An extradiol-cleaving catecholic dioxygenase, 2,3-dihydroxybiphenyl dioxygenase, plays important roles in the catabolism of biphenyl/polychlorinated biphenyl aromatic contaminants in the environment. To better elucidate the biodegradable pathway, a theoretical investigation of the ring-opening degradation of 2,3-dihydroxybiphenyl (DHBP) was performed with the aid of quantum mechanical/molecular mechanical calculations. A quintet state of the DHBP-iron-dioxygen group adducts was found to be the reactive state with a substrate radical-FeII-superoxo (DHBP•↑-FeII-O2•-↓) character. The HOO• species was the reactive oxygen species responsible for the subsequent attack of DHBP. Among the whole reaction energy profile, the first step in proton-coupled electron transfer was determined to be the rate-determining step with a potential energy barrier of 17.2 kcal/mol, which is close to the experimental value (14.7 kcal/mol). Importantly, the residue His194 shows distinct roles in the catalytic cycle, where it acts as an acid-base catalyst to deprotonate the hydroxyl group of DHBP at an early stage, then stabilizes the negative charge on the dioxygen group, and, at the final stage, promotes the semialdehyde product formation as a proton donor.