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

Katlyn K. Meier, Melanie S. Rogers, Elena G. Kovaleva, Michael M. Mbughuni, Emile L. Bominaar, John D. Lipscomb, Eckard Münck

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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 FeII. 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 kcat 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 (t1/2 = 1.5 min) intermediate, H200C-HPCAInt1 (Int1), to be trapped. Mössbauer and parallel mode electron paramagnetic resonance (EPR) studies show that Int1 contains an S1 = 5/2 FeIII center coupled to an SR = 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)FeIII(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 FeIII-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.

Original languageEnglish (US)
Pages (from-to)10269-10280
Number of pages12
JournalInorganic Chemistry
Volume54
Issue number21
DOIs
StatePublished - Nov 2 2015

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3,4-dihydroxyphenylacetate 2,3-dioxygenase
Density functional theory
Paramagnetic resonance
electron paramagnetic resonance
density functional theory
Substrates
Hydroxyl Radical
Ground state
Catalysis
catalysis
Tensors
Protons
Rate constants
electron transfer
reactivity
Chemical activation
Electric fields
activation
tensors
chemistry

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A long-lived FeIII-(hydroperoxo) intermediate in the active H200C variant of homoprotocatechuate 2,3-dioxygenase : Characterization by mössbauer, electron paramagnetic resonance, and density functional theory methods. / Meier, Katlyn K.; Rogers, Melanie S.; Kovaleva, Elena G.; Mbughuni, Michael M.; Bominaar, Emile L.; Lipscomb, John D.; Münck, Eckard.

In: Inorganic Chemistry, Vol. 54, No. 21, 02.11.2015, p. 10269-10280.

Research output: Contribution to journalArticle

Meier, Katlyn K. ; Rogers, Melanie S. ; Kovaleva, Elena G. ; Mbughuni, Michael M. ; Bominaar, Emile L. ; Lipscomb, John D. ; Münck, Eckard. / A long-lived FeIII-(hydroperoxo) intermediate in the active H200C variant of homoprotocatechuate 2,3-dioxygenase : Characterization by mössbauer, electron paramagnetic resonance, and density functional theory methods. In: Inorganic Chemistry. 2015 ; Vol. 54, No. 21. pp. 10269-10280.
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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 FeII. 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 kcat 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 (t1/2 = 1.5 min) intermediate, H200C-HPCAInt1 (Int1), to be trapped. M{\"o}ssbauer and parallel mode electron paramagnetic resonance (EPR) studies show that Int1 contains an S1 = 5/2 FeIII center coupled to an SR = 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•)FeIII(OOH) complex, in which OOH is protonated at the distal O and the substrate hydroxyls are deprotonated. By combining M{\"o}ssbauer 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 FeIII-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.",
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T1 - A long-lived FeIII-(hydroperoxo) intermediate in the active H200C variant of homoprotocatechuate 2,3-dioxygenase

T2 - Characterization by mössbauer, electron paramagnetic resonance, and density functional theory methods

AU - Meier, Katlyn K.

AU - Rogers, Melanie S.

AU - Kovaleva, Elena G.

AU - Mbughuni, Michael M.

AU - Bominaar, Emile L.

AU - Lipscomb, John D.

AU - Münck, Eckard

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N2 - The extradiol-cleaving dioxygenase homoprotocatechuate 2,3-dioxygenase (HPCD) binds substrate homoprotocatechuate (HPCA) and O2 sequentially in adjacent ligand sites of the active site FeII. 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 kcat 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 (t1/2 = 1.5 min) intermediate, H200C-HPCAInt1 (Int1), to be trapped. Mössbauer and parallel mode electron paramagnetic resonance (EPR) studies show that Int1 contains an S1 = 5/2 FeIII center coupled to an SR = 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•)FeIII(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 FeIII-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.

AB - The extradiol-cleaving dioxygenase homoprotocatechuate 2,3-dioxygenase (HPCD) binds substrate homoprotocatechuate (HPCA) and O2 sequentially in adjacent ligand sites of the active site FeII. 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 kcat 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 (t1/2 = 1.5 min) intermediate, H200C-HPCAInt1 (Int1), to be trapped. Mössbauer and parallel mode electron paramagnetic resonance (EPR) studies show that Int1 contains an S1 = 5/2 FeIII center coupled to an SR = 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•)FeIII(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 FeIII-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.

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