Fluorescent Properties And Physiological Functions Of Reactive Sulfane Sulfur

Hydrogen sulfide(H2S)is a new gasotransmitter that serves many important regulatory roles in biological systems.It is involved in apoptosis,vascular homeostasis,revascularization,neuromodulation,cytoprotection,and anti-inflammation.However,accumulating evidences imply that H2S is converted to reactive sulfane sulfur(RSS),which plays the observed roles.Sulfane sulfur is common inside cells,playing both regulatory and antioxidant roles.Reactive sulfane sulfur includes organic persulfides(R-SSH),organic polysulfides(R-SSnH or R-SSnR,n?2),and inorganic hydrogen polysulfides(H2Sn,n?2).Sulfane sulfur is different from thiols,as it often possesses both nucleophilic and electrophilic characteristics while thiols mainly function as nucleophiles.The reactive sulfane sulfur can be produced from specific and nonspecific enzymatic oxidations of H2S or from the metabolism of cysteine and N-Acetyl cysteine(NAC).GSSH is a key form of reactive sulfane sulfur in the sulfide oxidation pathway of heterotrophic bacteria and human mitochondria.Reactive sulfane sulfur can modify cysteine residues in a large number of proteins by S-persulfidation(R-SSH),which can alter enzyme activity and influence biological processes via signaling.For instance,rhodanese(thiosulfate:cyanide sulfurtransferase)that is present in almost all living organisms catalyzes the transfer of the sulfane sulfur from thiosulfate to cyanide via an intermediate(R-SSH)at its catalytic Cys residue.Collectively,previous reports have revealed the significance of reactive sulfane sulfur in biological processes.Thus,a better understanding of the chemical and biochemical properties of biologically relevant reactive sulfane sulfur will help to advance the fieldCurrent methods used for the detection of reactive sulfane sulfur include sulfur chemiluminescence detection,ion chromatography,HPLC analysis of the monobromobimane derivative of H2Sn,and the use of H2Sn-sensitive fluorescent dyes in living cells or in vitro.All of these methods are reaction-based.A reaction-free method that can real-timely probe reactive sulfane sulfur has not been developedMaintaining sulfide in a low range is critical because sulfide at high concentrations is toxic,probably through reversible binding and inhibition of the cytochrome c oxidase.Some heterotrophic bacteria can oxidize sulfide(H2S,HS,and S2-)by using SQR and PDO to polysulfides,sulfite,and then thiosulfate.The oxidized forms are less toxic than sulfide.Cupriavidus pinatubonensis JMP 1 34(formerly Ralstonia eutropha JMP134)is a Gram-negative bacterium of?-Proteobacteria.It is best known for the ability to degrade a variety of aromatic compounds and for its applications in biotechnology.The genes coding for the oxidation in Cupriavidus pinatubonensis JMP 134 have recently been identified.A regulator gene is adjacent to the operon of the sulfide-oxidizing genes,encoding a?54-dependent transcriptional factor(FisR)with three domains:an R domain,an AAA+ domain,and a DNA-binding domain.It has been reported that the regulator responds to the presence of sulfide and activates the sulfide-oxidizing genes.However,the regulation model of FisR and the control mechanism and the cooperation among the three domains are not very clearIn order to solve the above-mentioned problems,we studied the reactive sulfane sulfur using a rapid and sensitive method.1.We report the discovery that reactive sulfane sulfur can be detected via resonance synchronous spectroscopy(RS2)with a conventional spectrofluorometer by simultaneously scanning the excitation and emission.We discovered reactive sulfane sulfur species have RS2 properties only when the molecules contain an electrophilic sulfane sulfur.It can be applied to reactive sulfane sulfur analyses,such as pKa determination,reaction kinetics,pH-dependent sulfane reactivity of small and protein persulfides,etcWith RS2,we showed that inorganic polysulfides at low concentrations were unstable with a half-life about 1 min under physiological conditions due to reacting with glutathione.The protonated form of glutathione persulfide(GSSH)was electrophilic and had RS2 signal.GSS-was nucleophilic,prone to oxidation,but had no RS2 signal.Using this phenomenon,pKa of GSSH was determined as 6.9 GSSH/GSS-was 50-fold more reactive than H2S/HS-towards H2O2 at pH 7.4,supporting reactive sulfane sulfur species like GSSH/GSS-may act as antioxidants inside cells.Further,protein persulfides were shown to be in two forms:at pH 7.4 the deprotonated form(R-SS-)without RS2 signal was not reactive toward sulfite,and the protonated form(R-SSH)in the active site of a rhodanese had RS2 signal and readily reacted with sulfite to produce thiosulfate.These data suggest that RS2 of sulfane sulfur is likely associated with its electrophilicity.For whole cell analysis,sulfane sulfur showed species-specific RS2 spectra and intensities at physiological pH,which may reveal the relative abundance of a reactive sulfane sulfur species inside cellsThe RS2 method is rapid,sensitive and convenient,allowing us to reveal several new