The reaction mechanism of the dioxygen activation by homoprotocatechuate 2,3-dioxygenase (HPCD) with the substrate 4-nitrocatechol was investigated by quantum mechanical/molecular mechanical calculations. Our results demonstrated that the experimentally determined side-on iron oxygen complex in crystallo is a semiquinone substrate radical (SQ(center dot))-Fe-III-hydroperoxo species, which could not act as the reactive species. In fact, the Fe-III-superoxo species with a hydrogen bond between His200 and the proximal oxygen is the reactive oxygen species. The second-sphere His200 residue was found to play an important role in manipulating the orientation of the superoxide in the Fe-O-2 adduct for the further reaction. The rate-limiting step is the attack of the superoxo group on the substrate with a barrier of 17.2 kcal/mol, in good agreement with the experimental value of 16.8 kcal/mol. The reaction mechanism was then compared with the one for HPCD with its native substrate homoprotocatechuate studied recently by the same methods, in which a hybrid SQ(center dot)-Fe-II-O-2(center dot-)/Fe-III-O-2(center dot-) was suggested to be the reactive species. Therefore, our studies suggested that the substrate plays important roles in the dioxygen activation by HPCD.