expressed in periphral tissues, including vascular and nonvascular smooth muscle.
2. H2S CHEMISTRY
Hydrogen sulfide (H2S) is also known as hydrogen sulfuric acid, dihydrogen monosulfide,
sulfurated hydrogen, hepatic gas, stink damp, sewer gas. It is a toxic and malodorous gas with
characteristic rotten eggs smell, soluble in water (40 gL -1), however, its high vapor pressure
(1740 kPa at 21 °C), which is not always appreciated, can halve its concentration in open flasks
or tissue culture vessels within several minutes [6].
Dissolved H2S is a weak acid with the equilibrium: H2S ⇄ HS- + H+ ⇄ S2- + H+, (collectively
termed total sulfide). it is common to regard dissolved H2S and hydrosulfide anions (HS- ) as the
only biologically significant species, however, as all three are in equilibrium, theoretically there
is as much ‘reactive’ S2- as total sulfide [9].
There is variations in pH in different tissue compartments of the body with 81% HS in
extracellular fluid (pH 7.4) and 63% in cytosol (pH ~7.0), and less than 2% in lysosomes (pH ~5).
The pH gradient between the mitochondrial matrix (pH 7.8) and intermembrane space (pH 6.8)
will also affect the proportion of HS from over 90% to only 55% HS - , respectively. How these pH
gradients affect total sulfide movement is unknown; H2S gas readily diffuses across membranes
[12], whereas. HS- may be restricted to anion channel. Sulfur oxidation states range from -2 to
+6. Even integers are the most stable, although thiyl radicals (-1) are also recognized for their
considerable biological potential. Sulfur oxidation/reduction reactions are common in the
environment and in cells, however, animals cannot reduce fully oxidized sulfur (+6) and
therefore depend on plants and microorganisms for their needs. These are made mainly in the
form of the essential amino acid, methionine, and cysteine to provide fully reduced sulfur (-2)
which can be liberated as H2S . [8] H2S and Polysulfide Metabolism: Conventional and
Unconventional Pathways Kenneth R. Olson Indiana University School of Medicine - South Bend, South
Bend, Indiana 46617 USA.

Chemical properties

Dissolved H2S is a weak acid with the equilibrium: H2S ⇄ HS- + H+ ⇄ S2- + H+, (collectively
termed total sulfide). it is common to regard dissolved H2S and hydrosulfide anions (HS- ) as the
only biologically significant species, however, as all three are in equilibrium, theoretically there
is as much ‘reactive’ S2- as total sulfide [8] H2S and Polysulfide Metabolism: Conventional and
Unconventional Pathways Kenneth R. Olson Indiana University School of Medicine - South Bend, South
Bend, Indiana 46617 USA.

Under physiologically relevant conditions, i.e. in aqueous solutions and at pH 7.4, one third of H2S is un
dissociated and two thirds dissociate into H+ and HS– (hydrosulfide ion), which subsequently may
decompose to H+ and sulfide ion (S2–). However, the latter reaction occurs only at high pH, thus S2–
Does not occur in vivo at substantial amounts. Sodium hydrosulfide (NaHS) is commonly used as an H2S
donor since it dissociates to Na+ and HS–; the latter then partially binds H+ to form undissociated H2S.
Similarly to NO and CO, H2S is lipophilic and freely permeates plasma membranes, although due to
partial dissociation membranes are relatively less permeable to H2S than to both other gases. H2S is
detectable in serum and most tissues at a concentration of about 50 µM. Its physiological level in the
brain is up to three-fold higher than in serum and is in fact close to toxic concentration. [9] Hydrogen
sulfide (H2S) – the third gas of interest for pharmacologists Ewelina £owicka, Jerzy Be³towski.

Synthesis of H2S

H2S is produced at significant amounts in most tissues. The highest rate of production was noted in the
brain, cardiovascular system, liver and kidney [25].

