Mutational analysis of the tetrahydrobiopterin-binding site in inducible nitric-oxide synthase

Inducible nitric-oxide synthase (iNOS) is a hemeprotein that requires tetra hydrobiopterin (H4B) for activity. The influence of H4B on iNOS structure-f unction is complex, and its exact role in nitric oxide (NO) synthesis is un known. Crystal structures of the mouse iNOS oxygenase domain (iNOSox) revea led a unique H4B-binding site with a high degree of aromatic character loca ted in the dimer interface and near the heme. Four conserved residues (Arg- 375, Trp-455, Trp-457, and Phe-470) engage in hydrogen bonding or aromatic stacking interactions with the H4B ring. We utilized point mutagenesis to i nvestigate how each residue modulates H4B function. All mutants contained h eme ligated to Cys-194 indicating no deleterious effect on general protein structure. Ala mutants were monomers except for W457A and did not form a ho modimer with excess H4B and Arg. However, they did form heterodimers when p aired with a full-length iNOS subunit, and these were either fully or parti ally active regarding NO synthesis, indicating that preserving residue iden tities or aromatic character is not essential for H4B binding or activity. Aromatic substitution at Trp-455 or Trp-457 generated monomers that could d imerize with H4B and Arg. These mutants bound Arg and H4B with near normal affinity, but Arg could not displace heme-bound imidazole, and they had NO synthesis activities lower than wild-type in both homodimeric and heterodim eric settings. Aromatic substitution at Phe-470 had no significant effects. Together, our work shows how hydrogen bonding and aromatic stacking intera ctions of Arg-375, Trp-457, Trp-455, and Phe-470 influence iNOSox dimeric s tructure, heme environment, and NO synthesis and thus help modulate the mul tiple effects of H4B.