nitrogen monoxide and dihydrogen sulfide 
 are two gaseous transmitters 
 that regulate numerous physiological functions .
although the chemistry , biochemistry , and physiology of no have been widely studied since its identification 
 as the endothelium - derived relaxing factor , h2s has only recently been recognized as an important 
 signaling molecule with physiological effects similar to those of 
 nitrogen monoxide . a ( bio)chemical understanding 
 of its mechanism of action is minimal , however .
although some authors have proposed interplay between no and h2s signaling pathways , it is widely accepted that the physiological consequences 
 of h2s , unlike no , do not directly involve cyclic guanosine 
 monophosphate ( cgmp ) .
one of the 
 modes of no signaling is activation 
 of soluble guanylate cyclase to produce cgmp .
two 
 decades after characterization of the first s - nitrosothiols 
 in biological systems , s - nitrosation of 1000 proteins has been reported , and this 
 posttranslational modification is increasingly considered to be as 
 important as phosphorylation .
the mechanism 
 of s - nitrosation and signaling through formal transfer 
 of the  no  moiety is still a matter of 
 debate , however .
it has been recently 
 proposed that h2s may play 
 a role in modulating the s - nitrosothiol profile in 
 the cells , and we hypothesized that this mechanism may involve the 
 reaction of hydrogen sulfide with low - molecular weight and/or protein s - nitrosothiols ( rsnos ) to form the smallest s - nitrosothiol , thionitrous acid ( hsno ) .
although extensively examined by computational methods , hsno has never been proved to exist in aqueous solution or characterized 
 under physiologically relevant conditions .
it has only been isolated 
 and spectroscopically identified in an argon matrix at 12 k. in the reaction of elementary sulfur and bis(triphenylphosphine)iminium 
 nitrite , ( pnp)(no2 ) , upon 
 addition of triphenylphosphine under vacuum in pure organic water 
 free solvents , ( pnp)(sno ) has been 
 characterized . here , we prove the 
 existence of hsno under physiologically relevant 
 conditions and demonstrate in vitro and at the cellular level that 
 hsno can serve as a source of no , no , and no entities , all of which evoke their own 
 distinctive physiological responses .
furthermore , we show that hsno 
 can freely diffuse through membranes facilitating transnitrosation 
 of proteins .
all chemicals were of the highest purity 
 available and purchased from sigma - aldrich .
buffers were prepared 
 with nanopure water and further purified and stored over chelex-100 
 to remove traces of transition metals .
all experiments were performed 
 using anhydrous na2s ( sigma aldrich ) , which was stored in a glovebox under argon ( < 1 ppm o2 and < 1 ppm h2o ) .
stock solutions ( 100 mm ) of 
 sodium sulfide were prepared in the glovebox using argon - saturated 
 nanopure water and stored in glass vials with ptfe septa at 4 c 
 for no longer than 1 week .
gas - tight hamilton syringes were used to 
 transfer these solutions throughout our studies .
the fate of hydrogen sulfide and nitrogen monoxide during the course 
 of the reaction was monitored with a 2 mm shielded h2s 
 sensor and the iso - no probe ( world precision instruments ) , connected 
 to a free radical analyzer ( world precision instruments ) .
experiments were performed in a four - channel 
 chamber ( wpi ) with both electrodes running either simultaneously or 
 individually .
a 2 ml portion of 50 mm ph 7.4 potasium phosphate ( kpi ) 
 buffer was added to the reaction chamber before immersion of the electrodes . 

depending on the type of measurement , different concentrations of 
 sodium sulfide ( 50500 m ) were injected followed by 
 addition of s - nitrosoglutathione ( gsno ) or s - nitrosoalbumin .
all spectrophotometric 
 studies were performed on a hewlett - packard 8452a diode array spectrophotometer 
 connected to a dell computer operating with olis spectralworks software . a 5 mm gsno solution in 50 mm
ph 7.4 potassium phosphate buffer was 
 degassed with argon and kept in dark glass vials sealed with ptfe 
 septa .
sodium sulfide was added to yield a final concentration of 
 5 or 17.5 mm .
the gas chromatograph was equipped with 
 a capillary column varian , vf-5 m. a 50 l volume of headspace 
 gas was injected ( splitless mode ) , and the following oven temperature 
 program was used with helium as carrier gas : 5 min at 50 c , 
 then increased to 155 c at a rate of 10 c / min , and then 
 increased to 260 c at a rate of 30 c / min .
detector multiplier voltage 
 was set to 1400 v , and the detection was performed by selected ion 
 monitoring of m / z 46 ( n2o ) and m / z 31 ( no ) using a dwell time of 50 ms and scan width for sim of 
 0.7 au .
a 100 l 
 sample of a hydroxylamine standard solution in 50 mm phosphate buffer 
 at ph 7.4 was treated with 200 l of cyclopentanone , 2 l 
 of concentrated sulphuric acid , and 1 ml of hplc 
after 1 h of incubation , the samples were diluted 10 times in methanol 
 and injected into the gc  ms .
a 5 l volume of solution 
 was injected ( splitless mode ) and the sample eluted using a 15 c / min 
 ramp from 50 to 250 c .
the detector multiplier voltage was set to 1400 v , 
 and the detection was performed by selected ion monitoring of m / z 84 ( cyclopentanone ) and m / z 99 ( cyclopentanone oxime ) using a dwell time 
 of 50 ms and scan width for sim of 0.3 au .
linear dependence of the 
 area under the peak versus concentration of hydroxylamine was observed 
 in the 2100 m range .
spectra of n - labeled s - nitrosoglutathione 
 ( gsno , 25 mm ) , either with or without addition of sodium 
 sulfide ( 25 mm ) , in 300 mm potassium phosphate buffer ( ph 7.4 ) were 
 recorded using a bruker 400 mhz spectrometer ( reference n - nitromethane , at 50.67 mhz , 35 pulse width , 5 s relaxation 
 delay ) .
gsno or gsno ( 120 mm ) in 300 mm potassium phosphate 
 buffer , ph 7.4 , was monitored in solution ( reactir 45 m , mettler toledo ) 
 utilizing a dicomp agx fiber probe with diamond atr element and mct 
 detector for 25 min to confirm stability prior to addition of na2s ( 100 mm ) .
thereafter , spectra were recorded for 10 min , 
 with each spectrum representing an average of 256 scans collected 
 over 1 min in the range 9001900 cm .
a 
 difference spectrum was obtained by subtracting the initial gsno spectrum 
 from all subsequent spectra .
briefly , these free radicals 
 were generated by irradiation of 1.2 mm na2s and 0.12 mm 
 kno2 in ar - saturated water ph = 11 with electron pulses 
 of 2 mev energy and 2050 ns duration generated by a febetron 
 705 accelerator ( titan , san leandro , ca ) .
a 300 l capacity quartz cell was used , and a flow system 
 ensured that each sample was exposed only to a single electron pulse .
human umbilical 
 vein endothelial cells ( huvecs , passage 23 ) were obtained 
 from promocell gmbh ( heidelberg , germany ) and cultured in 35 mm -dishes 
 ( ibidi , martinsried , germany ) in endothelial cell growth medium 2 
 ( promocell gmbh ) at 37 c and 5% co2 . for hno detection , 
 huvecs
were incubated with 10 m of a nitroxyl - responsive dye 
 ( cubot1 ) for 20 min .
cells were washed 
 of excess fluorescent dye three times , placed into new medium , and 
 further treated as specified in the figure legends .
fluorescence microscopy 
 was carried out using an inverted microscope ( axiovert 40 clf , carl 
 zeiss ) , equipped with green fluorescent filters and icm1 axiocam . 

