Second Half-Reaction of Nitric Oxide Synthase:  Computational Insights into the Initial Step and Key Proposed Intermediate

Density functional theory methods have been employed to investigate possible first steps in the second half-reaction of the mechanism of nitric oxide synthases (NOSs). In particular, reactions and complexes formed via transfer of either or both hydrogens of the substrates (NHA) −NHOH group to the Fe-bound O₂ were considered. For each of these pathways, the effect of adding an extra electron from tetrahydrobiotperin (H₄B) was also examined. The preferred initial pathway involves the simultaneous transfer of both hydrogens of the −NHOH group to the Fe_heme−O₂, without an additional electron, to give the Fe_heme−HOOH species which lies only marginally higher in energy, 2.5 kcal mol^-1 or less, than the initial bound active site. An alternative mechanism in which only the −NH− proton of the −NHOH group is transferred to the Fe_heme−O₂ to give an Fe_heme−OOH derivative is found to require only slightly more energy, approximately 2 kcal mol^-1. However, transfer of the proton back to the −NOH nitrogen occurs without a barrier at 298.15 K. Tetrahedral intermediates in which the Fe_heme−O₂ has attached at the guanidinium carbon (C_guan) of NHA, that is, forms an Fe_heme−O₂−C_guan link, have also been investigated. All examples of such species considered, that is, with or without hydrogen or electron transfers, lie significantly higher in energy by at least 29.0 kcal mol^-1 than the initial bound active site. Thus, it is suggested that such complexes are not mechanistically feasible. The implications of the present findings for the second half-reaction are also discussed.