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 FeO2 species is best described as a Fe(III)O-2(center dot) state, which is triplet O-2 binding to quintet Fe(II), leading to a one-electron transfer from Fe(II) to O2. Subsequently, the O-2(center dot) 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 acidbase catalyst, providing a proton to the substrate.