The theory of "proton-assisted process" can well explain the catalytic mechanism of homoprotocatechuate 2,3-dioxygenase (2,3-HPCD) with a monoanionic substrate (homoprotocatechuate, HPCA). Here a "non-proton-assisted process" is presented to interpret catalytic mechanism of 2,3-HPCD with a dianionic substrate (4-nitrocatechol, 4NC). The ONIOM calculation is performed to investigate the reaction pathway of a wild-type 2,3-HPCD with 4NC (H200H-4NC system). The catalytic reaction is comprised of four steps: (1) A dioxygen attacks the aromatic ring to produce an alkylperoxo species. (2) O-O bond cleavage and the formation of an epoxide species occur. (3) A seven-membered O-heterocyclic compound is generated by the extinction of the epoxy structure. (4) The seven-membered ring undergoes ring opening to form the final product (C2-C3 cleavage product). The effective free energy barrier of the catalytic reaction of the H200H-4NC system is 26.2kcalmol-1, which is much higher than that of the H200H-HPCA system. Furthermore, two calculated electronic configurations (Fe(III)-O2•- and Fe(III)-SQ•) have a high similarity to previously detected ones, which demonstrates that the Asn200 variant (H200N-4NC variant system) employs a C4 (para-carbon) pathway to produce a C4-C5 cleavage product. Our findings provide an in-depth understanding of the catalytic mechanisms of dianionic catechol and its derivatives.