The extradiol-cleaving dioxygenase homoprotocatechuate 2,3-dioxygenase (HPCD) binds substrate homoprotocatechuate (HPCA) and O-2 sequentially in adjacent ligand sites of the active site Fe-II. Kinetic and spectroscopic studies of HPCD have elucidated catalytic roles of several active site residues, including the crucial acid-base chemistry of His200. In the present study, reaction of the His200Cys (H200C) variant with native substrate HPCA resulted in a decrease in both k(cat) and the rate constants for the activation steps following O2 binding by >400 fold. The reaction proceeds to form the correct extradiol product. This slow reaction allowed a long-lived (t(1/2) = 1.5 min) intermediate, H200C-HPCA(Int1) (Int1), to be trapped. Mossbauer and parallel mode electron paramagnetic resonance (EPR) studies show that Int1 contains an S-1 = 5/2 Fe-III center coupled to an S-R = 1/2 radical to give a ground state with total spin S = 2 (J > 40 cm(-1)) in . Density functional theory (DFT) property calculations for structural models suggest that Int1 is a (HPCA semiquinone center dot)Fe-III(OOH) complex, in which OOH is protonated at the distal O and the substrate hydroxyls are deprotonated. By combining Mossbauer and EPR data of Int1 with DFT calculations, the orientations of the principal axes of the Fe-57 electric field gradient and the zero-field splitting tensors (D = 1.6 cm(-1), E/D = 0.05) were determined. This information was used to predict hyperfine splittings from bound 17OOH. DFT reactivity analysis suggests that Int1 can evolve from a ferromagnetically coupled Fe-III-superoxo precursor by an inner-sphere proton-coupled-electron-transfer process. Our spectroscopic and DFT results suggest that a ferric hydroperoxo species is capable of extradiol catalysis.