Nitric oxide-induced autoinhibition of neuronal nitric oxide synthase in the presence of the autoxidation-resistant pteridine 5-methyltetrahydrobiopterin

Nitric oxide synthase (NOS) catalysis results in formation of NO or superoxide (O₂^-−) depending on the presence or absence of the cofactor tetrahydrobiopterin (BH4). In the absence of O₂^-− scavengers, net NO formation cannot be detected even at saturating BH4 concentrations, which is thought to be due to O₂^-− production by BH4 autoxidation. Because the N-5-methylated analogue of BH4 (5-Me-BH4) sustains NOS catalysis and is autoxidation-resistant, net NO formation by the neuronal isoform of NOS (nNOS) can be observed at saturating 5-Me-BH4 concentrations. Here we compare the effects of 5-Me-BH4 on L-citrulline formation, NADPH oxidation, H₂O₂ production and soluble guanylate cyclase (sGC) stimulation. All activities were stimulated biphasically (EC₅₀ approx. 0.2 μM and more than 1 mM), with an intermediate inhibitory phase at the same pterin concentration as that required for net NO generation and sGC stimulation (4 μM). Concomitantly with inhibition, the NADP⁺/L-citrulline stoichiometry decreased from 2.0 to 1.6. Inhibition occurred only at high enzyme concentrations (IC₅₀ approx. 10 nM nNOS) and was antagonized by oxyhaemoglobin and by BH4. We ascribe the first stimulatory phase to high-affinity binding of 5-Me-BH4. The inhibitory phase is due to low-affinity binding, resulting in fully coupled catalysis, complete inhibition of O₂^-− production and net NO formation. At high enzyme concentrations and thus high NO levels, this causes autoinhibition. NO scavenging by 5-Me-BH4 at concentrations above 1 mM, resulting in the antagonization of inhibition of NOS, explains the second stimulatory phase. In agreement with these assignments 5-Me-BH4 was found to stimulate formation of a haem-NO complex during NOS catalysis. The observation of inhibition with 5-Me-BH4 but not with BH4 implies that, unless O₂^-− scavengers are present, a physiological role for NO-induced autoinhibition is unlikely.