chemical and biochemical properties of biologically relevant reactive sulfane sulfur.The results that were reported here,such as the pKa of GSSH,the reaction parameters,the distribution of H2Sn species at different pH,may fill some gaps in the field2.The sulfide oxidation pathway present in C.pinatubonensis JMP134 to serve as a source of RSS.We discovered a new ?54-dependent TF,FisR,working as both a sulfide sensor and a transcriptional regulator of the sulfide oxidation pathway in JMP134.When sensing polysulfides,FisR activates ?54-dependent transcription of the operon for actively removing sulfide.We identified the amino acid residues(C53,C64,and C71)that are essential for the sensing polysulfides.FisR binds to its cognate operator at-114 bp to-135 bp of the transcription start of the pdo-sqr operon and regulates the transcription through PR via its ATPase activity,which is stimulated upon exposure to polysulfidesThe dependent of regulator oligomerization is different,such as D domain binds operon upstream sequence or phosphorylation of its R domain.For FisR,its oligomerization is R-domain dependent,and once translated,it forms a hexamer and binds to its cognate binding site in pdo-sqr operon of C.pinatubonensis JMP134.Negative regulation of the R domain on the AAA+ domain is a dominant mechanism for ?54-dependent TFs.However,the FisR AAA+ domain is positively regulated by the R domain and polysulfides stimulate the activity.The key process in this transcriptional activation model is the conversion of sulfide to polysulfides that activate the ?54-dependent transcription of sqr and pdo through PR.The base level of SQR,with a low Km value for sulfide,ensures that sulfide can be rapidly converted to polysulfides.The standby FisR hexamer guarantees the ATPase activity can be rapidly activated by sulfide via polysulfides.Therefore,the transcriptional activation process is always in the "ready to go" state,conferring a quick response to sulfide stress on C.pinatubonensis JMP134.Further,bioinformatics analysis suggested that FisR-like regulators are often involved in the regulation of sqr-pdo gene clusters in Proteobacteria.3.Saccharomyces cerevisiae is a well studied model organism and known to grow with thiosulfate as a sulfur source producing more ethanol than using sulfate.S.cerevisiae has five rhodaneses,Rdll,Rd12,Tuml,Ychl and Uba4,among which Rdlland Rd12 can convert thiosulfate with GSH to a persulfide and sulfite and tRNA-thiouridine modification protein 1(Tuml)plays essential role in the sulfur transfer process of Urml system.DUF442,a domain of Cupriavidus pinatubonensis JMP1 34 sulfide:quionone oxidoreductase(GeneBank:AAZ62946.1),has rhodanese activity and catalyzes the reaction of GSSH with sulfite to produce thiosulfate.The DUF442 domain consists of 128 amino acid residues with two cysteine residues,Cys34 and Cys94,and only C94 is conserved and functionally essentialGSSH-reacted DUF442 was subjected to LC-MS/MS to analyze the modification.Both C34-SSH and C94-SSH modifications were detected.The modified DUF442 displayed significant RS2,which was not observed from unmodified protein at pH 7.4 The pKa of C34-SSH protein was determined by using R2S2 method to be 6.29.Thus,only C94-SSH in the DUF442 wild type or the DUF442 C34S mutant is protonated at pH 7.4 and the sulfane sulfur can be transferred to sulfite to produce thiosulfate3D structures of Rd12 and DUF442 were modeled by using SWISS-MODEL.A putative RhoD from Saccharomyces cerevisiae(PDB ID:3DlP)at 0.98 A resolution and a putative RhoD from Neisseria meningitidis z2491(PDB ID:2F46)at 1.41 A resolution were chosen as templates for Rd12(40%sequence similarity)and DUF442(39%sequence similarity),respectively.The sulfur atom of catalytic cysteine residue is located at the bottom of a cradle-like pocket in both Rd12 and DUF442 models.The surrounding basic residues generate a strongly positive electrostatic field in the pocket Therefore,the protein-SSH is not dissociated in the pocket or protein-SS-forms a hydrogen bond with one of these groups as revealed by RS2.In contrast,sulfur atom of C34 is not located in a positively electrostatic field,and it should exist in the deprotonated form C34-SS-at pH 7.4,which showed no RS2 and electrophilicityWe first report the crystal structures of Rd12 persulfides(Rho D-SSH)form the yeast S.cerevisiae at 2.47 A,the most important intermediate in which the transferring sulfur is bound to the invariant catalytic Cys residue.The Arg residue is import to the active site and we observed that the Arg mutant protein decreased enzymatic activity.The thiosulfate-reacted Rdl2 was subjected to LC-MS/MS to analyze the modification.Rdl-SSH modifications were detected,which is consistent to the crystal structures.In this study,the biochemical fluorescence properties of sulfane sulfur,including polysulfides and persulfides,are analyzed.The physiological functions of the representative proteins(FisR and RhoD),which are closely related to RSS,were also reported in this paper.It provides theoretical basis and technical support for revealing regulation and signaling mechanism of RSS in the further.