Nitric oxide plays a crucial role in cellular signaling and innate immune response, and is implicated in many of the world’s most important diseases. It is produced by NO-Synthases (NOS) from L-arginine in two steps through formation of NG-hydroxy-L-arginine (NHA), which is converted to L-citrulline and NO·. Both steps involve synchronous rotation of the P450-like reaction cycle and the redox cycle of the cofactor tetrahydrobiopterin (H4B). Despite experimental and theoretical efforts, their mechanisms remain unknown. We study the second step of the NO· biosynthesis by Density Functional Theory calculations, in which the structural features of the NOS active site are preserved, with the goal to capture the electron and proton transfer (ET and PT) events that drive the reaction the designated way. It is demonstrated that the reaction cycle is controlled by four ET and five PT events, with only one electron and one proton being transferred from the external sources. The π-HOMO of H4B interacts with the P450-like reaction cycle through the coplanar filled π*(NO) orbitals of two NHA-derived intermediates, namely the transient iminoxy radical (IOR) and the singlet tetrahedral adduct of protonated L-citrulline and NO–. Results suggest that the central feature of the mechanism is the PT-coupled spin-selective hole tunneling from the σp*-orbital of the peroxide bond of the ferric-dihydroxyperoxide oxidant species (FeIIIO(H)OH) to the π-HOMO of H4B through the intervening π*(NO) bridge orbital of IOR leading to the late homolytic cleavage of this bond. The resulting HO· recombines with IOR forming the singlet adduct. Electron tunneling from the π*(NO) orbital of this adduct to the π-SOMO of H4B+· releases NO· with 100% catalytic efficiency. Unnatural NHA analogues disrupt the delicate orbital balance inherent in the inter-cyclic ping-pong electron tunneling, which changes the mechanism or slows down the reaction cycle, thereby leading to unintended products including reactive nitrogen and oxygen species.

Theoretical studies of the synchronization of the catalytic cycles in the second half-reaction step of nitric oxide synthase

249th National Meeting (2015)

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