Proton–Electron Transfer to the Active Site Is Essential for the Reaction Mechanism of Soluble Δ9-Desaturase

A full understanding of the catalytic action of non-heme iron (NHFe) and non-heme diiron (NHFe 2 ) enzymes is still beyond the grasp of contemporary computational and experimental techniques. Many of these enzymes exhibit fascinating chemo-, regio-, and stereoselectivity, in spite of employing highly reactive intermediates which are necessary for activations of most stable chemical bonds. Herein, we study in detail one intriguing representative of the NHFe 2 family of enzymes: soluble Δ 9 desaturase (Δ 9 D), which desaturates rather than performing the thermodynamically favorable hydroxylation of substrate. Its catalytic mechanism has been explored in great detail by using QM(DFT)/MM and multireference wave function methods. Starting from the spectroscopically observed 1,2-μ-peroxo diferric P intermediate, the proton-electron uptake by P is the favored mechanism for catalytic activation, since it allows a significant reduction of the barrier of the initial (and rate-determining) H-atom abstraction from the stearoyl substrate as compared to the "proton-only activated" pathway. Also, we ruled out that a Q -like intermediate (high-valent diamond-core bis-μ-oxo-[Fe IV ] 2 unit) is involved in the reaction mechanism. Our mechanistic picture is consistent with the experimental data available for Δ 9 D and satisfies fairly stringent conditions required by Nature: the chemo-, stereo-, and regioselectivity of the desaturation of stearic acid. Finally, the mechanisms evaluated are placed into a broader context of NHFe 2 chemistry, provided by an amino acid sequence analysis through the families of the NHFe 2 enzymes. Our study thus represents an important contribution toward understanding the catalytic action of the NHFe 2 enzymes and may inspire further work in NHFe (2) biomimetic chemistry.