L-Arginine-derived nitric oxide (NO) acts as an inter- and intra-cellular signal molecule in many mammalian tissues including brain, where it is formed by a flavin-containing Ca2+/calmodulin-requiring NO synthase with NADPH, tetrahydrobiopterin (H4biopterin) and molecular oxygen as cofactors. We found that purified brain NO synthase acted as a Ca2+/calmodulin-dependent NADPH:oxygen oxidoreductase, catalysing the formation of hydrogen peroxide at suboptimal concentrations of L-arginine or H4biopterin, which inhibited the hydrogen peroxide formation with half-maximal effects at 11 microM and 0.3 microM respectively. Half-maximal rates of L-citrulline formation were observed at closely similar concentrations of these compounds, indicating that the NO synthase-catalysed oxygen activation was coupled to the synthesis of L-citrulline and NO in the presence of L-arginine and H4biopterin. N omega-Nitro-L-arginine, its methyl ester and N omega-monomethyl-L-arginine inhibited the synthesis of L-citrulline from L-arginine (100 microM) with half-maximal effects at 0.74 microM, 2.8 microM and 15 microM respectively. The N omega-nitro compounds also blocked the substrate-independent generation of hydrogen peroxide, whereas N omega-monomethyl-L-arginine did not affect this reaction. According to these results, activation of brain NO synthase by Ca2+ at subphysiological levels of intracellular L-arginine or H4biopterin may result in the formation of reactive oxygen species instead of NO, and N omega-nitro-substituted L-arginine analogues represent useful tools to effectively block NO synthase-catalysed oxygen activation.

Brain nitric oxide synthase is a Ca2~/ca1modulin-re~lat~ enzyme which converts L-arginine into NO. Enzymatic activity of this enzyme essentially depends on NADPH and is stimulated by tetrahydrobiopterin (H,biopterin). We found that purified NO synthasc contains enzyme-bound H.,biopterin, explaining the enzymatic activity observed in the absence of added cofactor. Together with the finding that H.,biopterin was effective at substoichiomctrical concentrations, these results indicate that NO synthase essentially depends on H_,biopterin as a cofactor which is recycled during enzymatic NO formation. We found that the purified enzyme also contains FAD, FMN and non-hcme iron in equimolar amounts and exhibits striking activities. including a Ca2"/calmodulin-depcndent NADPH oxidase activity, leading to the formation of hydrogen peroxide at suboptimal concentrations of L-arginine or H_,biopterin.

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