Stoichiometric arginine binding in the oxygenase domain of inducible nitric oxide synthase requires a single molecule of tetrahydrobiopterin per dimer

In addition to its catalytic roles, the nitric oxide synthase (NOS) cofacto r tetrahydrobiopterin (H4B) is required for substrate binding and for stabi lization of the dimeric structure. We expressed and purified the core of th e iNOS oxygenase domain consisting of residues 75-500 (CODiNOS) in the pres ence (H4B(+)) and absence (H4B(-)) of this cofactor. Both forms bound stoic hiometric amounts of heme (>0.9 heme per protein subunit). H4B(-) CODiNOS w as unable to bind arginine, gave an unstable ferrous carbonyl adduct, and w as a mixture of monomer and dimer. H4B(+) CODiNOS bound arginine, gave a st able ferrous carbonyl adduct, and was exclusively dimeric. The H4B cofactor content of this species was only one per dimer yet this was sufficient to form two competent arginine binding sites as determined by optical stoichio metric titrations. (C) 1999 Academic Press.