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

Nitric oxide syntheses (NOSs) catalyze two mechanistically distinct, tetrah ydrobiopterin (H4B)dependent, heme-based oxidations that first convert L-ar ginine (L-Arg) to N-omega-hydroxy-L-arginine (NHA) and then NHA to L-citrul line and nitric oxide. Structures of the murine inducible NOS oxygenase dom ain (iNOS(ox)) complexed with NHA indicate that NHA and L-Arg both bind wit h the same conformation adjacent to the heme iron and neither interacts dir ectly with it nor with H4B. Steric restriction of dioxygen binding to the h eme in the NHA complex suggests either small conformational adjustments in the ternary complex or a concerted reaction of dioxygen with NHA and the he me 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 init ial reaction, but support an Fe(III)-peroxo-NHA radical conjugate as an int ermediate. 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 tra nsfers both an electron and a 3,4-amide proton to the heme during the first step of NO synthesis.