Studies On The Mechanism Of Nitrite And Nitric Oxide Affecting The Content Of Cytochrome C In Shewanella Oneidensis

Cytochromes c(cyts c),ubiquitous heme-containing proteins present in all domains of life,are essential for many biological processes,such as respiration and photosynthesis.This group of proteins are characterized by the covalent bonds between apocyt c and heme.With the help of cyts c,bacteria can respire on a variety of non-oxygen electron acceptors,among which Shewanella oneidensis is renowned for its respiratory diversity,encoding at least 42 different cyts c.The process of covalent binding of apocyt c and heme is called maturation of cyt c.In many bacteria,both apocyt c and heme are synthesized intracellularly and transported to the periplasmic space,where the covalent linkage is catalyzed by the cyt c maturation system(Ccm).As important nitrogen species in nature,both nitrite and NO are bacteriostatic agents.Because nitrite can be spontaneously transformed to nitric oxide(NO)under reducing conditions,and enzymes that convert nitrite into NO are diverse in living organisms,the functional mechanism of nitrite has long been considered to be via NO.However,previous studies of this group have recently revealed that the cellular targets of nitrite and NO in S.oneidensis differ substantially.This dissertation moves a step further into the cellular targets of nitrite and NO in S.oneidensis.The research starts from the phenomenon that both nitrite and NO compromise the content of cyt c,and focuses on how these two nitrogen oxides impact the content of cyt c.By systematically analyzing the data,multiple hypotheses are brought up: damaging cyt c directly,repressing transcription of the gene encoding cyt c,impairing the intracellular level of heme,and affecting the maturation of cyt c.All of these hypotheses are explored and the results are summarized as following:1.Nitrite and NO only affect the cyt c content of living cells.When the cells die or stop growing,the inhibitory effect of Nitrite/NO on cyt c content disappears.Although modification of cyt c by NO at high concentrations is observed,neither nitrite nor NO impairs cyt c at physiological concentrations.2.Nitrite and NO down-regulate the activity of c AMP-CRP complex by reducing the level of c AMP,thereby affecting the expression of a variety of cyt c regulated by Crp.However,exogenous addition of c AMP cannot fully eliminate the effect of nitrite and NO on the content of cyt c.We managed to eliminate the influence of the c AMP-CRP regulatory system on the cyt c content by expressing a c AMP-independent Crp mutant,and found that nitrite and NO still had a repressing effect on cyt c levels.3.Cyt c is composed of apocyt c and heme,and the decrease in cellular heme levels will lead to reduction in the cyt c content.The data showed that neither nitrite nor NO had significant effect on the expression of key enzymes in the heme synthesis pathway and the intracellular heme level.Interestingly,increasing the level of heme can alleviate the decrease of cyt c content caused by nitrite/NO,suggesting that some biological processes involving heme in cyt c synthesis may be influenced by nitrite and NO.4.S.oneidensis has a Ccm system containing 9 proteins,Ccm ABCDEFGHI.We overexpressed all individual components of the Ccm system in S.oneidensis wild type in different combination and examined the effect of nitrite/NO on cyt c content.The results showed that the compromise of nitrite/NO on cyt c content could be alleviated only by overexpression of heme transporter CcmE.5.By analyzing the UV-Vis spectra of CcmE before and after nitrite/NO treatment,we concluded that nitrite/NO can nitrosylate heme on CcmE and damage its activity.It is worth mentioning that overexpression of CcmE proteins from Escherichia coli and Pseudomonas aeruginosa in S.oneidensis also had a similar effect,suggesting that this phenomenon is general in bacteria.In summary,this study identifies CcmE a common target of nitrite and NO,and reveals that nitrite and NO affect the maturation of cyt c by interfering with the ability of CcmE.As a result,heme transfer is compromised,leading to decrease in the overall cyt c content,which eventually negatively impacts diverse physiological pathways,including anaerobic respiration capacity.This study provides new insights into the mechanism of antibacterial mechanisms of nitrite and NO.