First 11 pages of thesis. - OPUS - Universität Würzburg

of NADPH, FAD and FMN, whereas the oxygenase domain binds heme, BH4
and the substrate L-arginine. As shown in figure 4, between these two domains
lies the calmodulin binding site, which plays an important role in both structure
and function of the enzyme.
The biosynthesis of nitric oxide involves a two step oxidation reaction and
consumes 1.5 mol of NADPH and 2 mol of oxygen including the formation of the
intermediate product, N G -hydroxy-L-arginine. The reductase domain transfers
the electrons from NADPH via the flavins: FAD and FMN to the heme molecule
in the oxygenase domain, where the substrate L-arginine is oxidized to L-
citrulline and nitric oxide. Hence, the two domains perform catalytically distinct
functions. Despite the fact that each monomer consists of both domains,
dimerisation of the enzyme is essential for its catalytic activity since the
electrons are transferred from the flavins in the reductase domain of one
subunit to the heme centre in the oxygenase domain of the second subunit 88
(Figure 4). Heme plays a key role in dimerisation of both the subunits in all three
NOS isoforms and is also required for the interaction between the reductase
and oxygenase domains. Calcium dependence is the key feature that
distinguishes constitutive and inducible isoforms of NOS. eNOS and nNOS are
activated by elevation of intracellular calcium levels, followed by subsequent
binding of calcium/ calmodulin. In contrast, iNOS contains irreversibly bound
calmodulin and thus its activation is independent of intracellular calcium
concentration.
Under conditions of either substrate L-arginine or cofactor BH4
deficiency, all the isoforms of NOS can “uncouple”. The term “uncoupling”
defines a situation during which the electrons flowing from the reductase
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