The first direct characterization of a high-valent iron intermediate in the reaction of an α-ketoglutarate-dependent dioxygenase: A high-spin Fe(IV) complex in taurine/α-ketoglutarate dioxygenase (TauD) from Escherichia coli

John C. Price, Eric W. Barr, Bhramara Tirupati, Joseph M. Bollinger, Jr., Carsten Krebs

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Abstract

The Fe(II)- and α-ketoglutarate(αKG)-dependent dioxygenases have roles in synthesis of collagen and sensing of oxygen in mammals, in acquisition of nutrients and synthesis of antibiotics in microbes, and in repair of alkylated DNA in both. A consensus mechanism for these enzymes, involving (i) addition of O2 to a five-coordinate, (His)2(Asp)-facially coordinated Fe(II) center to which αKG is also bound via its C-1 carboxylate and ketone oxygen; (ii) attack of the uncoordinated oxygen of the bound O2 on the ketone carbonyl of αKG to form a bicyclic Fe(IV)-peroxyhemiketal complex; (iii) decarboxylation of this complex concomitantly with formation of an oxo-ferryl (Fe(IV)=O2-) intermediate; and (iv) hydroxylation of the substrate by the Fe(IV)=O2- complex via a substrate radical intermediate, has repeatedly been proposed, but none of the postulated intermediates occurring after addition of O2 has ever been detected. In this work, an oxidized Fe intermediate in the reaction of one of these enzymes, taurine/α-ketoglutarate dioxygenase (TauD) from Escherichia coli, has been directly demonstrated by rapid kinetic and spectroscopic methods. Characterization of the intermediate and its one-electron-reduced form (obtained by low-temperature γ-radiolysis of the trapped intermediate) by Mössbauer and electron paramagnetic resonance spectroscopies establishes that it is a high-spin, formally Fe(IV) complex. Its Mössbauer isomer shift is, however, significantly greater than those of other known Fe(IV) complexes, suggesting that the iron ligands in the TauD intermediate confer significant Fe(III) character to the high-valent site by strong electron donation. The properties of the complex and previous results on related αKG-dependent dioxygenases and other non-heme-Fe(II)-dependent, O2-activating enzymes suggest that the TauD intermediate is most probably either the Fe(IV)-peroxyhemiketal complex or the taurine-hydroxylating Fe(IV)=O2- species. The detection of this intermediate sets the stage for a more detailed dissection of the TauD reaction mechanism than has previously been reported for any other member of this important enzyme family.

Original languageEnglish (US)
Pages (from-to)7497-7508
Number of pages12
JournalBiochemistry
Volume42
Issue number24
DOIs
StatePublished - Jun 24 2003

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Dioxygenases
Taurine
Escherichia coli
Iron
Enzymes
Oxygen
Ketones
Electrons
Dissection
Radiolysis
Hydroxylation
Decarboxylation
Mammals
Electron Spin Resonance Spectroscopy
Substrates
Viperidae
DNA Repair
Isomers
Nutrients
Paramagnetic resonance

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

@article{466a1911c1494949819beded97d8730a,
title = "The first direct characterization of a high-valent iron intermediate in the reaction of an α-ketoglutarate-dependent dioxygenase: A high-spin Fe(IV) complex in taurine/α-ketoglutarate dioxygenase (TauD) from Escherichia coli",
abstract = "The Fe(II)- and α-ketoglutarate(αKG)-dependent dioxygenases have roles in synthesis of collagen and sensing of oxygen in mammals, in acquisition of nutrients and synthesis of antibiotics in microbes, and in repair of alkylated DNA in both. A consensus mechanism for these enzymes, involving (i) addition of O2 to a five-coordinate, (His)2(Asp)-facially coordinated Fe(II) center to which αKG is also bound via its C-1 carboxylate and ketone oxygen; (ii) attack of the uncoordinated oxygen of the bound O2 on the ketone carbonyl of αKG to form a bicyclic Fe(IV)-peroxyhemiketal complex; (iii) decarboxylation of this complex concomitantly with formation of an oxo-ferryl (Fe(IV)=O2-) intermediate; and (iv) hydroxylation of the substrate by the Fe(IV)=O2- complex via a substrate radical intermediate, has repeatedly been proposed, but none of the postulated intermediates occurring after addition of O2 has ever been detected. In this work, an oxidized Fe intermediate in the reaction of one of these enzymes, taurine/α-ketoglutarate dioxygenase (TauD) from Escherichia coli, has been directly demonstrated by rapid kinetic and spectroscopic methods. Characterization of the intermediate and its one-electron-reduced form (obtained by low-temperature γ-radiolysis of the trapped intermediate) by M{\"o}ssbauer and electron paramagnetic resonance spectroscopies establishes that it is a high-spin, formally Fe(IV) complex. Its M{\"o}ssbauer isomer shift is, however, significantly greater than those of other known Fe(IV) complexes, suggesting that the iron ligands in the TauD intermediate confer significant Fe(III) character to the high-valent site by strong electron donation. The properties of the complex and previous results on related αKG-dependent dioxygenases and other non-heme-Fe(II)-dependent, O2-activating enzymes suggest that the TauD intermediate is most probably either the Fe(IV)-peroxyhemiketal complex or the taurine-hydroxylating Fe(IV)=O2- species. The detection of this intermediate sets the stage for a more detailed dissection of the TauD reaction mechanism than has previously been reported for any other member of this important enzyme family.",
author = "Price, {John C.} and Barr, {Eric W.} and Bhramara Tirupati and {Bollinger, Jr.}, {Joseph M.} and Carsten Krebs",
year = "2003",
month = "6",
day = "24",
doi = "10.1021/bi030011f",
language = "English (US)",
volume = "42",
pages = "7497--7508",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "24",

