Tetrahydrobiopterin-dependent inhibition of superoxide generation from neuronal nitric oxide synthase

The binding of calcium/calmodulin stimulates electron transfer between the reductase and oxygenase domains of neuronal nitric oxide synthase (nNOS). H ere, we demonstrate using electron spin resonance spin-trapping with 5-diet hoxyphosphoryl-5-methyl-1-pyrroline N-oxide that pterin-free nNOS generates superoxide from the reductase and the oxygenase domain by a calcium/calmod ulin-dependent mechanism. Tetrahydrobiopterin (BH4) diminishes the formatio n of superoxide by a mechanism that does not cause inhibition of NADPH cons umption. In contrast, BH4 analogs 7,8-dihydrobiopterin and sepiapterin do n ot affect superoxide yields. L-Arginine alone inhibits the generation of su peroxide by nNOS but not by C331A-nNOS mutant that has a low affinity for L -arginine. A greater decrease in superoxide peroxide yields is observed whe n nNOS is preincubated with L-arginine. This effect is in accordance with t he slow binding rates of L-arginine to NOS in the absence of BH4. L-Arginin e alone or in combination with BH, decreases the rates of NADPH consumption . The effect of L-arginine on superoxide yields, however, was less dramatic than that caused by BH4 as much higher concentrations of L-arginine are ne cessary to attain the same inhibition. In combination, L-arginine and BH4 i nhibit the formation of superoxide generation and stimulate the formation o f L-citrulline. We conclude that, in contrast to L-arginine, BH4 does not i nhibit the generation of superoxide by controlling electron transfer throug h the enzyme but by stimulating the formation of the heme-peroxo species.