CONVENTIONAL PATHWAYS OF H2S PRODUCTION AND CATABOLISM

3.1. Production

Three enzymes, cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE aka CGL) and 3-
mercaptopyruvate sulfurtransferase (3-MST) are commonly regarded as the primary mechanisms for H2S
biosynthesis (Fig. 1). CBS and CSE are components of the transsulfuration pathway, where as 3-MST
usually requires the transfer of sulfur from cysteine to pyruvate which is catalyzed by cysteine
aminotransferase (CAT). CBS catalyzes the β-replacement reaction of homocysteine with serine to form
cystathionine, and in so doing commits sulfur into the transsulfuration pathway [15]. CSE then catalyzes
the α, γ-elimination of cystathionine to form cysteine, α-ketobutyrate and ammonia(NH3). Both CBS and
CSE can then generate H2S from cysteine via β elimination reactions. CAT transfers the amine group from
cysteine to α-keto glutarate producing 3-mercaptopyruvate (3-MP). 3 MST removes the sulfur from 3-MP
forming a persulfide on the enzyme (3-MST-SSH). Two endogenous reductants thioredoxin (Trx) or
dihydrolipoic acid (DHLA), then liberate the terminal sulfur as H2S [16-18]. The relative promiscuity of
CBS and CSE [19] allows for additional pathways for H2S synthesis from cysteine [20-22]. H2S can be
produced by CBS-catalyzed β-replacement of two molecules of cysteine, generating lanthionine in the
process, or β-replacement of one molecule of homocysteine and one molecule of cysteine resulting in
cystathionine, the latter tending to predominate. CSE can form H2S and homoserine by γ-elimination of
homocysteine, H2S and lanthionine by β-replacement of two molecules of cysteine, H2S and
homolanthionine by γreplacement of two molecules of homocysteine and H2S and cystathionine by β- or
γreplacement of homocysteine and cysteine. Elevated homocysteine increases CSE and this may be
important clearance pathways in hyperhomocysteinemia; it has also been proposed that the excess H2S
may contribute to the cardiovascular pathology associated with severe hyperhomocystenemia [20]

CBS, CSE and CAT are pyridoxal 5'phosphate (PLP)-dependent, enzymes. CBS also has a regulatory heme-
binding N-terminal domain and a C-terminal domain that binds S-adenosyl L-methionine. CBS activity is
increased by S-adenosyl L-methionine (AdoMet; [15]), whereas CO or NO binding to the heme domain
inhibits the enzyme [23,24]. The two regulatory domains also interact; AdoMet binding enhances the
inhibitory effect of both CO and NO [24]. CBS heme also can reduce nitrite to NO, the latter then
inhibiting H2S production [25]. Redox potential or Ca-calmodlin may also affect activity. CAT activity is
inversely sensitive to calcium and as it is completely inhibited at 2.9 μM Ca2+ this could be an important
physiological regulator of H2S [26]. CBS is generally considered to be predominant enzyme in the brain
and CSE in the heart and vasculature, although more variability in enzyme distribution is being reported,
i.e., CBS has been found in vascular endothelium, CAT and 3-MST in endothelium and brain and MST, but
not CAT, in vascular smooth muscle [27,28]. CBS and CSE have also been found in human plasma where
they may metabolize circulating homocysteine and the H2S produced has been proposed to protect the
endothelium from oxidative stress [29]. CAT and 3-MST have been found in both mitochondria and
cytosol, although 3-MST may be more localized to the mitochondrial matrix [16,30-33]. CBS and CSE are
considered to be cytosolic enzymes, however, under certain circumstances they may be translocated to
the mitochondria to take advantage of the three-fold higher cysteine concentrations [34]. The
mitochondrial heat shock protein (mtHsp 70) transports CBS from the cytosol to the mitochondrial
matrix. Under normoxic conditions, where the CBS heme is oxygenated the enzyme is degraded by Lon
protease. CBS is not degraded in hypoxia and within 10 min its concentration doubles, in one hour it is
increased six-fold and it returns to normal within 5 min upon reoxygenation [35]. Although the resultant
increase in H2S production has been proposed to sustain energy production during hypoxia [35], this is
unlikely. First, ATP production from H2S also requires oxygen. Second, ATP production from H2S is less
efficient than from carbon-based substrate; the number of protons pumped across the inner
mitochondrial membrane per atom of oxygen decreases from 10 with NADH to 8 with FADH2, to 2 with
H2S [36]. Stress also stimulates CSE translocation from the cytosol to the mitochondria [34], although
this can take hours. Although L-cysteine is conventionally considered as the precursor for H2S
biosynthesis, under certain circumstances H2S can also be derived from D-cysteine after the latter is
oxidized to 3-MP by peroxisomal D-amino acid oxidase (DAO; [37]. The 3-MP is then delivered to the
mitochondrion by vesicular transfer where it is further metabolized by 3-MST to form H2S (Fig. 1, also
see above), or polysulfides (as described below). This process is cytoprotective in the kidney, brain and
stomach [37, 38] and potentiates hypoxic vasoconstriction in isolated rat pulmonary arteries [39].
Over a decade, hydrogen sulfide (H2 S) has been recognized as a crucial signalling molecule with a wide
range of physiological functions that can profoundly affect most organ systems in animals and humans.
The realization of the biological importance of H2 S in numerous cells, tissues and organs is now
shedding light on the pathogenesis of various human diseases, and paving the way for innovative
therapeutic interventions. [9] Hydrogen sulfide-based therapeutics: exploiting a unique but ubiquitous
gasotransmitter John L. Wallace1,2 and Rui Wang3.

