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 O-2 reduction is the primary outcome of NOS activation, H4B promotes the generation of H2O2 at the expense of O-2(.-). 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 O-2 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 O-2(.-) 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 O-2(.-) we found that the initial rate of H2O2 production by H4B-bound NOS I was considerably greater than that for H4B-free NOS I.