NO synthesis
Nitric Oxide

Author: Francesca Bar
Date: 28/10/2007


NO-Related species

Although NO is a radical, it lacks the reactivity normally inherent to other radicals.
This makes NO fairly innocuous to cells, but key chemical reactions can lead to the production of more reactive species, potentially more toxic than NO itself.

Biologically significant NO redox and additive reactions include those with oxygen in its various redox forms and with transition metals.

1. Oxidation by O2 (aerobic decomposition) alone can lead to numerous nitrogen oxide species existing simultaneously in aqueous solution, irrespective of redox reactions with other biochemical components.
Such NO-related species (NOx) include: NO, •OONO, NO2, (NO)2, N2O3, N2O4, NO2-and NO3-

2. In turn, NO can undergo radical-radical interactions with other oxygen- and nitrogen-centered radicals. The former includes its near-diffusion controlled reaction:
• with O2•– (k=1.9×1010 M– 1s– 1) to yield peroxynitrite (ONOO–)
• with the hydroxyl radical (HO•) (k=1×1010 M– 1s– 1) to yield HNO2
• with the peroxyl radical (ROO•) (k=2×109 M– 1s– 1) to yield ROONO

3. The latter, nitrogen-centered radicals, include a reaction of NO:
• with NO2 (k=2×109 M– 1s– 1) to yield N2O3;
• with ONOO– to yield nitrosating species.

The nature of NOx can thus be significantly altered by the presence of other oxyradicals, which are generally ubiquitous and highly diffusible in the cytosol.

Alternatively, the fate of NO can be shifted if it is produced by NOS in close proximity to sources of O2•– or H2O2 (e.g. NADPH oxidase and/or iNOS). The contribution of O2•– and H2O2 by the NOS enzymes is particularly relevant under conditions of substrate and co-factor limitation.

The ability of NO to bind or react with transition metals or metal-containing proteins may be of the most biological significance with respect to NO and cell signalling pathways. The classic example is the role of NO in cGMP-dependent signaling pathways where the activation of guanylate cyclase arises through the ligation of NO to the iron heme of the enzyme. NO is capable of binding to both the ferric (FeIII) and ferrous (FeII) oxidation states of iron.

• The reaction with FeIII is reversible, catalyzing a process called reductive nitrosation: the reduction of the metal by NO leads to the formation of bound NO+, the nitrosonium ion. Reduction of non-heme transition metals has also been observed, including iron-sulfur centers in proteins (e.g. components of the mitochondrial respiratory chain and other mitochondrial enzymes).

• NO is also a high affinity ligand for FeII, forming a stable nitrosyl complex in competition with O2. Free FeII ions or FeII-containing proteins can thus reduce NO to the nitroxyl anion (NO–), which can oxidize sulfhydryl (thiol) groups. Oxymyoglobin and haemoglobin are important NO scavengers in this regard.

Ultimately, cellular NO chemistry has the potential to generate steady-state concentrations of a variety of NOx species that are in dynamic equilibrium.
In particular, NO+ is a redox species of NO with potential for regulation of cell signaling pathways.

NO+ can undergo addition or substitution reactions with nucleophiles, resulting in the nitrosation of -S, -N, -O and –C centers.
However, under physiological conditions, the direct oxidation of NO to NO+ is very unlikely, and NO+ reacts rapidly with H2O. Rather NO+ carrier species, such as N2O3 and metal-nitrosyl complexes, transfer NO+ to nucleophilic centers through bimolecular nitrosation reactions. The latter reactions create a series of new NO+ donors, such as nitrosamines and nitrosothiols, which can then participate in further trans-nitrosation reactions.


The propensity for S-nitrosothiol formation, through the S-nitrosation of free or protein thiols, seems to have the most biological significance.
S-nitrosothiols act as a bioactive pool serving as a source and sink of NO, buffering free NO. S-nitrosothiols are relatively stable, prolonging the half-life of NO and protecting against generation of more toxic NOx species. Furthermore S-nitrosation of proteins occurs favorably under physiological conditions and is reversible, capable of trans-nitrosation reactions: two criteria that point to S-nitrosation as a potential cellular regulatory mechanism.

It can be seen that the formation of nitrosating species and the process of S-nitrosation show a sensitive dependence on:
• the O2 tension
pH, redox state
• the transition metal content of the local microenvironment.

The cellular redox state is the net balance between the oxidative and reductive potentials within a cell.
The steady-state levels of oxygen- and nitrogen-centered radicals are a key contributor to the oxidative potential, whereas intracellular GSH is a major determinant of the reductive potential.


Mitochondria are the main generators of cellular H2O2. Superoxide anion, the stoichiometric precursor of H2O2, is formed:
• predominantly by ubisemiquinone auto-oxidation during electron transfer
• secondarily, as a by-product of NADH-dehydrogenase (Complex I) activity.
Once generated, O2•– is vectorially released into the matrix where it undergoes disproportionation to H2O2 by Mn-SOD. H2O2 that escapes matrix glutathione peroxidase activity can freely diffuse from the mitochondria to the cytosol, contributing to the cellular [H2O2]ss and redox state.


Clinton S. Boyd and Enrique Cadenas (2002) Biol. Chem. Vol. 383, pp 411-423

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