H2 S synthesis and metabolism

H2 S synthesis.

H2S is produced in mammalian cells mostly through the reverse trans-

sulfuration pathway, this involve the enzymatic action of pyridoxal 5ʹ-
phosphate (PLP) (vitamin B6) –dependent enzymes called cystathionine
β-synthase (CBS) and cystathionine γ-lyase (CSE). However, CBS and CSE
have different tissue distributions; cystathionine β-synthase is primarily
responsible for production of H2S in the central nervous system, while
cystathionine γ-lyase is primarily expressed in periphral tissues including
cardiovascular system, liver and kidney. Thus, L-cysteine may be hydrolyzed
by CBS to produce H2S, with L-serine as the by-product, or hydrolyzed by
CSE to produce H2S, pyruvate and ammonia. In an alternative pathway, CSE
may catalyze the conversion of cystine to thiocysteine, which is then
hydrolyzed by CSE to cysteine and H2S. These two key cytosolic enzymes are
also involved in the trans-sulfuration pathway of methionine and
homocysteine metabolism, with CBS catalyzing the condensation of
homocysteine and serine to form cystathionine in an irreversible reaction.
Cystathionine may then be hydrolyzed by CSE to cysteine with α-
ketobutyrate and ammonia as by-products. [50] Hughes MN, Centelles MN, Moore
KP. Making and working with hydrogen sulfide: the chemistry and generation of hydrogen
sulfide in vitroand its measurement in vivo: a review. Free Radic Biol Med 2009; 47: 1346–53.
Beside CSE and CBS, a recent investigation also suggests the presence of
another enzyme called 3-mercaptopyruvate sulfurtransferase (3-MST) that in
coordination with cysteine aminotransferase (CAT) can generate endogenous
H2S: 3-MST is located in the liver, kidney, heart, lung, thymus, testis,
thoracic aorta, and brain. CAT uses PLP as a cofactor and transfer the amine
group from cysteine to α-ketoglutarate producing 3-mercaptopyruvate (3-
MP). 3 MST removes the sulfur from 3-MP forming a persulfide on the enzyme
(3-MST-SSH). Two endogenous reductants thioredoxin (Trx) or dihydrolipoic
acid (DHLA), then liberate the terminal sulfur as H2S. In essence, 3-MST acts
as a sulfur carrier, rather than a H2S producer, as the sequential reactions
that are catalyzed by CAT and MST generate sulfane sulfur. This bound sulfur
has to be released or reduced to liberate H2S. Another sulfur-carrying
enzyme in our bodies is rhodanese (also known as thiosulfate
sulfurtransferase). However, the biological and physiological importance of
this enzyme in endogenous H2S metabolism has not been fully determined.
In contrast to MST, which is localized both in the cytosol and mitochondria,
rhodanese is a true mitochondrial protein. 11] Hydrogen Sulfide: From Physiology to
Pharmacology Stefano Fiorucci.

H2 S metabolism and excretion.

Unlike NO, H2 S is relatively stable in body fluids. In the circulation and in the cytoplasm, free H2S can be
scavenged by methaemoglobin or by metallo- or disulfide-containing macromolecules that function as
sulfane-sulfur and bound-sulfate pools. Oxidation and methylation are another two mechanisms of H2 S
metabolism.