images were postprocessed using imagej software , nih , for semiquantitative 
 determination of fluorescence intensity .
huvecs were exposed to medium supplemented with 1 mm l - name , 1 mm 
 propargylglycine , or both for 2 h and then fixed with 4% formaldehyde . 

cells were permeabilized , blocked , and incubated with primary monoclonal , 
 mouse anti - s - nitrosocysteine antibodies ( abcam , cambridge , 
 uk ) following the instructions of the manufacturer .
fitc - labeled , 
 goat antimouse fc - igg antibodies ( sigma aldrich , st .
gsno ( 1 mm ) was incubated 
 with ( 1 mm ) sodium sulfide in 300 mm kpi , ph 7.4 , for 3 min .
a 200 
 m portion of bovine serum albumin ( bsa ) was added to the solution 
 and incubated for 15 min , dialyzed against water for 24 h , and prepared 
 for ms analysis in acetonitrile / methanol ( 1:1 , v / v ) containing 0.1% 
 formic acid . to test the possibility that hsno can serve as a small , easily diffusible 
 trans - nitrosating agent
poly - s - nitrosoalbumin ( bsa - sno ) was prepared by mixing 
 300 m bsa with 1 mm gsno in the dark and incubation for 15 
 min , followed by 4 h dialysis against chelex - resin 100-containing 
 50 mm kpi , ph 7.4 , with constant exchange , and divided into two dialysis 
 bags ( cut off 5 kda ) .
a 1.5 ml portion of this material , serving as 
 a control , was placed into a 15 ml falcon tube containing 30 m 
 bsa and incubated there for 5 min .
to prove hsno formation and that 
 diffusion through the membrane takes place , 500 m na2s was added into the other 1.5 ml of 300 m bsa - sno , and the 
 solution was dialyzed against 30 m bsa for 5 min .
subsequently , the 
 dialysis bag was removed and the external solution was analyzed for 
 rsno content using no and h2s electrodes , saville s method , and the biotin - switch assay .
red 
 blood cells ( rbcs ) were obtained fresh from venous blood of healthy 
 volunteers by centrifugation at 5000 g for 5 min and 
 washed three times with sterile pbs ( sigma aldrich ) .
rbcs were then 
 resuspended in buffer to a cell count comparable to that of blood 
 and used immediately .
s - nitrosoalbumin was prepared 
 by mixing 300 m bsa with 1 mm cysno in the dark and incubated 
 for 15 min , followed by 4 h dialysis against chelex resin-100-containing 
 50 mm kpi , ph 7.4 , with constant exchange .
a 30 m portion of 
 this polynitroso - albumin was injected into 1 ml of rbc suspension , 
 with or without further addition of 50 m na2s or 
 glutathione .
after 5 min of incubation at room temperature , rbcs were 
 centrifuged at 10  000
rbc hemolysis 
 was performed by addition of 3 volumes of nanopure water containing 
 100 m neocuproine , and obtained hemoglobin was further purified 
 on sephadex g-10 columns protected from light .
all samples were adjusted 
 to the same protein concentration and directly analyzed by saville s 
 assay and/or esi - tof - ms . additionally , 20 m washed rbc was diluted 
 100 with pbs and exposed to 100 m poly bsa - sno ( prepared 
 by acidification of bsa / nitrite mixture and then further purified 
 on micro bio spin column ) without or with addition of 100 m 
 h2s , 100 m gsh , or 100 m cys .
two minutes 
 after the addition of bsa - sno , the samples were centrifuged for 3 
 min at 3000 g and then washed with pbs three times 
 prior to the hemolysis with nanopure water supplemented with 100 m 
 neocuproine .
samples ( 20 l ) were separated on lc and subsequently 
 analyzed by esi - tof - ms .
lc separation was done using following protocol : 
 column was equilibrated with a 50:50 mixture of buffer a ( 80:20 , water : acetonitrile ; 
 0.1% trifluoroacetic acid ) and buffer b ( 40:60 , water : acetonitrile ; 
 0.1% trifluoroacetic acid ) at a flow rate of 1 ml / min .
twenty microliters 
 of each sample was loaded and eluted with a 2 min hold at 50% b , followed 
 by a linear gradient to 66% b over 40 min .
all samples were 
 analyzed using maxis ( bruker daltonics ) , an ultrahigh - resolution esi - tof 
 mass spectrometer operating in the positive ion mode .
data analysis , 
 mass deconvolution , and spectra simulation were performed using data 
 analysis software , provided by the manufacturer .
geometries of all structures 
 were fully optimized at the b3lyp level of theory using the aug - cc - pvtz basis set .
stationary points were confirmed to be minima or transition 
 states by calculating the normal vibrations within the harmonic approximation . 

