The role of tetrahydrobiopterin in the regulation of neuronal nitric-oxide synthase-generated superoxide

Tetrahydrobiopterin (H₄B) is a critical element in the nitric-oxide synthase (NOS) metabolism of L-arginine to L-citrulline and NO^.. It has been hypothesized that in the absence of or under nonsaturating levels of L-arginine where O₂ reduction is the primary outcome of NOS activation, H₄B promotes the generation of H₂O₂ at the expense of O₂^._. The experiments were designed to test this hypothesis. To test this theory, two different enzyme preparations, H₄B-bound NOS I and H₄B-free NOS I, were used. Initial rates of NADPH turnover and O₂ utilization were found to be considerably greater in the H4B-bound NOS I preparation than in the H₄B-free NOS I preparation. In contrast, the initial generation of O₂^.- from the H₄B-free NOS I preparation was found to be substantially greater than that measured using the H₄B-bound NOS I preparation. Finally, by spin trapping nearly all of the NOS I produced O₂^.-, we found that the initial rate of H₂O₂ production by H₄B-bound NOS I was considerably greater than that for H₄B-free NOS I.