Nitric Oxide Synthesis

Nitric oxide is produced by a group of enzymes called nitric oxide synthases. These enzymes convert arginine into citrulline, producing NO in the process. Oxygen and NADPH are necessary co-factors. There are three isoforms of nitric oxide synthase (NOS) named according to their activity or the tissue type in which they were first described. The isoforms of NOS are neuronal NOS (or nNOS), endothelial NOS (or eNOS) and inducible NOS (or iNOS). These enzymes are also sometimes referred to by number, so that nNOS is known as NOS1, iNOS is known as NOS2 and eNOS is NOS3. Despite the names of these enzymes, all three isoforms can be found in a variety of tissues and cell types. The general mechanism of NO production from NOS is illustrated below.


Two of the enzymes (nNOS and eNOS) are constitutively expressed in mammalian cells and synthesise NO in response to increases in intracellular calcium levels. In some cases, however, they are able to increase NO production independently of calcium levels in response to stimuli such as shear stress.

iNOS activity is independent of the level of calcium in the cell, however its activity - like all of the NOS isoforms - is dependent on the binding of calmodulin. Increases in cellular calcium lead to increases in levels of calmodulin and the increased binding of calmodulin to eNOS and nNOS leads to a transient increase in NO production by these enzymes. By contrast iNOS is able to bind tightly to calmodulin even at very low cellular concentration of calcium. Consequently iNOS activity doesn't respond to changes in calcium levels in the cell. As a result the production of NO by iNOS lasts much longer than from the other isoforms of NOS, and tends to produce much higher concentrations of NO in the cell.

The production of NO by iNOS can, however, be controlled through transcription. In most cell types iNOS protein levels are either very low or undetectable. However, stimulation of these cells with, for example, cytokines or growth factors, can lead to increased transcription of the iNOS gene, with subsequent production of NO.

The general structure of the NOS enzymes is illustrated below.



The functional NOS protein is a dimer formed of two identical sub-units. There are three distinct domains in each NOS sub-unit: a reductase domain, a calmodulin-binding domain and an oxygenase domain.

1) The reductase domain: This domain contains the FAD and FMN moieties and it acts to transfer electrons from NADPH to the oxygenase domain. It should be noted that the reductase domain transfers electrons to the oxygenase domain of the opposite sub-unit of the dimer, and not to the domain on the same sub-unit.

2) Calmodulin binding: The binding of calmodulin is required for the activity of all the NOS isoforms. It detects changes in intracellular calcium levels, although its precise function is slightly different in each of the three isoforms.

3) The oxygenase domain: This domain contains the binding sites for tetrahydrobiopterin, haem (heme) and arginine. The oxygenase domain catalyses the conversion of arginine into citrulline and NO.

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