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Abstract

A detailed QM/MM study on the reaction mechanism of Cytochrome P450 NO reductase is reported. Two reaction pathways connecting the two well‐characterized intermediates as well as two putative intermediates that represent the unknown third intermediate are explored, with emphasis on the unusual direct reduction of the enzymatic active site by the cofactor NADH. Activation barriers and kinetic isotope effect are calculated and reveal that reduction of the NO‐bound species occurs in form of a hydride ion transfer. Furthermore, the impact of different hydrogen bonds in the active site to binding and reactivity of NADH is explored. The calculated kinetic and thermodynamic properties for both modelled pathways are used for the kinetic simulation of the entire reaction course. It is thus shown that the unknown key intermediate is the singlet diradical Fe^III‐NHOH^⋅. It is also found that the mechanism of the N-N bond formation is spin‐recoupling, which is only possible due to the diradical character of the key intermediate.