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

Abstract

A full understanding of the catalytic action of non-heme iron (NHFe) and non-heme diiron (NHFe₂) 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 NHFe₂ family of enzymes: soluble Δ⁹ desaturase (Δ⁹D), 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 <b>P</b> intermediate, the proton-electron uptake by <b>P</b> 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 <b>Q</b>-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 Δ⁹D 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 NHFe₂ chemistry, provided by an amino acid sequence analysis through the families of the NHFe₂ enzymes. Our study thus represents an important contribution toward understanding the catalytic action of the NHFe₂ enzymes and may inspire further work in NHFe₍₂₎ biomimetic chemistry.