H2 S is oxidized sequentially in the mitochondrion to thiosulfate and then to sulfite, with the end-
product under physiological conditions being sulfate. Local tissue H2S concentrations are determined by
the rate of formation and the rate of degradation. The major routes of degradation are via non-
enzymatic oxidation of sulphide to sequentially thiosulfate, sulfite, and sulfate, the latter which is
excreted in urine. The mitochondrial enzyme sulfide-quinone reductase has a crucial role in the oxidation
of H2S. The cytosol is the major intracellular site of H2 S methylation. Thiol-S -methyltransferase
catalyses the methylation of H2 S to yield methanethiol and dimethylsulfide. H2 S is excreted in urine
and flatus as free sulfate, thiosulfate or free sulfide, and is also exhaled in breath. [56] Hydrogen Sulfide:
From Physiology to Pharmacology Stefano Fiorucci

In mammalian cells, endogenous H2S is synthesized by four enzymes: cystathionine γ-lyase (CSE, EC
4.4.1.1), cystathionine βsynthase (CBS, EC 4.2.1.22), cysteine aminotransferase (CAT, EC 2.6.1.3) and 3-
mercaptopyruvate sulfurtransferase (MST, EC 2.8.1.2). These enzymes are involved in the
transsulfuration and reverse transsulfuration pathways as described earlier [10,18,34–37]. CBS (the main
H2S-producing enzyme in the central nervous system) and CSE (presents in the vasculature, liver and
kidney) are the enzymes using amino acids: L-cysteine (which is synthesized from L-methionine through
the transsulfuration), L-homocysteine and L-cystathionine to produce hydrogen sulfide with pyridoxal 5′
phosphate (vitamin B6) as a cofactor [10,37–42]. The level of CBS is low in the embryonic brain, but it
significantly increases from the late prenatal to the early postnatal period and then declines in the adult
brains [43,44]. CBS expression is associated with the generation and differentiation of the lineage in
brain development [43,44]. Its expression is up-regulated in reactive astrocytes [44]. Recent different
results have reported that H2S production can use also D-cysteine [17,45]. D-Cysteine is metabolized by
D-amino acid oxidase (DAO) to an achiral 3-mercaptopyruvate, which is also produced by CAT from L-
cysteine in the presence of α-ketoglutarate [17]. Fig. 2 demonstrates the known enzymatic pathways of
endogenous H2S biosynthesis from L-cysteine and D-cysteine. There are two possible sources of D-
cysteine: racemase-induced hiral change of L-cysteine and absorption from food. Cysteine is structurally
similar to serine with an OH replaced by an SH. Aspartate racemase is homologous to CAT, which has an
affinity for both aspartate and cysteine [45,46]. It is possible that serine racemase or aspartate racemase
changes L-cysteine to D-cysteine [45].

D-Cysteine-dependent pathway operates predominantly in the brain (especially in the cerebellum) and
the kidney [17,45]. Moreover, the production of H2S from D-cysteine is higher in the cerebellum than in
other regions of the brain; but the production of hydrogen sulfide in the kidney is 7 times greater than in
the cerebellum. The generation of H2S from D-cysteine is 80 times greater than from L-cysteine in the
kidney [17,45]. D-Cysteine may have therapeutic potential. Moreover, it is less toxic than L-cysteine [17].
Administration of D-cysteine protects primary cultures of cerebellar neurons from the oxidative stress by
hydrogen peroxide (H2O2) and attenuates ischemia–reperfusion injury in the kidney more than L-
cysteine [45]. Recently, Kimura [17] has demonstrated that the production of H2S by CSE and the
3MST/CAT pathway may be regulated by Ca2+. In the steady state, low intracellular concentrations of
Ca2+, CSE and 3MST/ CAT pathway produce H2S. When cells are stimulated and the intracellular level of
Ca2+ is increased by Ca2+ influx and/or Ca2+ release from the intracellular stores, the generation of
hydrogen sulfide by CSE is decreased by approximately 50% and that via 3MST/CAT pathway is stopped
[17].