all computed relative energies are corrected for zero - point vibrational 
 energies ( zpe ) .
thermochemical 
 arguments are based on the group additivity approach described by 
 benson ( see the supporting information ) 
 and standard electrode potentials .
to test whether hsno could be generated in an aqueous solution , 
 we performed pulse radiolysis experiments in which hs and no ( scheme 1 , eq 1 ) 
 were cogenerated from hs / nitrite - containing solutions 
 ( ph 11 ) . a broad peak around 370 nm is formed transiently after 
 irradiation , reaching a maximum 5 s after irradiation ( see 
 figure 1a )
however , this spectrum comprises 
 not only hsno / sno but also precursors of hsno / sno and side products formed during pulse radiolysis of 
 nitrite and sulfide solutions .
initially , ho add to hs , reaction 7 , with a rate constant k1 = 5.4  
 10 ms and , 
 given the concentrations used , with a half - life of 110 ns .
this oxidation 
 step is followed by addition of hydrogen sulfide anion to yield disulfanuidyl , 
 or dihydrogen disulfide radical anion ( hssh).789 generation of hsno / sno by pulse radiolysis in 
 argon - saturated water ph 11 .
( a ) time - resolved absorbance spectral 
 investigation of hsno / sno generated by pulse radiolysis . 

the spectra reveal formation of a peak at 370 nm consisting of hssh / hss and hsno / sno .
( b ) the actual spectrum of generated hsno / sno , obtained by subtracting the first spectrum from that 
 acquired after 5 s .
the reaction given in eq 8 has a half - life of 300 
 ns ( k2  2  10 m s ) .
the hssh product has an absorption 
 maximum near 380 
 nm ( figure 1a , 
 red dots ) and equilibrates very rapidly with hs / s ( eq 9 ) , with a half - life of 100 
 ns ( k3  
 5  10 s , k3 = 5.4  10 m s , k3  10 ) . with 1.2 mm hs present ,
we expect 
 90% of the sulfur radicals to be present in the form of disulfanuidyl , 
 which decays by mixed order .
therefore , 
 we have to assume that at least two different decay mechanisms compete 
 with the desired reaction with no .
dominant at 
 high radiation doses and , consequently , large radical concentrations 
 is radical recombination leading to disulfide , which may trigger polysulfide 
 production with an ill - defined reaction order .
nitrogen monoxide 
 is formed from no2 according to eqs 
 10 and 11:1011the rate constant of reaction 10 is 5  
 10 m s , or , given 
 the concentration of nitrite , that reaction has a half - life of approximately 
 1.2 s . under first - order conditions ,
therefore , we observe initially ( 1 s 
 after pulse ) the spectrum of the disulfide radical anion with max  380 nm ( figure 1a ) . only 
 after the production of no2 or no does the desired radical recombination 
 product , hsno / sno , appear ( figure 1a ) .
we assign the difference between the spectra before quantitative 
 formation of no ( 1 s after pulse ) and after 
 its complete formation ( 5 s after pulse ) to thionitrite , as 
 nitrogen monoxide itself is colorless .
as shown in figure 1b , this spectrum has an absorbance maximum at 340 
 nm , a feature characteristic of rsnos that corresponds well to calculated 
 values for sno .
very 
 rapid disappearance of the reaction product is expected because hs is in great excess under these experimental conditions 
 and reacts with hsno / sno to generate h2s2 and its deprotonated forms together with hno ( eq 5 , 
 scheme 1 ) .
furthermore , under the applied alkaline 
 conditions , hsno / sno could hydrolyze according 
 to reaction 12 .
these processes all contribute to the 
 short half - life of hsno / sno in the pulse radiolysis 
 experiments .
mass spectrum of hsno , that is , [ hsno + h ] , prepared by acidification of nitrite in the presence of 
 sulfide 
 and then neutralized with the 300 mm potassium phosphate ph 7.4 buffer . as shown in scheme 1 , eq 2 , s - nitrosothiols could be generated from 
 the acidified mixture of corresponding thiol and nitrite .
an attempt to synthesize hsno by mixing sodium 
 sulfide with acidified solution of sodium nitrite resulted in a transient 
 color change , which was rapidly followed by decoloration and sulfur 
 formation .
fast neutralization of the observed brownish reaction mixture 
 gave a uv  vis spectrum with broad absorbance maximum at 370 
 nm , similar to that observed in pulse - radiolysis experiments ( figure s1a ) consisting of dihydrogen disulfide 
 radical anion , unreacted nitrite , and hsno / sno .
when analyzed by esi - tof - ms in the positive ion mode , the acidic 
 reaction mixture showed a dominant peak at m / z 64 ( figure s1b ) .
neutralization 
 of the colored solution and subsequent ms analysis revealed the existence 
 of the same peak at m / z 63.9898 
 ( 100% abundance ) , which we ascribe to [ hsno + h ] ( figure 2 ) .
this might suggest that , at ph = 7.4 , some 
 hsno present in solution . 

