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.
The citric acid cycle is inhibited by NO production by macrophages.
HOONO Reaction with Methionine

HOONO + Methionine
sulfoxide + HONO

HOONO is oxidized.

Methionine sulfoxide is not biologically active.
Source: Pryor et al., 1994
Methionine
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 Microbiology 2: 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 PhagocyteDerived 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.