Theoretical studies of the second step of the nitric oxide synthase reaction: Electron tunneling prevents uncoupling

Nitric oxide (NO·) is a messenger molecule with diverse physiological roles including host defense, neurotransmission and vascular function. The synthesis of NO· from l-arginine is catalyzed by NO-synthases and occurs in two steps through the intermediary N^ω-hydroxy-l-arginine (NHA). In both steps the P450-like reaction cycle is coupled with the redox cycle of the cofactor tetrahydrobiopterin (H₄B). The mechanism of the second step is studied by Density Functional Theory calculations to ascertain the canonical sequence of proton and electron transfer (PT and ET) events. The proposed mechanism is controlled by the interplay of two electron donors, H₄B and NHA. Consistent with experimental data, the catalytic cycle proceeds through the ferric-hydroperoxide complex (Cpd 0) and the following aqua-ferriheme resting state, and involves interim partial oxidation of H₄B. The mechanism starts with formation of Cpd 0 from the ferrous-dioxy reactant complex by PT from the C-ring heme propionate coupled with hole transfer to H₄B through the highest occupied π-orbital of NHA as a bridge. This enables PT from NHA⁺· to the proximal oxygen leading to the shallow ferriheme-H₂O₂ oxidant. Subsequent Fenton-like peroxide bond cleavage triggered by ET from the NHA-derived iminoxy-radical leads to the protonated Cpd II diradicaloid singlet stabilized by spin delocalization in H₄B, and the closed-shell coordination complex of HO^- with iminoxy-cation. The complex is converted to the transient C-adduct, which releases intended products upon PT to the ferriheme-HO^- complex coupled with ET to the H₄B⁺·. Deferred ET from the substrate or undue ET from/to the cofactor leads to side products.