recent experimental and computational work indicates that the pka value of an iron - coordinated hsno intermediate 
 is 10.5 . on the basis of
that , free hsno 
 should have pka > 10.5 and hsno would 
 be 
 the major form under physiological conditions .
however , comparing 
 hsno with hno2 , one would expect a pka < 3.2 and thus the anion as the predominant species at 
 physiological ph . this issue remains to be resolved by future work .
although 
 indicative of hsno formation , the foregoing generation of the molecule 
 under very acidic conditions is unlikely to be of physiological relevance 
 except perhaps for the stomach or acidic cell compartments .
we therefore 
 investigated the ability of h2s to participate in transnitrosation 
 reactions ( scheme 1 , eq 3 ) .
addition of equimolar 
 amounts of na2s to buffered gsno ( s - nitrosoglutathione ) 
 solutions led to rapid ( < 1 min ) formation of a yellow color ( see figure s2 ) .
a very similar uv  vis spectrum 
 reported in the literature was ascribed 
 to ssno as a product of this reaction . however
, the presence of other polysulfides 
 in the reaction mixture can also be anticipated .
we analyzed 
 the reaction mixture using ultrahigh - resolution esi - tof mass spectrometry . 
 when mixed with na2s
, the intensities of the main gsno 
 peaks decreased ( see figure s3 ) and new 
 peaks appeared .
the most intense is a peak at m / z 345.0358 , which we assign to [ gs + 
 k ] ( or [ gssg + 2k ] ) ( see figure s3 ) .
more importantly , low - mass range spectral analysis 
 revealed the formation of a new peak at m / z 63.9902 ( 5% of maximal intensity , similar to the 
 spectrum observed in figure 2 ) .
simulation 
 of the isotopic distribution for [ hsno + h ] and comparison 
 of the calculated molecular weight ( 63.9852 ) with 
 the obtained value unambiguously established its identity as hsno 
 ( figure 3a ) .
the same experiment with gsno additionally confirmed generation of hsno 
 ( see figure s4 ) in the reaction of gsno and h2s ( scheme 1 , eq 
 3 ) .
no peaks of the putative [ hssno + h ] cation were detected , 
 suggesting that this adduct is not present in solution .
difference ftir spectra ( figure 3b ) clearly 
 show the disappearance of the no vibration from 
 gsno at 1515 cm with the appearance of 
 a new signal of similar intensity at 1568 cm .
this frequency is slightly lower than that calculated for hsno 
 in the gas phase and measured in an argon matrix ( 1596 cm for the trans- and 1569 cm for 
 the cis - isomer ) , due 
 to solvation effects .
characterization of hsno / sno generated 
 by transnitrosation 
 of s - nitrosoglutathione ( gsno ) and h2s . 

( a ) esi - tof - ms spectrum of hsno generated in transnitrosation reaction 
 between gsno and h2s .
experimental ( black ) and theoretical 
 ( red ) isotope distribution of the detected m / z 64 peak of [ hsno + h ] .
( b ) real - time ftir 
 confirms formation of a new nitrosothiol product .
differential ir 
 spectrum of the reaction of 120 mm gsno and 100 mm na2s 
 in 300 mm potassium phosphate buffer ph 7.4 ( black , after 1 min ; red , 
 after 10 min ) .
the reaction was performed in 300 mm potassium 
 phosphate buffer , ph 7.4 . when gsno was used to prepare hsno ,
the 
 corresponding n = o vibration shifted to lower values ( 1549 
 cm as compared to the calculated value of 1540 
 cm from the simple harmonic oscillator model ) 
 ( inset to figure 3b ) .
finally , hsno prepared 
 in the reaction of n - enriched 
 gsno and h2s was characterized by using n 
 nmr spectroscopy .
addition of an equimolar amount of na2s led to the immediate disappearance of the gsno peak ( 409 ppm ) and 
 appearance of a previously unidentified n resonance at 
 322 ppm ( figure 3c ) , which we assign to hsno . 

this new chemical entity was stable for less than 1 h at ph 7.4 and 
 21 c , with subsequent measurements showing only the nitrite 
 signal at 247 ppm .
a linear relationship exists between chemical shifts 
 of s - nitrosothiols and the pka values of the corresponding thiols ; a lower pka of a starting thiol implies smaller ppm values of n chemical shifts of the resulting s - nitrosothiol .
therefore , the lower pka of h2s in comparison to r - substituted thiols implies 
 a smaller chemical shift of hsno as compared to those of the substituted 
 analogs .
furthermore , less electron density on s in the case of hsno 
 when compared to rsno speaks in favor of a higher n   * 
 transition energy and a smaller n chemical shift .
the yellow coloration of the solution , 
 corresponding to the absorbance 
 maximum at 412 nm ( figure s2 ) , remains 
 even after the n nmr peak and characteristic n = o 
 vibration disappear from the solution spectra .
this result , in agreement 
 with our ms findings , excludes the presence of ssno , which as a nitrogen atom - containing species would have displayed 
 a corresponding n peak and n = o vibration band . 

