How the Diiron Cofactor in HDO Enzymes Collaboratively Promotes the Complex Oxidative Rearrangement via Multistep H/H ⁺ Transfer: The Mechanism of AetD-Catalyzed Nitrile Synthesis

Nitriles are prized motifs in medicines and natural products, yet enzymatic synthesis routes are extremely scarce. The cyanobacterial enzyme AetD can install a −CN on 5,7-dibromo-l-tryptophan via an oxidative rearrangement with nitrogen migration and C−C/C−N cleavage. To elucidate the catalytic mechanism, we constructed the computational reactant model of AetD based on its crystal structure and performed extensive QM/MM calculations. Our results revealed that the dioxygen activation proceeds by spin crossover and direct O−O cleavage to form a diiron(IV,IV)-oxo species. This powerful oxidant affects hydrogen atom transfer (HAT)-triggered, proton-coupled electron transfer (PCET)-driven aziridine formation: Fe1(IV)O abstracts the Cβ−H and Fe2(IV)O abstracts the N−H via PCET, leading to ring closure. The aziridine intermediate further undergoes H abstraction and a diiron-mediated PCET to form an azirine, whose ring then opens by homolytic Cα−N scission accompanied by proton transfer from metal (Fe1) to the substrate. The following OH rebound to Cα and heterolytic Cα−Cβ cleavage produce 5,7-dibromo-indole-3-carbonitrile. The three H abstractions and Cα−Cβ scission are necessary for the N migration, and each of these steps corresponds to a barrier of 20−23 kcal/mol. Overall, the diiron site functions as (i) an HAT/PCET oxidant, (ii) an ET relay station during PCET and Cα−N homolysis, and (iii) a Lewis-acidic catalyst for OH rebound and proton relays. These findings provide a detailed mechanistic picture of AetD catalysis and highlight the cooperative redox between the two iron centers, offering mechanistic insights applicable to the design of biomimetic catalysts for nitrile synthesis.