Sulfane Sulfur Quantification In Biological Samples And Its Metabolism By Rhodanese

Sulfur is an important element in living organisms.It exists mainly in the form of sulfide and sulfate in nature.Hydrogen sulfide(H2S),as a new gasotransmitter plays important roles in many physiological activities,including vasorelaxation,cardioprotection,resistance to oxidative stress,neurotransmission,and anti-inflammatory action.However,excessive sulfide is toxic to cells.It inhibits cellular respiration by poisoning cytochrome c oxidase or raises oxidative stress with various reactive sulfur species.Sulfide homeostasis keeps intracellular H2S in a desired range.Many heterotrophic bacteria produce H2S in rich medium.Sulfide:quinone oxidoreductases(SQR)and flavocytochrome c-sulfide dehydrogenases(FCSDs)are two main enzymes for H2S removal.SQR,more active than FCSDs for sulfde oxidation,often forms an operon with persulfide dioxygenase(PDO)in bacteria.The functions of SQR-PDO pathways in humans,Staphylococcus aureus and Cupriavidus pinatubonensis JMP134 has been characterized.SQR oxidizes H2S to zero valence sulfur with glutathione(GSH),H2S,and sulfite as sulfur acceptors to produce different sulfane sulfur species.PDO oxidizes glutathione persulfide(GSSH)into sulfite.The latter spontaneously reacts with zero valeance sulfur to form thiosulfateRhodaneses(thiosulfate:cyanide sulfurtransferases or TSTs),may also participate in sulfide oxidation by SQR and PDO.Rhods catalyze the reaction of thiosulfate with cyanide via a ping-pong mechanism,with an active site cysteine residue carrying the transferring sulfur as a covalent intermediate.GSH,sulfite,and thioredoxin can also be used as sulfur acceptors besides cyanide,and polysulfides,GSSH as sulfur donors besides thiosulfate.TSTD1,a Rhod that functions in the SQR-PDO pathway inside human cells,catalyzes the transfer of sulfane sulfur from GSSH to sulfite to produce GSH and thiosulfate.A Rhod domain at the C-terminal of PDO-Rhod fusion protein CstB in S.aureus promotes thiosulfate formation during sulfide oxidation.Reversely,the Rhod domain of a PDO2-Rhod fusion protein in Burkholderia phytofirmans transfers sulfane sulfur in thiosulfate to GSH to produce GSSH.Although Rhod was discovered more than 80 years ago,new physiological functions of various Rhods are being elucidated.However,the physiological significance of Rhod in the SQR-PDO pathway has not been clarified.In addition,Rhods also widely distributed in a large number of bacteria without the sulfide oxidation pathway.E.coli does not have the SQR-PDO pathway,but contains nine Rhods.The function of some Rhods have been revealed,including the production of Fe-S cluster,the biosynthesis of thiamin and selenium metabolism.However,the functions of many Rhods remain to be characterized.Sulfur sulfane is a regular cellular component in living organisms,palying roles in cyanide detoxification,antioxidation,signal regulation.Sulfane sulfur includes persulfide(RSSH),polysulfide(RSS.R,RSS,H,and HS,H,n?2),and elemental sulfur(S8).Sulfane sulfur and H2S often coexist inside cells,and it is increasingly accepted that sulfane sulfur,a product of H2S oxidation,is mainly responsible for protein persulfidation.Several methods have been developed to detect sulfane sulfur normally based on three theories.One of these sulfane sulfur detection methods baded on the reaction with a nucleophile or electrophile.The detection limit is generally high,and it is not sensitive enough for the detection of endogenous sulfane sulfur in live cells.The second method uses fluorescent probes,which are good for relative comparison and real-time monitoring,but not for quantification.The third method detects specific sulfane sulfur species,such as protein-bound sulfane sulfur from cells,by using liquid chromatography and mass spectrometry,which has high cost and unsuitable for the detection of total sulfane sulfur in biological samples.Therefore,the detection of total sulfane sulfur in biological samples is still a challenge.In order to solve above mentioned problems,a rapid and sensitive method was developed to investigate sulfane sulfur contents in biological samples,and the physiological functions of Rhods in sulfur metabolism were also studied.The main research contents are as follows1.A sensitive method to detect total sulfane sulfur in biological samples was developed.Sulfite in a solution containing 1%TritonX-100,50?M DTPA,50 mM Tris base(pH 9.5)reacted with unstable sulfane sulfur at 95? for 10 min to produce thiosulfate,which was derivatized with monobromobimane to form thiosulfate bimane.This reaction condition was also fit for cell disruption even with complex cell walls,further simplifying this detection method.Different sulfur compounds including elemental sulfur(Ss),inorganic polysulfide(H2Sn),tetratetrasulfate,persulfide(GSSH