thus , the band at 412 nm results predominantly from the mixture of 
 polysulfides .
collectively , these experiments unambiguously 
 confirm the presence 
 of hsno / sno in aqueous solution at ph 7.4 and demonstrate 
 that it can be formed in a transnitrosation reaction between h2s and rsnos ( scheme 1 , eq 3 ) .
rsnos may spontaneously decompose to give no , nitrosate a thiol , or form nitroxyl ( hno ) , as originally proposed .
following the same reasoning ( scheme 1 , eqs 46 ) , we focused on characterizing 
 the metabolic fate of hsno .
evidence for no production from hsno was obtained by following the previously characterized 
 transnitrosation reaction between gsno and h2s , as a source 
 of hsno , in combination with simultaneous amperometric monitoring 
 with the use of h2s- and no - sensitive 
 electrodes . to minimize metal ion - catalyzed decomposition of s - nitrosothiols ,
addition of gsno 
 to a buffered na2s solution caused removal of free h2s with concomitant no formation ( 7  
 3% based on total nitrogen ) ( figure 4a ) .
similar 
 results were obtained for the reaction of s - nitrosoalbumin 
 and h2s ( see figure s5 ) , confirming 
 that the reaction also proceeds with high - molecular - weight rsnos .
the release of no is a consequence of hsno homolysis , 
 as shown in eq 4 of scheme 1 . taking into account 
 the existence of the zwitterionic and ion - pair resonance structures 
 ( r 
s = n  o and rs / no , respectively ) , resulting in multireference 
 character of the hsno wave function , s 
n bond length and dissociation 
 energy for hsno were computed to be 1.85  and 29.2 kcal / mol , 
 respectively .
as compared to other s - nitrosothiols , hsno should be slightly less stable and more prone 
 to homolysis .
the kinetics of hsno homolytic 
 bond dissociation ( eq 4 , scheme 1 ) can be determined 
 on the basis of amperometric detection of no release ( figure 4b ) .
kinetics of h2s consumption and no generation 
 in the 
 reaction of gsno with h2s .
( a ) representative recordings 
 by h2s ( black ) and no ( red ) electrodes , 
 illustrating two processes described by eqs 3 and 4 . a 400 m 
 na2s solution was prepared in 50 mm potassium phosphate 
 buffer , ph 7.4 .
when the electrode response reached its maximum , an 
 equimolar amount of gsno was added , triggering an immediate drop in 
 current at the h2s electrode and a rise in the no signal .
kinetic traces of no release ( b ) and 
 h2s consumption ( c ) from the reaction mixture containing 
 250 m gsno and 250 m h2s at ph 7.4 at 25 
 c .
red lines represent a first - order fit for ( b ) and a second - order 
 fit for ( c ) . for this first - order reaction ,
the corresponding 
 rate constant 
 was determined to be 0.12 + 0.01 s. conversely , 
 the kinetics of the hsno / sno formation reaction 
 ( eq 3 in scheme 1 ) , assuming that the side 
 reactions and the spontaneous release 
 of no from gsno are slower than the 
 main transnitrosation reaction , were monitored following h2s consumption ( figure 4c ) under second - order 
 reaction conditions ( gsno : h2s ratio was 1:1 , 250 m 
 each ) .
the corresponding rate constant is 84  7 m s at 25 c . in comparison with other transnitrosation 
 reactions ( for s - nitroso - n - acetylpenicillamine 
 ( snap ) + gsh ,
k = 9 m s ; for snap + cys , k = 21 m s ) , higher reactivity 
 of h2s is expected .
recent studies showed that sulfide is produced in tissues at a high 
 metabolic rate , the concentration in 
 plasma being in the micromolar range and therefore in excess of what has been proposed to represent the 
 physiological concentration range for rsnos .
considering such concentrations , cellular hsno / sno formation would be expected to yield nitroxyl ( hno / no ) ( scheme 1 , eq 5 ) , the reduced form of no with distinct signaling properties . to prove its existence , reductive nitrosylation with methemoglobin 

was used . when a mixture of gsno and 
 h2s ( 1:2 molar ratios ) was added to a buffered methemoglobin 
 solution , immediate formation of nitrosyl - hemoglobin occurred ( figure 5a ) , whereas no spectral changes were observed with 
 gsno or h2s alone ( see figure s6 ) .
nitroxyl also converts to nitrous oxide , n2o ( k = 8  10 m s ) , reaction 13.13 we therefore wanted to confirm the 
 generation of nitroxyl along with that of no from 
 hsno using gc 
fifteen minutes after addition of an equimolar amount of h2s to gsno , a moderate increase of the no amount 
 was detected in the gas phase above the reaction mixture , in agreement 
 with the no release detected by no electrode ( figure 2a ) .
more importantly , 
 an increased h2s concentration caused a large increase 
 in the peak area of n2o ( yield of 17  5% calculated 
 on the basis of total n content in starting gsno solution ) ( see figure s7 ) .
hno also reacts rapidly with 
 thiols to give hydroxylamine , reaction 
 14.14assuming that excess h2s does react 
 further with hno generated in this manner , we wished to detect hydroxylamine . 

however , the use of standard colorimetric assays proved to be of no value for this purpose , because free 
 thiols interfere with this assay , resulting in false negative results . 

