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

Abstract

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 <i>k</i>_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 (<i>t</i>_1/2 = 1.5 min) intermediate, H200C-HPCA_Int1 (<i>Int1</i>), to be trapped. Mössbauer and parallel mode electron paramagnetic resonance (EPR) studies show that <i>Int1</i> contains an <i>S</i>₁ = 5/2 Fe^III center coupled to an <i>S</i>_R = 1/2 radical to give a ground state with total spin <i>S</i> = 2 (<i>J</i> > 40 cm^–1) in Hexch=JŜ1·ŜR. Density functional theory (DFT) property calculations for structural models suggest that <i>Int1</i> 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 <i>Int1</i> with DFT calculations, the orientations of the principal axes of the ⁵⁷Fe electric field gradient and the zero-field splitting tensors (<i>D</i> = 1.6 cm^–1, <i>E</i>/<i>D</i> = 0.05) were determined. This information was used to predict hyperfine splittings from bound ¹⁷OOH. DFT reactivity analysis suggests that <i>Int1</i> 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