and Cys-SSH),organic polysulfide(Me2S3 and Tsp2S4)were used to check the specificity of the reaction.All tested sulfane sulfur species were quantitatively transformed into thiosulfate in the reaction buffer.Thus,all types of sulfane sulfur reacted with sulfite to quantitatively produce thiosulfate under the assay condition.A standard curve of thiosulfate revealed a detection limit of 200 nM.We further detected the sulfane sulfur contents according to growth stages in liquid media for bacteria and yeast.The sulfane sulfur contents increased in the early logarithmic growth phase,kept for a steady state in the middle to late of the logarithmic phase,and decreased sharply in the late logarithmic phase or early stationary phage.The sulfane sulfur contents in mammalian cells and zebrafish at various growth phases were also detected.We checked the sulfane sulfur content of mammalian cells in one single batch of the nutrient fed process.The sulfane sulfur contents increased in the early stage and decreased in the late stage.The sulfane sulfur contents of zebrafish reached the highest level in the sixth day right before hatching and decreased after growing for several weeks as adult fish.Further,we detected sulfane sulfur in different organs from arabidopsis,mouse and Chinese white shrimp.All samples had sulfane sulfur,but its concentrations varied from about 1 to 10 nmol/mg of weight or protein in different organs.Therefore,this method is suitable for the quantitative analysis of total sulfane sulfur in biological samples.2.The physiological functions of fused Rhod domain(DUF442)of SQR in C.pinatubonensis JMP134 during sulfide oxidationA heterogenous expression cassette in E.coli have been constracted to study the functions of DUF442 domain of CpSQR.It was showed that freely expressed DUF442 could accelerate GSSH and thiosulfate formation.Here we found the integrated DUF442 domain in the SQR-PDO pathway has new physiological functions in maintaining redox homeostasis and oxidative stress.We balanced the expression level of recombinant CpSQR and CpPDO to keep metabolic flow as the same as that in native C.pinatubonensis JMP134.Then,the eight native Rhod were deleted to generate the E.coli RHOD-8K strain.The integrated DUF442 domain decelerated the H2S oxidation catalyzed by CpSQR to avoid sulfane sulfur accumulation in E.coli 8K,and the integrated DUF442 domain could also protect GSH from persulfidation.The excessive GSSH reacts with GSH sponancely to form GSSG and H2S.The produced H2S was further oxidized by CpSQR with GSSG reduced by glutathione reductase.In this futile cycle,NADPH was consumed and GSSG would accumulate.The integrated DUF442 domain prevented this cycle and the waste of NADPH.Sulfite,as the direct product of PDO,speeded up sulfide oxidation rate in this pathway.Sulfite as an inorganic micromolecule got into the active center of fused DUF442 to react with the covalently bound S0 to produce thiosulfate.This reaction released the repression effect to the sulfide oxidation by CpSQR.The integrated DUF442 and sulfite could dynamically controlled the H2S oxidation rate and product generation.The fused DUF442 worked in a different manner from freely expressed DUF442 domain,though both forms could decrease the CpSQR activity and avoid sulfane sulfur formation.Freely DUF442 accelerated GSSH and thiosulfate formation in the assistant of CpPDO,and the function could be complemented by overexpression of GlpE and YgaP in E.coli 8K strain.These results indicated that the functions of freely expressed DUF442 to SQR-PDO pathway could be replaced by other RHODs with rhodanese activity.However,the fused DUF442 did not show any rhodanese activity and worked independent of CpPDO.In contrast with its free form,the fused DUF442 slowed the GSSH formation and protect GSH in its reduced form.In addition,removing DUF442 domain affects the stability and membrane location of CpSQR.3.The physiological functions of Rhods in E.coli.As model microorganism,E.coli has nine Rhods but not the SQR-PDO pathway.The role of Rhods in E.coli were explored by using the Rhod knocked-out mutants.Rhod knocked-out mutant assimilated thiosulfate normally as wild type when CysM present.When cysM deleted,Rhod played a synergistic role in the thiosulfate utilization,by transferring the sulfur sulfane from thiosulfate to GSH with the production of GSSH,which was further reduced by GSH to produce GSSG and H2S.Further,sulfite could be reduced by sulfite reductase(CysI and CysJ)to H2S.CysK,cysteine synthase,uses H2S to produce cysteine.In the absence of CysM,the expression of CysK in E.coli was upregulated.It could be an emergency mechanism when cells could not metabolize thiosulfate by CysM.Meanwhile,The Rhods also play a positive role in resisting oxidative pressure and osmotic pressure.