for the present purpose , we therefore developed a rapid and reproducible 
 gc  ms method for detecting hydroxylamine based on its ability 
 to react with ketones , cyclopentanone in the present case , to give 
 corresponding oximes .
selected 
 ion monitoring for the m / z 99 ( cyclopentanone 
 oxime ) peak was applied . the maximal yield , observed for 10-fold excess 
 of h2s over gsno , was 71  4% ( figure 5b ) .
together with the detected n2o and no , this 
 value corresponds to 112  12% of total nitrogen consumed in 
 the reaction .
some hydroxylamine ( 2  1% ) was detected when a 
 1:1 ratio was used , presumably due to the side reactions .
although 
 endogenous generation of hno has never been detected directly 
 in vivo , the recent development of a nitroxyl - responsive dye , cubot1 , enabled us to do so .
when human umbilical vein 
 endothelial cells , loaded with cubot1 , were incubated with either 
 gsno or h2s ( figure s8 ) , there 
 was no change in fluorescence .
nitrogen monoxide releasing donor , 
 diethylammonium ( z)-1-(n , n - diethylamino)diazen-1-ium-1,2-diolate ( deanonoate ) , also 
 did not have any significant effect on cubot1 fluorescence , confirming 
 the previous study of its selectivity .
addition of both gsno and h2s provided clear proof of 
 hno generation ( figure 5c , d ) .
the effect was 
 even more pronounced when cells were pretreated with gsno to increase 
 intracellular rsno content , washed , and then loaded with the probe 
 before exposure to h2s ( figure 5c , d ) .
100 m gsno and 200 
 m na2s solution were added to 50 m methb 
 in 50 mm potassium phosphate buffer ph 7.4 , and the reaction was followed 
 every 10 s for a total of 5 min .
a 60 m gsno solution was mixed , at different ratios indicated 
 in the figure , with h2s at ph 7.4 , and after 10 min the 
 reaction mixture was treated with cyclopentanone and methanol . at 
 1 h after the incubation , the corresponding mixtures were analyzed 
 by gc 
human umbilical vein endothelial 
 cells , loaded with 10 m cubot1 , were treated with either 100 
 m na2s , 100 m gsno , or both for 20 min .
some 
 cells were pretreated with 100 m gsno for 20 min to increase 
 intracellular nitrosothiol content and then exposed to 100 m 
 na2s .
( d ) fluorescence intensity was quantified using imagej , nih 
 ( n = > 30 cells ) .
these experiments strongly support the hypothesis 
 that cellular 
 hsno is involved in the formation of nitroxyl , but they can not exclude 
 the possibility that hno is produced directly in the reaction of gsno 
 and h2s ( eq 15).15 in particular , previous studies suggest 
 that gsno decomposes in 
 the presence of gsh with the formation of gssg , which would imply the direct formation of hno via eq 15 . 

we found no evidence for such direct formation of hno , for neither 
 n2o nor nh2oh levels were significantly changed 
 when gsno was mixed with h2s in a 1:1 molar ratio ( figure 5b and figure s7 ) .
furthermore , 
 no gssh was detected in the esi - tof ms spectra ( figure s3 ) .
we further explored the possibility , however , 
 by carrying out dft calculations for the reaction of ch3sno , as a model for s - nitrosothiols , with h2s / hs . figure 6 ( table s1 ) shows the lowest - energy profile 
 for the reaction of protonated 
 ( a ) and deprotonated hydrogen sulfide ( b ) . in both cases ,
the reaction 
 can proceed by two pathways , h2s / hs attack 
 on either the n or the s atom of ch3sno .
h2s attack on the n atom leads to the formation of ch3sh 
 and hsno molecules ( final 1 ) via transition 
 state , ts1 , with the activation barrier of 23.8 kcal 
 mol ( figure 6a ) .
the reaction 
 via attack on the s atom of ch3sh is characterized by a 
 much higher activation barrier ( ts2 , eact = 47.5 kcal mol ) and leads to 
 the formation of ch3s  sh and hno ( final 2 ) .
formation of ch3s  sh and hno , final 2 , is also thermodynamically less favorable than formation of ch3sh and hsno , final 1 ( 13.9 and 7.3 kcal mol , respectively , figure 6a ) . with hs
, the reaction proceeds in the same manner : 
 the activation barrier for the n attack ( ts3 ) is lower 
 than that for the s attack ( ts4 ) ( 5.8 vs 39.8 kcal mol , respectively ) , with favorable formation of the n attack products 
 ( final 3 , figure 6b ) . in 
 addition
, we estimate that hno generation by eq 15 is energetically 
 unfavorable by 40 kj / mol by using standard electrode potentials and 
 known bond energies ( see the supporting information ) .
b3lyp / aug - cc - pvtz computed energy profile for the reaction between 
 ch3sno and h2s / hs . energies 
 are in kcal mol ; e+zpe ( first 
 entry ) , g ( second entry ) . whereas rsno can exist as two isomers ( cis / trans , 
 with an energy 
 difference of 0.8 kcal mol ) , regioselective attack 
 on the n atom leads only to cis - hsno , in agreement 
 with the observed ir spectra .
whether the same kinetics and 
 thermodynamics operate for larger 
 intracellular proteins remains to be determined . in the light of recent 
 suggestions
that h2s acts though sulfhydrylation of protein 
 thiol residues , it is possible that direct reaction 
 of rsnos and h2s , with concomitant formation of hno , takes 
 place in vivo .
sulfhydrylation of the proteins in vivo can be 
 envisioned via another 
 reaction mechanism .
the reaction of hydroxylamine formation , given 
 in eq 14 , goes stepwise , and , for the case where h2s is 
 reacting thiol , it would proceed as follows:1617 it is possible , however , that the product 
 of the initial reaction , 
 hs(o)nh2 , reacts further with protein thiols , particularly 
 when h2s is not present in large excess ( eq 18 ) , and form 
 sulfhydrylated protein with elimination of hydroxylamine.18 hsno serves as a shuttle for no . 

