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oxidative injury. High levels of NO disrupt DNA and cause both genotoxic and cytotoxic
damage. If there are changes in the pH, in the thiol content or in the redox state, this can result
in increased damaging and carcinogenic potential. Of interest is the reaction of NO with
amino acids such as tyrosine, which in itself is now used as a marker in several inflammatory
diseases.
Finally, there is now discussion that the original EDRF concept is made up of the actions of
NO and the actions of some of the post NO production of other compounds, in particular the
s-nitrosothiols (Myers, Minor et al. 1990; Vanin l99l; Jia, Bonaventura et al. 1996; Liu,
Miller et al. 1998).
3.4
Nitric oxide synthase isoenzymes
The nitric oxide synthases are large complex proteins that unusually contain both oxidative
and reductive domains. They have homology with cytochrome P+so which is also unique
(Bredt, Hwang et al. l99l; Vallance and Moncada 1994). They are synthases rather than
synthetases as they do not use adenosine triphosphate (ATP) in their reactions (Knowles and
Moncada 1994). NOS needs the substrate (L-arginine), the co-substrates (O2 and NADpI{)
and the co-factors (8H4, FAD, FMN) (see Chapter 3.2 above). The reaction is a five electron
mono-oxygenate oxidation pathway which involves two separate mono-oxygenation steps in
sequence (see Figure 3.2, Knowles, 1994 #712). In step one N*-hydroxyarginine is an
intermediate species formed (Stuehr, Kwon et al. l99l) in the first reaction by aquiring one
02 molecule and one NADPH molecule (Kwon, Nathan et al. 1990) and the presence of BII+
(Kwon, Nathan et al. 1989; Tayeh and Marletta 1989). The second step is the oxidation of N*-
hydroxyarginine to form citrulline and *NO. Flavin coenzymes are involved in the transfer of
electrons to form a reduced oxygen species (Stuehr, Cho et al. 1991; Stuehr, Fasehun et al.
1991). There are consensus binding sites for FAD, FMN and NADPH located in the carboxyl
terminal portion of the NOS protein, and also a consensus binding site for calmodulin. The
other co-factor BFIa binds to NO on a 1:1 stoichiometry basis and has a redox role in enzyme
activity (Hevel and Marletta 1992; Knowles and Moncada 1994). All three isoenzymes are
phosphorylated (Nathan and Xie 1994).
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