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Cell Signalling Biology Michael J. Berridge Module 2 Cell Signalling Pathways 2 67

Module 2: Figure NO synthase mechanism

Reductase domain

O
2
Oxidase domain CAM
Arginine
Oxidase domain
Haem BH 4
CAM
FMN FAD NADPH
Reductase domain

Ca 2+ S 1177

Citrulline NO

Nitric oxide synthase (NOS) reaction mechanism.
The two nitric oxide synthase (NOS) monomers are lined up alongside each other so that the reductase domain of one functions together with
the oxidase domain of its neighbour. The enzyme dimer also functions as a scaffold to organize the other components of the reaction mechanism
such as the bound cofactors [flavin--adenine dinucleotide (FAD), flavin adenine mononucleotide (FMN), haem and tetrahydrobiopterin (BH 4 )] and
the tightly bound prosthetic group calmodulin (CaM). The formation of NO is driven by an NADPH-dependent electron flux that passes from the
reductase towards the oxidase domain. The attached haem is the terminal electron acceptor, which binds the oxygen that is inserted into arginine to
form the hydroxyarginine that decays to release NO. One of the important regulators of NOS is calmodulin, which is constitutively active in inducible
NOS (iNOS), but requires an elevation of Ca 2 + for both neuronal NOS (nNOS) and endothelial NOS (eNOS). One consequence of increasing the
concentration of Ca 2 + in cells is therefore to increase the formation of NO.

reactive nitrogen species (RNS) signalling pathways, NO synthetic reaction mechanism
whereby the NO alters the activity of a variety of protein The different nitric oxide synthase (NOS) enzymes func-
targets through a nitrosylation reaction. This diverse tion as homodimers, which are arranged in a head-to-head
NO/cyclic GMP signalling pathway operates to control orientation with the N-terminal oxidase domain of one
the following cellular processes: monomer lined up alongside the C-terminal reductase do-
• NO/cyclic GMP and smooth muscle relaxation main of its neighbour. The substrates for the enzymatic
• NO/cyclic GMP and synaptic plasticity reaction mechanism are L-arginine, oxygen and NADPH,
• NO/cyclic GMP and cardiac hypertrophy which combine to form citrulline with the liberation of
NO (Module 2: Figure NO synthase mechanism). NOS
NO synthesis regulation is complicated because each isoform appears to
Nitric oxide (NO) synthesis is carried out by NO syn- be regulated by different mechanisms.
thase (NOS), of which there are three isoforms named
either after the tissues where they were first discovered,
i.e. neuronal nitric oxide synthase (nNOS) and endothelial Endothelial nitric oxide synthase (eNOS)
nitric oxide synthase (eNOS) or by the way in which they As its name implies, endothelial nitric oxide synthase
are controlled, i.e. inducible nitric oxide synthase (iNOS) (eNOS) was first described in endothelial cells, where it
(Module 2: Figure NO and cyclic GMP signalling). The generates NO in response either to agonists such as acet-
expression of these enzymes is not as restricted as their ylcholine and bradykinin that elevate Ca 2 + or to blood
names imply, but are widely expressed and can coexist in flow-induced shear stress. It is now evident that eNOS is
many cell types. Even though these isoforms are regulated expressed in many different cell types (lung epithelial cells,
differently and have different cellular locations, they all blood platelets, cardiac myocytes and hippocampal neur-
seem to use the same NO synthetic reaction mechanism. ons). It has a complex regulation, which is very dependent
The excessive production of NO can have patholo- on its attachment to caveolin, one of the proteins in cave-
gical consequences and has been linked to various disease olae (Module 6: Figure caveolae molecular organization),
states such as Huntington’s disease, Alzheimer’s disease where it contributes to their signalling function. One of
and hypertension. the key regulators of eNOS is Ca 2 + , which acts through

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