( a ) experimental design 
 for the protein - to - protein trans - nitrosation mediated by hsno ( see experimental section ) .
( b ) s - nitrosothiol 
 content in the sample obtained from protein - to - protein transnitrosation 
 experiment .
no electrode responses upon subsequent 
 addition of aliquots of the control ( black ) or h2s - treated 
 samples ( red ) into 500 m ascorbate / cu containing 
 solution .
( c ) total amount of rsnos generated by h2s in protein - to - protein transnitrosation experiment ( n = 4 ) .
( d ) the results of the biotin switch assay for the same samples . 
 ( e , f ) hsno facilitated nitrosation of hemoglobin in human red blood 
 cells .
deconvoluted mass spectra ( e ) of hemoglobin beta subunit isolated 
 from rbc after the treatment with synthetic poly - s - nitrosoalbumin ( bsa - sno ) in the presence or absence of h2s .
sample treated with poly - s - nitrosoalbumin in 
 the presence of h2s exhibit another peak shifted by mass 
 of 58 corresponding to [ hb 
given its 
 small size and reactive nature , hsno might serve as a shuttle for 
 nitrosonium equivalents by supporting sequential transnitrosation 
 reactions .
10 kj mol , uphill energetically , which is 
 readily calculated from the minor difference 
 ( ca .
12 kj stronger ) between bond strengths of gs  no and hs  no , and the electrode 
 potentials , e(gs , h / gsh ) = 0.94 v and e(s,2h / h2s ) = e(s , h / hs ) = 0.92 v at ph 7 . consequently , eq 6 in scheme 1 is energetically favorable by the same amount . to examine 
 the feasibility of this scenario , we first investigated transnitrosation 
 reactions between gsno and bovine serum albumin ( bsa ) as a model . 

albumin is proposed to be one of the carriers / storages of bioactive 
 no in human plasma . as a proof - of - concept experiment ,
h2s was incubated with 
 gsno at 37 c for 5 min to allow complete gsno decomposition 
 prior to the addition of albumin .
after addition of bsa and a further 
 15 min incubation , the reaction mixture was analyzed by esi - tof ms . 

the peak shifts imply that four nitroso equivalents per protein were 
 added ( see figure s9 ) . when the dialyzed 
 hsno - treated albumin sample was placed into buffer and irradiated 
 with light , release of no
was induced ( see figure s9 ) , confirming s - nitrosation 
 of bsa .
s - nitrosoproteins are relatively stable 
 and unless 
 exposed to light or traces of heavy metals , they can not exchange no equivalents with other proteins through the membrane .
considering the potential significance of the 
 shuttle function of hsno , we designed an experiment specifically to 
 address this notion ( figure 7a ) .
poly nitrosated 
 albumin was placed inside a dialysis bag and inserted into a buffered 
 solution of non - nitrosated albumin . in one of the samples ,
h2s was injected into the dialysis bag containing bsa - sno , while the 
 untreated one served as a control .
if hsno were to be formed as a 
 small neutral species , it should have been able to freely diffuse 
 through the membrane and reach the outside solution of bsa . without 
 such intermediate ,
the exchange of no equivalents between 
 the compartments would be unlikely . after 5 min of incubation
, the 
 dialysis bags were removed and the exterior albumin solution was dialyzed 
 for 2 h against water to remove traces of small molecules .
subsequent 
 addition of aliquots of these solutions into cu - containing 
 buffer ( figure 7b ) revealed a clear no signal for h2s - treated samples as detected 
 by an no electrode , whereas control incubates showed 
 only minor signals , most likely the result of metal contamination . 

rsno quantification by the saville assay showed that 11  1% 
 of bsa became nitrosated in h2s - treated samples as compared 
 to only 2  1% in the controls ( figure 7c ) , and these results were qualitatively confirmed with the biotin - switch 
 assay ( figure 7d ) .
additional experiments 
 were carried out using human red blood cells 
 ( rbcs ) .
s - nitrosation of hemoglobin ( hb ) is important 
 for the regulation of oxygen delivery , but the chemical basis for this process is still a matter of debate . to test whether hsno could be a carrier from plasma s - nitrosoproteins to hb , washed rbcs were incubated for 5 min in pbs 
 supplemented with 30 m poly - s - nitrosoalbumin 
 ( bsa - sno ) , in the absence or presence 
 of 50 m na2s .
hemoglobin was obtained after extensive 
 washing of cells with pbs followed by hemolysis in nanopure water 
 containing 100 m neocuproine and analyzed by esi - tof - ms and 
 saville assay .
deconvoluted mass spectra ( figure 7e ) revealed that the hemoglobin -subunits ( obtained 
 mass 15  866.3  1 , expected 15  867.2 ) of rbcs treated 
 with nitrosoalbumin and na2s exhibit an additional peak 
 shifted by a mass of 58 , indicative of the addition of 2no moieties / hb 
 ( [ hb 
, the total s - nitrosoprotein concentration was determined in the samples 
 of hb treated with gsno , bsa - sno , and bsa - sno + h2s ( figure 7f ) .
there are several pathways by which gsno 
 or cysno could traverse 
 biological membranes and deliver the  no  
 moiety .
some of them require a specific transporter or a thiol - to - thiol 
 cascade transfer of  no  .
we wanted to test the observed superiority 
 of hsno over other low - molecular weight thiols to pass freely through 
 membranes and s - nitrosate the targets faster .
a 20 
 l portion of washed packed rbc was diluted 100 times with pbs 
 and exposed to 100 m poly bsa - sno in the presence or absence 
 of 100 m gsh , cysteine , or h2s for 2 min .
after 
 hemolysis , hb was analyzed by lc - esi - tof - ms . only in the case of h2s treatment
these data imply that , unlike gsno 
 and cyssno , hsno can freely diffuse to reach its intracellular target 
 and increase the intracellular hbsno content . given the universal abundance 
 of h2s and rsnos in biological systems , it is plausible 
 that hsno is produced in vivo .
the direct in vivo detection of hsno , 
 however , seemed challenging considering its demonstrated reactivity 
 and relative instability .
if produced in cells , hsno would be capable of further trans - nitrosation , 
 so it is reasonable to assume that daf-2 dye could serve as a sensor 
 for its formation as well .
mechanistically , the principle of this 
 imaging technique involves a nitrosation step of daf-2 to produce 
 the fluorescence .
we therefore tested the role of intracellularly 
 produced h2s on transnitrosation of daf-2 induced by gsno .
cells were incubated in buffer supplemented with the inhibitor 
 of h2s producing enzyme ( cse ) , propargyl glycine ( pg ) , 
 washed and then exposed to gsno .
whereas endothelial cell pretreatment 
 with pg had no significant effects on basal daf-2 fluorescence , it 
 markedly blocked the gsno - induced fluorescence increase ( figure 8a ) .
cse inhibition followed by sulfide treatment 
 before exposure to gsno restored gsno - induced fluorescence , confirming 
 that the effects were not due to unspecific quenching .
huvecs were incubated for 2 h in medium with 
 or without cse - inhibitor propargylglycine ( 1 mm ) .
some cells were additionally incubated 
 with 100 m na2s for 20 min prior to gsno addition . 