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TY - JOUR

T1 - The first direct characterization of a high-valent iron intermediate in the reaction of an α-ketoglutarate-dependent dioxygenase

T2 - A high-spin Fe(IV) complex in taurine/α-ketoglutarate dioxygenase (TauD) from Escherichia coli

AU - Price, John C.

AU - Barr, Eric W.

AU - Tirupati, Bhramara

AU - Bollinger, Jr., Joseph M.

AU - Krebs, Carsten

PY - 2003/6/24

Y1 - 2003/6/24

N2 - The Fe(II)- and α-ketoglutarate(αKG)-dependent dioxygenases have roles in synthesis of collagen and sensing of oxygen in mammals, in acquisition of nutrients and synthesis of antibiotics in microbes, and in repair of alkylated DNA in both. A consensus mechanism for these enzymes, involving (i) addition of O2 to a five-coordinate, (His)2(Asp)-facially coordinated Fe(II) center to which αKG is also bound via its C-1 carboxylate and ketone oxygen; (ii) attack of the uncoordinated oxygen of the bound O2 on the ketone carbonyl of αKG to form a bicyclic Fe(IV)-peroxyhemiketal complex; (iii) decarboxylation of this complex concomitantly with formation of an oxo-ferryl (Fe(IV)=O2-) intermediate; and (iv) hydroxylation of the substrate by the Fe(IV)=O2- complex via a substrate radical intermediate, has repeatedly been proposed, but none of the postulated intermediates occurring after addition of O2 has ever been detected. In this work, an oxidized Fe intermediate in the reaction of one of these enzymes, taurine/α-ketoglutarate dioxygenase (TauD) from Escherichia coli, has been directly demonstrated by rapid kinetic and spectroscopic methods. Characterization of the intermediate and its one-electron-reduced form (obtained by low-temperature γ-radiolysis of the trapped intermediate) by Mössbauer and electron paramagnetic resonance spectroscopies establishes that it is a high-spin, formally Fe(IV) complex. Its Mössbauer isomer shift is, however, significantly greater than those of other known Fe(IV) complexes, suggesting that the iron ligands in the TauD intermediate confer significant Fe(III) character to the high-valent site by strong electron donation. The properties of the complex and previous results on related αKG-dependent dioxygenases and other non-heme-Fe(II)-dependent, O2-activating enzymes suggest that the TauD intermediate is most probably either the Fe(IV)-peroxyhemiketal complex or the taurine-hydroxylating Fe(IV)=O2- species. The detection of this intermediate sets the stage for a more detailed dissection of the TauD reaction mechanism than has previously been reported for any other member of this important enzyme family.

AB - The Fe(II)- and α-ketoglutarate(αKG)-dependent dioxygenases have roles in synthesis of collagen and sensing of oxygen in mammals, in acquisition of nutrients and synthesis of antibiotics in microbes, and in repair of alkylated DNA in both. A consensus mechanism for these enzymes, involving (i) addition of O2 to a five-coordinate, (His)2(Asp)-facially coordinated Fe(II) center to which αKG is also bound via its C-1 carboxylate and ketone oxygen; (ii) attack of the uncoordinated oxygen of the bound O2 on the ketone carbonyl of αKG to form a bicyclic Fe(IV)-peroxyhemiketal complex; (iii) decarboxylation of this complex concomitantly with formation of an oxo-ferryl (Fe(IV)=O2-) intermediate; and (iv) hydroxylation of the substrate by the Fe(IV)=O2- complex via a substrate radical intermediate, has repeatedly been proposed, but none of the postulated intermediates occurring after addition of O2 has ever been detected. In this work, an oxidized Fe intermediate in the reaction of one of these enzymes, taurine/α-ketoglutarate dioxygenase (TauD) from Escherichia coli, has been directly demonstrated by rapid kinetic and spectroscopic methods. Characterization of the intermediate and its one-electron-reduced form (obtained by low-temperature γ-radiolysis of the trapped intermediate) by Mössbauer and electron paramagnetic resonance spectroscopies establishes that it is a high-spin, formally Fe(IV) complex. Its Mössbauer isomer shift is, however, significantly greater than those of other known Fe(IV) complexes, suggesting that the iron ligands in the TauD intermediate confer significant Fe(III) character to the high-valent site by strong electron donation. The properties of the complex and previous results on related αKG-dependent dioxygenases and other non-heme-Fe(II)-dependent, O2-activating enzymes suggest that the TauD intermediate is most probably either the Fe(IV)-peroxyhemiketal complex or the taurine-hydroxylating Fe(IV)=O2- species. The detection of this intermediate sets the stage for a more detailed dissection of the TauD reaction mechanism than has previously been reported for any other member of this important enzyme family.

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