A long-lived Fe^III-(hydroperoxo) intermediate in the active H200C variant of homoprotocatechuate 2,3-dioxygenase: Characterization by mössbauer, electron paramagnetic resonance, and density functional theory methods

The extradiol-cleaving dioxygenase homoprotocatechuate 2,3-dioxygenase (HPCD) binds substrate homoprotocatechuate (HPCA) and O₂ 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. Mössbauer and parallel mode electron paramagnetic resonance (EPR) studies show that Int1 contains an S₁ = 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 Hexch=J͈1·͈R. Density functional theory (DFT) property calculations for structural models suggest that Int1 is a (HPCA semiquinone^•)Fe^III(OOH) complex, in which OOH is protonated at the distal O and the substrate hydroxyls are deprotonated. By combining Mössbauer and EPR data of Int1 with DFT calculations, the orientations of the principal axes of the ⁵⁷Fe 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.