no - induced fluorescence was detected by fluorescence microscopy and 
 quantified using imagej ( n = 2030 cells ) . 

( b ) the effect of inhibitors of endogenous no and h2s production 
 on intracellular s - nitrosation .
huvecs were exposed 
 to medium supplemented without or with 1 mm l - name , 1 mm pg , or both 
 for 2 h. cells were fixed and intracellular rsnos visualized by immunocytochemistry 
 using anti - s - nitrosocysteine antibodies .
we demonstrate that endogenous h2s is 
 indeed involved 
 in regulation of the intracellular s - nitrosation 
 by an experiment in which huvecs were exposed to the inhibitor of 
 no production ( l - n - nitroarginine methyl ester , l - name ) 
 or / and inhibitor of h2s production ( propargylglycine , pg ) .
exposure to pg lowered the level of intracellular snos 
 as detected using specific s - nitrosocysteine antibodies 
 ( figure 8b ) .
l - name had a much stronger effect , 
 as expected , but did not completely block s - nitrosation 
 because it is a competitive inhibitor of nitric oxide synthase .
an 
 inhibitory effect was , however , most obvious when cells were exposed 
 to both l - name and pg ( figure 8b ) .
the unique physicochemical properties of hsno make 
 it an excellent 
 candidate for redox sensing and regulation of metabolic activities . 

in particular , hsno could lead to the generation of hno , which has 
 significant cardio - pharmacological potential , and herein we provide 
 the first direct intracellular detection of its generation .
additionally , hsno / sno might 
 serve to facilitate further trans - nitrosation / denitrosation of other 
 targets in the cell or even intercellularly , due to the facile diffusion 
 ability of hsno ( as the neutral form of the hsno / sno acid 
hemoglobin is one possible target , and 
 the chemical mechanism of its s - nitrosation is still 
 a matter of debate .
s - nitrosoproteins ( rsnos ) can not 
 cross cell membranes by free diffusion , and  no  transport has long been proposed to be facilitated 
 by formation of smaller s - nitrosothiols , especially 
 those of cysteine and/or glutathione , which may require specific transporters .
formation of hsno / sno might 
 link plasma s - nitrosothiols to intracellular s - nitrosohemoglobin .
circulating h2s could react 
 with rsnos forming hsno / sno by transnitrosation , 
 where hsno would then freely diffuse across the rbc membrane to transfer 
 its  no  moiety to the -subunit of 
 hemoglobin forming s - nitrosohemoglobin ( figure 9 ) , known to have important physiological functions . proposed reaction scheme for hsno - induced nitrosation 
 of hemoglobin .
three key proteins presently accepted as major 
 targets for h2s and its subsequent regulation of blood 
 pressure , neuronal activity , and cardioprotection , the katp channel , nmda receptor , and nrf-2 , respectively , 
 are shown to be 
 regulated by s - nitrosation .
early and recent work suggests that activity of no synthase 
 is a prerequisite for h2s to exhibit its effect . 

the present study thus offers a chemical perspective into the metabolic 
 fate of h2s and opens a new chapter in biological chemistry 
 of redox signaling .
 dihydrogen sulfide recently emerged as a biological signaling 
 molecule 
 with important physiological roles and significant pharmacological 
 potential . 
 chemically plausible explanations for its mechanisms of 
 action have remained elusive , however . here , we report that h2s reacts with s - nitrosothiols to form thionitrous 
 acid ( hsno ) , the smallest s - nitrosothiol . 
 these results 
 demonstrate that , at the cellular level , hsno can be metabolized to 
 afford no+ , no , and no species , all 
 of which have distinct physiological consequences of their own . 
 we 
 further show that hsno can freely diffuse through membranes , facilitating 
 transnitrosation of proteins such as hemoglobin . 
 the data presented 
 in this study explain some of the physiological effects ascribed to 
 h2s , but , more broadly , introduce a new signaling molecule , 
 hsno , and suggest that it may play a key role in cellular redox regulation . 
Introduction
Experimental Section
Results and Discussion