H2S METABOLISM

H2S exerts a variety of physiological functions (Fig. 1) and is produced endogenously via specific cysteine
desulfhydration pathways which are widely found in the body (Fig. 2). The two major enzymatic
pathways function under physiological conditions to catalyze the desulfhydration of cysteine are CBS or
CSE (cystathionase) [1]. In the brain, H2S synthesis is thought to occur via CBS [1-3] which condenses
homocysteine with serine to generate the thiol ether cystathionine. In the condensation, the hydroxyl
group of serine is replaced with the thiolate of homocysteine. The gene of human CBS is localized to
chromosome 21 at 21q22.3. In human and rat CBSexists primarily as a homotetramer with a subunit
molecular weight of 63 kDa. Each subunit also binds the cofactors pyridoxal 5'-phosphate, S-adenosyl
methionine and heme (Fig. 2). The heme appears to be a redox sensor, while S-adenosyl methionine is
an allosteric activator of the enzyme. The Cterminal portion of CBS contains a tandem repeat of two 'CBS
domains' which appear to act as inhibitors of enzymatic function, as their deletion activates CBS.
Recently CBS has been shown to be sumoylated at lysine 211 in the 'CBS domain' [7]. Heme binds to the
N-terminal portion of CBS comprising about 70 amino acids. In its ferrous state, this heme binds both CO
and NO. CO binds with higher affinity, with a Kiof about 5.6 μM, while NO (Ki360 μM) is only about two
percent as potent so that its binding probably is not physiologically relevant. The interaction of CO with
CBS leads to inhibition of CBS activity raising the possibility of cross-talk between CO and H2S as
messenger molecules. S-adenosyl methionine activates CBS several fold by binding to the CBS domain in
the carboxyl terminus of the enzyme [2, 3, 7]. The biologic rationale for activation of CBS by S-adenosyl
methionine suggest that CBS domain is an energy-sensing domain. CSE is a major route of H2S formation
in liver, is expressed in ileum, portal vein and thoracic aorta, and is present in vascular smooth muscle
cells [see ref. 8 for review]. The enzyme converts cystine to thiocysteine, pyruvate and ammonia, in a
-disulphide elimination reaction, with the thiocysteine then reacting with cysteine or other thiols to
produce H2S and cystine or the corresponding disulfide (Fig. 2). In most peripheral tissues CSE levels are
much higher than those of CBS.

CSE inhibitors have been employed to examine the enzyme's role in generating H2S physiologically. The
two principal inhibitors utilized are D, L-propargylglycine (PAG) and cyano-L-alanine (-CNA) however, are
poorly specific since they influence other enzymes such as cystathionine -synthetase, methionine -lyase,
aminotranferases, and D-amino acid oxidase. Thus, one must be cautious in interpreting results utilizing
such agents. However, it is of interest that PAG and -CNA do suppress H2S production in the liver and
kidney but not in the brain; fitting with other evidence that CBS is the predominant source of H2S in
brain tissue [7].

Like CBS, CSE is a pyridoxal 5'-phosphate-dependent and calcium calmodulin dependent enzyme.
Definitive evidence that CSE is a physiologic source for H2S comes from experiments employing CSE
knockout mice. H2S levels in aorta and heart of homozygous CSE knockout mice are reduced by about
80% with a 50% reduction in heterozygous knockouts [6]. Serum H2S levels in homozygous and
heterozygous CSE knockouts are reduced 50% and 20%, respectively. The studies with CSE knockouts
establish that H2S is a product of normal mammalian physiology. A third enzymatic route contributing to
H2S production from cysteine has been identified recently. Indeed it is possible to generate H2S from
cysteine by the coordinate actions of two enzymes, 3-mercaptopyruvate sulfurtransferase (Fig. 3) and
cysteine aminotransferase [8]. The physiologic significance of this pool of sulfur is unclear.

Finally, a non-enzymatic method for H2S formation has been described in astrocytes. In these cells
mobilization of bound sulphide occurs in response to intracellular alkalinization caused by cell
depolarization (Fig. 3). This latter route may constitute a rapidly responsive H2S generating system which
responds to neuronal activity. In addition, H2S-producing intestinal bacteria may provide unknown
quantities of H2S to the circulation.

Local tissue H2S concentrations are determined by the rate of formation and the rate of degradation.
Dissolved gaseous H2S is in a pH-dependent equilibrium with the sulfide anion (HS-), which can oxidize
to sulfite, sulfate, thiosulfate, sulphur (elemental), polysulphides, dithionate and polythionates. The
major routes of degradation are via non-enzymatic oxidation of sulphide to sequentially thiosulfate,
sulfite, and sulfate, the latter which is excreted in urine (Fig. 3). [11] Hydrogen Sulfide: From Physiology
to Pharmacology Stefano Fiorucci