A Long-Lived FeIII-(Hydroperoxo) Intermediate in the Active H200C Variant of Homoprotocatechuate 2,3-Dioxygenase: Characterization by M枚ssbauer, Electron Paramagnetic Resonance, and Density Functional

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• 作者：Katlyn K. Meier ; Melanie S. Rogers ; Elena G. Kovaleva ; Michael M. Mbughuni ; Emile L. Bominaar ; John D. Lipscomb ; Eckard M眉nck
• 刊名：Inorganic Chemistry
• 出版年：2015
• 出版时间：November 2, 2015
• 年：2015
• 卷：54
• 期：21
• 页码：10269-10280
• 全文大小：642K
• ISSN：1520-510X

文摘

The extradiol-cleaving dioxygenase homoprotocatechuate 2,3-dioxygenase (HPCD) binds substrate homoprotocatechuate (HPCA) and O<sub>2sub> sequentially in adjacent ligand sites of the active site Fe<sup>IIsup>. Kinetic and spectroscopic studies of HPCD have elucidated catalytic roles of several active site residues, including the crucial acid鈥揵ase chemistry of His200. In the present study, reaction of the His200Cys (H200C) variant with native substrate HPCA resulted in a decrease in both k<sub>catsub> and the rate constants for the activation steps following O<sub>2sub> binding by >400 fold. The reaction proceeds to form the correct extradiol product. This slow reaction allowed a long-lived (t<sub>1/2sub> = 1.5 min) intermediate, H200C-HPCA<sub>Int1sub> (Int1), to be trapped. M枚ssbauer and parallel mode electron paramagnetic resonance (EPR) studies show that Int1 contains an S<sub>1sub> = 5/2 Fe<sup>IIIsup> center coupled to an S<sub>Rsub> = 1/2 radical to give a ground state with total spin S = 2 (J > 40 cm<sup>鈥?sup>) in . Density functional theory (DFT) property calculations for structural models suggest that Int1 is a (HPCA semiquinone<sup>鈥?/sup>)Fe<sup>IIIsup>(OOH) complex, in which OOH is protonated at the distal O and the substrate hydroxyls are deprotonated. By combining M枚ssbauer and EPR data of Int1 with DFT calculations, the orientations of the principal axes of the <sup>57sup>Fe electric field gradient and the zero-field splitting tensors (D = 1.6 cm<sup>鈥?sup>, E/D = 0.05) were determined. This information was used to predict hyperfine splittings from bound <sup>17sup>OOH. DFT reactivity analysis suggests that Int1 can evolve from a ferromagnetically coupled Fe<sup>IIIsup>-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.