O₂ Activation in Salicylate 1,2-Dioxygenase: A QM/MM Study Reveals the Role of His162

Nonheme iron enzymes play an important role in the aerobic degradation of aromatic ring systems. Most enzymes can only cleave substrates with electron-rich substituents, e.g., with two hydroxyl groups. However, salicylate 1,2-dioxygenase (SDO) can cleave rings with only a single hydroxyl group. We investigated the oxygen-activation mechanism of the ring fission of salicylate by SDO by computational methods using combined quantum mechanical and molecular mechanical (QM/MM) geometry optimizations, large QM calculations with 493 atoms, and QM/MM free-energy perturbations. Our results demonstrate that the reactive Fe–O₂ species is best described as a Fe­(III)–O₂^•– state, which is triplet O₂ binding to quintet Fe­(II), leading to a one-electron transfer from Fe­(II) to O₂. Subsequently, the O₂^•– group of this species attacks the aromatic ring of substrate to form an alkylperoxo intermediate. Mutation studies suggested that His162 is essential for catalysis. Our calculations indicate that His162 plays a role as an acid–base catalyst, providing a proton to the substrate