The Production And Physiological Function Of Hydrogen Sulfide And Reactive Sulfane Sulfur In E.coli MG1655

Sulfur,which symbol is S,is abundant,multivalent and nonmetallic.Under normal conditions,sulfur atoms form cyclic octatomic molecules with a chemical formula S8.Elemental sulfur is a bright yellow,crystalline solid at room temperature.Sulfur is the fifth most common element on Earth and the tenth most common element in the universe.Sulfur on Earth is usually present in the form of sulfides and sulfate minerals,and could be used to make matches,insecticides and fungicides.In addition,sulfur is one of the core chemical elements needed for biochemical functioning and is an elemental macronutrient for all living organisms.In recent years,hydrogen sulfide(H2S)is considered to be the third type of gas signal molecule after NO and CO,which can participate in physiological functions such as signal transmission in cells.Appropriate concentrations of H2S can be involved in the regulation of vasodilation or contraction,anti-inflammatory and ant-oxidation,and even inhibit apoptosis.However,high concentrations of H2S can also inhibit the metabolism of the cells.In studies on mammalian cells,H2S can inhibit the respiratory chain and interfere with the normal function of the animal respiratory system and even the central nervous system.It can also inhibit the function of cytochrome c and affect the normal metabolism of organisms.Unfortunately,the direct involvement of H2S in protein sulfhydration has been questioned,as it does not have the ability to directly react with a thiol group(-SH).On the other hand,reactive sulfane sulfur(RSSH,RSSnH,n ?1;HSSnH and RS(S)nSR;n ?1),which does not include the insoluble elemental sulfur,can react with a protein thiol to generate protein-SSH.H2S and reactive sulfane sulfur often coexist,making it difficult to precisely distinguish their production and function.On the one hand,H2S can lead to the production of reactive sulfane sulfur via chemically reacting with a disulfide bond and a sulfenic acid or via oxidation by sulfide:quinone oxidoreductases(SQR)and flavocytochrome c-sulfide dehydrogenases(FCSD).On the other hand,reactive sulfane sulfur can be reduced by thiols to product H2S,especially in a cellular environment containing high concentrations of cellular thiols,such as GSH,thioredoxin and glutaredoxin.Three H2S generating enzymes have been characterized in mammals:cystathionine b-synthase(CBS),cystathionine g-lyase(CSE).and 3-mercaptopyruvate sulfurtransferase(MST).Reactive sulfane sulfur can also be generated from sulfur-containing amino acids by several enzymes.While recently reports suggested that CBS and CSE can also produce CysSSH from cystine(Cys-SS-Cys).Therefore,sometimes the effect of reactive sulfane sulfur was attributed to H2S,due to H2S is more easily observed and detected.In order to investigate the production and function of H2S and reactive sulfane sulfur in vivo,a series of studies were carried out on the model strain E.coli MG1655.First,we used E.coli MG 1655 resting cells to metabolize different sulfur compounds.The results showed that L-cysteine is the optimal metabolic substrate for endogenous biogenesis of H2S and reactive sulfane sulfur in E.coli MG 1655.High concentrations of L-cysteine are also toxic to cells,so their intracellular concentrations are tightly controlled.The concentration of L-cysteine in cells is generally around 200 ?M.In most cells,L-cysteine is stored in the form of GSH,which is up to 10 mM.Studies have shown that E.coli MG 1655 can use the cysteine desulfhydrases(CDs,including TnaA,MetC,MalY,CysK,CysM and YhaM)pathway and L-cysteine aminotransferase associated with 3-mercaptopyruvate sulfurtransferase(CAT/M ST)pathway metabolizes L-cysteine to produce H2S.We compared the importance of CDs and CAT/MST pathways on L-cysteine degradation under different culture conditions.We found that E.coli had different detoxification strategies for high concentrations of L-cysteine under different nutrient conditions.Under nutrient-rich conditions.E.coli mainly controls the intracellular concentration of L-cysteine through the amino acid degrading protein TnaA.Under minimal medium conditions,E.coli accelerates L-cysteine mainly conversion to other amino acids reduces its concentration through the CAT/MST pathway.We also tried to obtain E.coli that did not accumulate H2S by knocking out the L-cysteine metabolic gene,but not succeed.Indicating that E.coli has a complex H2S accumulation mechanism.After knocking out the relevant L-cysteine metabolism gene,we obtained strain E.coli ?cysK with significantly increased H2S accumulation,strain E.coli AcysM with significantly reduced H2S accumulation,and strain E.coli AsseA.CysK is the main pathway for the synthesis of L-cysteine by H2S,and its deletion leads to an increase in the accumulation of H2S.Interestingly,the deletion of the CysM pathway reduces the accumulation of H2S.and other L-cysteine degradation pathways have limited effects on H2S accumulation in E.coliWe compared the differences in the mechanism by which CDs and CAT/MST pathways metabolize L-cysteine to produce H2S.It was found that H2S production can be detected in both the CDs and CAT/MST pathways for the metabolism of L-cysteine,but the catalytic mechanisms of the two pathway are not the same.CDs convert L-cysteine to H2S,while the CAT/MST pathway produces reactive sulfane sulfur,due to different catalytic mechanisms.CDs catalyze L-cysteine metabolism through a mechanism of ?,?-elimination,?-replacement,or ?