Toxic Effects of Nitric Oxide

1. Toxic Effects of Nitric Oxide By Martha Gutierrez and Malerie Mock

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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

8. 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)

9. 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

10. 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

11. 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.

12. The citric acid cycle is inhibited by NO production by macrophages.

13. HOONO Reaction with Methionine • HOONO + Methionine Methionine sulfoxide + HONO • HOONO is oxidized. • Methionine sulfoxide is not biologically active. Source: Pryor et al., 1994

14. 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

15. 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

16. 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

17. 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

18. 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.

19. 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.