Tetrahydrobiopterin Inhibits Monomerization and Is Consumed during Catalysis in Neuronal NO Synthase*

The biosynthesis of nitric oxide (NO) is catalyzed by homodimeric NO synthases (NOS). For unknown reasons, all NOS co-purify with substoichiometric amounts of (6 R)-5,6,7,8-tetrahydrobiopterin (H4Bip) and require additional H4Bip for maximal activity. We examined the effects of H4Bip and pterin-derived inhibitors (anti-pterins) on purified neuronal NOS-I quaternary structure and H4Bip content. During l-arginine turnover, NOS-I dimers time dependently dissociated into inactive monomers, paralleled by a loss of enzyme-associated pterin. Dimer dissociation was inhibited when saturating levels of H4Bip were added during catalysis. Similar results were obtained with pterin-free NOS-I expressed in Escherichia coli. This stabilizing effect of H4Bip was mimicked by the anti-pterin 2-amino-4,6-dioxo-3,4,5,6,8,8a,9,10-octahydro-oxazolo[1,2f]-pteridine (PHS-32), which also displaced NOS-associated H4Bip in a competitive manner. Surprisingly, H4Bip not only dissociated from NOS during catalysis, but was only partially recovered in the solute (50.0 ± 16.5% of control at 20 min). NOS-associated H4Bip appeared to react with a NOS catalysis product to a derivative distinct from dihydrobiopterin or biopterin. Under identical conditions, reagent H4Bip was chemically stable and fully recovered (95.5 ± 3.4% of control). A similar loss of both reagent and enzyme-bound H4Bip and dimer content was observed by NO generated from spermine NONOate. In conclusion, we propose a role for H4Bip as a dimer-stabilizing factor of neuronal NOS during catalysis, possibly by interfering with enzyme destabilizing products.