Inducible nitric oxide synthase: Identification of amino acid residues essential for dimerization and binding of tetrahydrobiopterin

Nitric oxide synthases (NOSs) require tetrahydrobiopterin (BH₄) for dimerization and NO production. Mutation analysis of mouse inducible NOS (iNOS; NOS2) identified Gly-450 and Ala-453 as critical for NO production, dimer formation, and BH₄ binding. Substitutions at five neighboring positions were tolerated, and normal binding of heme, calmodulin, and NADPH militated against major distortions affecting the NH₂-terminal portion, midzone, or COOH terminus of the inactive mutants. Direct involvement of residues 450 and 453 in the binding of BH₄ is supported by the striking homology of residues 448-480 to a region extensively shared by the three BH₄-utilizing aromatic amino acid hydroxylases and is consistent with the conservation of these residues among all 10 reported NOS sequences, including mammalian NOSs 1, 2, and 3, as well as avian and insect NOSs. Altered binding of BH₄ and/or L-arginine may explain how the addition of a single methyl group to the side chain of residue 450 or the addition of three methylenes to residue 453 can each abolish an enzymatic activity that reflects the concerted function of 1143 other residues.