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Toxic Effects of Nitric Oxide. By Martha Gutierrez and Malerie Mock. Free Radical Toxicity. Free radicals are toxic to cells because of their reactivity with DNA, RNA, lipids, and proteins. Free radical cytotoxicity causes: Damage to cell membranes
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Toxic Effects of Nitric Oxide By Martha Gutierrez and Malerie Mock
Free Radical Toxicity • Free radicals are toxic to cells because of their reactivity with DNA, RNA, lipids, and proteins. • Free radical cytotoxicity causes: • Damage to cell membranes • Disruption of cellular activities, such as cellular respiration and protein synthesis
Host Defense and Toxicity • Human phagocytic cells produce free radicals to inhibit infectious bacteria. • Free radicals in high concentrations will exert toxic effects on all cells, including the cells that are producing the free radicals. Source: Fang, 2004
Nitric Oxide (NO) • NO is a free radical that is employed by macrophages for defense. • The toxicity of NO is attributed to its ability to bind to proteins that contain heme, iron, or copper, which will result in protein disruption. • In reaction with a protein, NO can either be oxidized (lose electrons) or reduced (gain electrons). This ability is responsible for the high reactivity of free radicals. • NO producing species are known as reactive nitrogen species (RNS). Source: Winstedt and Wachenfeldt, 2000
Reaction of NO Inside Host Cell • NO reacts with superoxide inside a host cell to give a highly reactive intermediate product. • NO ONOO- (peroxynitrite) • ONOO- ONOOH (peroxynitrous acid) • ONOOH is very unstable and a highly reactive oxidizing compound. • ONOOH oxidizes nearby bio-molecules. • Production of ONOOH is very rapid. Source: Rogstam et al., 2007
Reaction of Nitrosonium Ion (NO+) • NO+ reacts with different organic side groups, especially thiols. • Can also react with metals, lipids, and DNA. • NO+ reactivity will damage cell membranes and shut down cellular activities. • As a result of the high reactivity with a wide range of cellular components, NO+ is very toxic to cells. Source: Rogstam et al., 2007
NO+ Reaction • NO+ reacts with thiols to yield S-nitroso compounds. • Thiol containing peptides, amino acids, and proteins lose their biological activity. • Cysteine contains a thiol group, and two cysteine residues form a disulfide bond. • Cysteine thiol group replaced with S-nitroso disulfide bonds cannot be formed tertiary or quaternary protein structure is disrupted. Source: Rogstam et al., 2007
NO Production by Phagocytes • In phagocytes, the ability to kill microorganisms is attributed to phagocyte-derived RNS. • (Fang, 2004) • In the cytoplasm, inducible NO synthase (iNOS) is a protein responsible for NO production in macrophages. Lipopolysaccharide production by bacteria stimulates NO production. • NO is an immune response to pathogenic bacteria. • (Rothfork et al., 2004)
Inhibition by NO • NO inactivates key enzymes that play an important role in microbial growth or infection, such as: • Terminal respiratory oxidases • Iron/sulfur protein aconitase • NO reacts with Methionine to make it biologically inactive. Source: Ouellet et al., 2002
Terminal Respiratory Oxidase • Terminal respiratory oxidase produces water molecules. • If this enzyme is deactivated, then cell growth would be greatly reduced. Source: Ouellet et al., 2002
Iron-sulfur protein aconitase • Aconitase is the enzyme that is responsible for the isomerization of citrate to isocitrate in the citric acid cycle. • If inhibited by NO, the bacteria cannot produce ATP for energy.
HOONO Reaction with Methionine • HOONO + Methionine Methionine sulfoxide + HONO • HOONO is oxidized. • Methionine sulfoxide is not biologically active. Source: Pryor et al., 1994
Inhibition of Staphylococcus aureus • Auto inducing peptides (AIP) are secreted in large numbers by S. aureus. • Under high AIP conditions, the bacteria upregulates the production of toxins. • ONOOH inhibits AIP biologic function. Source: Rothfork et al., 2004
Inhibition of Staphylococcus aureus • Inactivation of AIP is accomplished by the oxidation of the C-terminal methionine in the autoinducing peptide by ONOOH. • Summary: • OHOOH produced by macrophages Inactivates AIP from S. aureus Staphylococcal toxin production is not activated Decreased S. aureus virulence Source: Rothfork et al., 2004
How Does Bacteria Respond to NO Toxicity? • Bacteria respond to NO toxicity by activating genes that encode for proteins that will detoxify NO, repair damage, and maintain homeostasis. Source: Justino et al., 2005
Escherichia coli Response to NO Toxicity • E. coli uses NO reductase and flavohemoglobin, which plays a big role in RNS detoxification. • Flavohemoglobins are enzymes that bind oxygen and reduce NO • NO N2O • NO NO3 • Flavohemoglobins successfully render NO free radicals un-reactive. Source: Justino et al., 2005
Conclusion • In macrophages, free radical production is important in innate immunity against numerous infections, such as S. aureus. • E. coli has evolved a mechanism to detoxify and protect against NO. • NO is extremely toxic to cells because it disrupts many cellular activities.
Works Cited Fang, F. C. 2004. Antimicrobial reactive oxygen and nitrogen species: concepts and controversies. Nature Reviews Microbiology2: 820-832. Justino, M., Vicente, J., Teixeira, M., and Saraiva, L. 2005. New Genes Implicated in the Protection of Anaerobically Grown Escherichia coli Against Nitric Oxide. Journal of Biological Chemistry 280: 2636-2643. Ouellet, H., Y. Ouellet, C. Richard, M. Labarre, B. Wittenberg, J. Wittenberg, and M. Guertin. 2002. Truncated hemoglobin HbN protects Mycobacterium bovis from nitric oxide. Proc. Natl. Acad. Sci. USA 99: 5902-5907. Pryor, W., Jin, X., and Squadrito, G. 1994. One and Two Electron Oxidations of Methionine by Peroxynitrite. PNAS 91: 11173-11177. Rogstam, A., Larsson, J. T., Kjelgaard, P., and von Washenfeldt, C. 2007. Mechanism of Adaptation to Nitrosative Stress in Bacillus subtilis. Journal of Bacteriology 189: 3063-3071. Rothfork, J., Timmins, G., Harris, M., Chen, X., Lusis, A., Otto, M., Ceung, A., and Gresham, H. 2004. Inactivation of Bacterial Virulence Pheromone by Phagocyte-Derived Oxidants: New Role for the NADPH Oxidase in Host Defense. PNAS 101: 13867-13872. Winstedt, L. and Wachenfeldt, C. 2000. Terminal Oxidases of Bacillus subtilis strain 168: One Quinol Oxidase, Cytochrome aa3 or Cytochrome bd, Is Required for Aerobic Growth. Journal of Bacteriology 182: 6557-6564.