Structures of the Nω-hydroxy-L-arginine complex of inducible nitric oxide synthase oxygenase dimer with active and inactive pterins

Nitric oxide syntheses (NOSs) catalyze two mechanistically distinct, tetrahydrobiopterin (H4B)dependent, heme-based oxidations that first convert L-arginine (L-Arg) to N-omega-hydroxy-L-arginine (NHA) and then NHA to L-citrulline and nitric oxide. Structures of the murine inducible NOS oxygenase domain (iNOS(ox)) complexed with NHA indicate that NHA and L-Arg both bind with the same conformation adjacent to the heme iron and neither interacts directly with it nor with H4B. Steric restriction of dioxygen binding to the heme in the NHA complex suggests either small conformational adjustments in the ternary complex or a concerted reaction of dioxygen with NHA and the heme iron. Interactions of the NHA hydroxyl with active center beta-structure and the heme ring polarize and distort the hydroxyguanidinium to increase substrate reactivity. Steric constraints in the active center rule against superoxo-iron accepting a hydrogen atom from the NHA hydroxyl in their initial reaction, but support an Fe(III)-peroxo-NHA radical conjugate as an intermediate. However, our structures do not exclude an ore-iron intermediate participating in either L-Arg or NHA oxidation. Identical binding modes for active H4B, the inactive quinonoid-dihydrobiopterin (q-H2B), and inactive 4-amino-H4B indicate that conformational differences cannot explain pterin inactivity. Different redox and/or protonation states of q-H2B and 4-amino-H4B relative to H4B likely affect their ability to electronically influence the heme and/or undergo redox reactions during NOS catalysis. On the basis of these structures, we propose a testable mechanism where neutral H4B transfers both an electron and a 3,4-amide proton to the heme during the first step of NO synthesis.