Structures of the N(ω)-hydroxy-L-arginine complex of inducible nitric oxide synthase oxygenase dimer with active and inactive pterins

Nitric oxide synthases (NOSs) catalyze two mechanistically distinct, tetrahydrobiopterin (H₄B)-dependent, heme-based oxidations that first convert L-arginine (L-Arg) to N(ω)-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 β-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 oxo-iron intermediate participating in either L-Arg or NHA oxidation. Identical binding modes for active H₄B, the inactive quinonoiddihydrobiopterin (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.