DELINEATION OF THE ARGININE-BINDING AND TETRAHYDROBIOPTERIN-BINDING SITES OF NEURONAL NITRIC-OXIDE SYNTHASE

Nitric oxide synthase (EC 1.14.13.39) catalyses the conversion of argi nine, NADPH and oxygen to nitric oxide and citrulline, using haem, (6R )-5,6,7,8-tetrahydro-L-biopterin (tetrahydro-biopterin), calmodulin, F AD and FMN as cofactors. The enzyme consists of a central calmodulin-b inding sequence flanked on the N-terminal side by a haem-binding regio n that contains the arginine and tetrahydrobiopterin sites and on the C-terminal side by a region homologous with NADPH:cytochrome P-450 red uctase. By using domain boundaries defined by limited proteolysis of f ull-length enzyme, recombinant haem-binding regions of rat brain neuro nal nitric oxide synthase were expressed and purified. Two proteins we re made in high yield: one, corresponding to residues 221-724, contain ed bound haem and tetrahydrobiopterin and was able to bind N-omega-nit ro-L-arginine (nitroarginine) or arginine; the other, containing resid ues 350-724, contained bound haem but was unable to bind tetrahydrobio pterin, nitroarginine or arginine. These results showed that rat brain neuronal nitric oxide synthase contains a critical determinant for ar ginine/tetrahydrobiopterin binding between residues 221 and 350. Limit ed proteolysis with chymotrypsin of the former protein resulted in a n ew species with an N-terminal residue 275 that retained the ability to bind nitroarginine, further defining the critical region for arginine binding as being between 275 and 350. Comparison of the sequences of nitric oxide synthase and the tetrahydrobiopterin-requiring amino acid hydroxylases revealed a similarity in the region between residues 470 and 600, suggesting that this might represent the core region of the pterin-binding site. The stoichiometries of binding of substrate and c ofactors to the recombinant domains were not more than 0.5 mol/mol of monomer, suggesting that there might be a single high-affinity site pe r dimer.