-hydrogen exchange with the formation of a double-bond intermediate,leading to co-elimination of H2S and NH3.In the CDs catalyzed reaction,the sulfur with a valence of-2 does not undergo oxidoreduction.In the CAT/MST pathway,MST metabolizes MP to pyruvate and produces sulfane sulfur,which is initially in the form of a persulfide as the active site;MST can pass the sulfane sulfur to GSH to produce GSSH.Although the produced sulfane sulfur can be reduced to H2S by thioredoxin and glutaredoxin inside cells,the CAT/MST pathway did not contribute to H2S production during normal growth of E.coli.From the catalytic mechanism,MST and SQR are more similar.The catalytic centers of MST and SQR are both cysteine residues.When catalyzing the corresponding substrate,cysteine persulfide can be formed in the active center.They can also transfer the reactive sulfane sulfur on them to the appropriate acceptor to form a wide variety of reactive sulfane sulfur containing compounds.Therefore,we believe that MST should be classified as reactive sulfane sulfide producing enzyme,but not an H2S producing enzyme,like SQR.In addition,in vitro experiments have shown that in the absence of other sulfur receptors,MST can catalyze the production of a certain amount of S8 by MP.However,when containing other small molecule thiol substances(RSH),a series of RSSnH is generated without accumulation of S8.Under physiological conditions.E.coli MG1655 is rich in thiol substances that can act as reactive sulfane sulfur receptors,so it does not have the objective conditions for the production of S8.In vitro enzymatic experiments and whole-cell metabolic L-cysteine experiments show that high concentrations of ?-KG inhibited CAT activity and reduced the rate of degradation of L-cysteine by the CAT/MST pathway.As a coenzyme,PLP did not exhibit a high concentration inhibitory effect on the CAT/MST pathway.Although the deletion pathway of CAT/MST does not affect the accumulation of E.coli H2S,the intracellular content of reactive sulfane sulfur per mg of protein in the supernatant of E.coli ?sseA lysate was clearly reduced.To assess the importance of MST,we evaluated the conservation and distribution of MST in bacteria.Our result showed that 48.2%the sequenced genomes contained MST,which is significantly more than the 20.6%for SQR.97%of the sequenced genomes had only one MST;most of MSTs(89.2%)were distributed in six classes:Gammaproteobacteria(45.4%),Betaproteobacteria(18.3%),Alphaproteobacteria(12.3%),Bacilli(7.3%),Corynebacteriales(6.0%),and Streptomycetales(1.9%).The phylogenetic tree of MSTs suggests that they are highly heterogeneous,but clearly separated from rhodaneses that are also highly heterogeneous.In the study,we obtained E.coli ?cysK with significantly increased H,S accumulation,E.coli ?cysM with significantly reduced H2S accumulation,and E.ccoli?sseA with no significant change in H2S accumulation.These strains provide research materials for studying the possible functions of H2S and reactive sulfane sulfur under physiological conditions.Our experimental results indicate that CysK and CysM are a cooperative and competition in the process of synthesizing L-cysteine in E.coli.Both CysK and CysM can synthesize L-cysteine by using H2S,so they have a certain competitive relationship in substrate utilization.In the synthesis of L-cysteine,the two synergistically maintain the intracellular L-cysteine content stable,and the deletion of either on of them does not significantly because the decrease of intracellular L-cysteine,but when both deletions result in a significant decrease in the intracellular L-cysteine content and even inhibit bacterial growth.However,E.coli ?cysK and E.coli AcysM show opposite results in the phenotype of H2S accumulation change.CysK is the main pathway for the synthesis of L-cysteine by using H2S.CysM is its complementary pathway.It is unresolved whether H2S or reactive sulfane sulfur is mainly responsible for protein sulfhydration and resistance to oxidative stress in live cells.Compared with H2S,reactive sulfane sulfur has better reducing ability and reacts more rapidly with H2O2 under certain conditions.Studies have shown that the antioxidant capacity of small molecule polysulfide(GSSH,Cys-SSH)is 10-100 times that of its sulfhydryl small molecule.The changes in H2S accumulation of E.coli AcysK and E.coli AcysM did not affect the antioxidant capacity of the strain.The E.coli ?sseA strain contained a significant decrease in the reactive sulfane sulfur content per mg of protein come with significantly reduced antioxidant capacity.The SQR which can oxidize H2S to form reactive sulfane sulfur was converted into E.coli,and the antioxidant capacity of the E.coli(CpSQR)strain is remarkably improved.These results indicate that reactive sulfane sulfur plays a pivotal role in the antioxidant process.In the process of GSSH and H2O2 reaction.we first discovered the formation of sulfite and thiosulfate.Based on this,we proposed a new antioxidant mechanism of reactive sulfane sulfur.In addition,by detecting the growth of strains with change of H2S accumulation under antibiotic stress.our results do not support H-2S as a universal defense against antibiotics in bacteria.By comparing the difference in the production and function of H2S and reactive sulfane sulfur in E.coli,the importance of reactive sulfane sulfur in anti-oxidation was clarified,and the antioxidant mechanism of reactive sulfane sulfur was proposed.Our findings provide a basis for further investigation of the cellular functions of reactive sulfane sulfur.