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 radicals are toxic to cells because of their reactivity with DNA, RNA, lipids, and proteins. Free radical cytotoxicity causes: Free radical cytotoxicity causes: Damage to cell membranes Damage to cell membranes Disruption of cellular activities, such as cellular respiration and protein synthesis 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. 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. 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. 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. 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. 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). 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 reacts with superoxide inside a host cell to give a highly reactive intermediate product. NO  ONOO- (peroxynitrite) NO  ONOO- (peroxynitrite) ONOO-  ONOOH (peroxynitrous acid) ONOO-  ONOOH (peroxynitrous acid) ONOOH is very unstable and a highly reactive oxidizing compound. ONOOH is very unstable and a highly reactive oxidizing compound. ONOOH oxidizes nearby bio-molecules. ONOOH oxidizes nearby bio-molecules. Production of ONOOH is very rapid. 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. NO+ reacts with different organic side groups, especially thiols. Can also react with metals, lipids, and DNA. Can also react with metals, lipids, and DNA. NO+ reactivity will damage cell membranes and shut down cellular activities. 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. 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. NO+ reacts with thiols to yield S-nitroso compounds. Thiol containing peptides, amino acids, and proteins lose their biological activity. 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 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. 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. In phagocytes, the ability to kill microorganisms is attributed to phagocyte-derived RNS. (Fang, 2004) (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. 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. NO is an immune response to pathogenic bacteria. (Rothfork et al., 2004) (Rothfork et al., 2004)

Inhibition by NO NO inactivates key enzymes that play an important role in microbial growth or infection, such as: NO inactivates key enzymes that play an important role in microbial growth or infection, such as: Terminal respiratory oxidases Terminal respiratory oxidases Iron/sulfur protein aconitase Iron/sulfur protein aconitase NO reacts with Methionine to make it biologically inactive. NO reacts with Methionine to make it biologically inactive. Source: Ouellet et al., 2002

Terminal Respiratory Oxidase Terminal respiratory oxidase produces water molecules. Terminal respiratory oxidase produces water molecules. If this enzyme is deactivated, then cell growth would be greatly reduced. 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. 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. If inhibited by NO, the bacteria cannot produce ATP for energy.

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

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. Inactivation of AIP is accomplished by the oxidation of the C-terminal methionine in the autoinducing peptide by ONOOH. Summary: Summary: OHOOH produced by macrophages  Inactivates AIP from S. aureus  Staphylococcal toxin production is not activated  Decreased S. aureus virulence 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. 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. E. coli uses NO reductase and flavohemoglobin, which plays a big role in RNS detoxification. Flavohemoglobins are enzymes that bind oxygen and reduce NO Flavohemoglobins are enzymes that bind oxygen and reduce NO NO  N2O NO  N2O NO  NO3 NO  NO3 Flavohemoglobins successfully render NO free radicals un-reactive. 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. 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. E. coli has evolved a mechanism to detoxify and protect against NO. NO is extremely toxic to cells because it disrupts many cellular activities. NO is extremely toxic to cells because it disrupts many cellular activities.

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