Trapping and spectroscopic characterization of an FeIII-superoxo intermediate from a nonheme mononuclear iron-containing enzyme

Michael M. Mbughuni, Mrinmoy Chakrabarti, Joshua A. Hayden, Emile L. Bominaar, Michael P. Hendrich, Eckard Münck, John D. Lipscomb

Research output: Contribution to journalArticle

103 Citations (Scopus)

Abstract

FeIII-O2 •- intermediates are well known in heme enzymes, but none have been characterized in the nonheme mononuclear FeII enzyme family. Many steps in the O2 activation and reaction cycle of FeII-containing homoprotocatechuate 2,3-dioxygenase are made detectable by using the alternative substrate 4-nitrocatechol (4NC) and mutation of the active site His200 to Asn (H200N). Here, the first intermediate (Int-1) observed after adding O2 to the H200N-4NC complex is trapped and characterized using EPR and Mössbauer (MB) spectroscopies. Int-1 is a high-spin (S1 = 5/2) FeIII antiferromagnetically (AF) coupled to an S2 = 1/2 radical (J ≈ 6 cm-1 in H = JS1•S2). It exhibits parallel-mode EPR signals at g = 8.17 from the S = 2 multiplet, and g = 8.8 and 11.6 from the S = 3 multiplet. These signals are broadened significantly by 17O2 hyperfine interactions (A17O ≈ 180 MHz). Thus, Int-1 is an AF-coupled FeIII-O2 •- species. The experimental observations are supported by density functional theory calculations that show nearly complete transfer of spin density to the bound O2. Int-1 decays to form a second intermediate (Int-2). MB spectra show that it is also an AF-coupled Fe III-radical complex. Int-2 exhibits an EPR signal at g = 8.05 arising from an S = 2 state. The signal is only slightly broadened by 17O2 (<3% spin delocalization), suggesting that Int-2 is a peroxo-FeIII-4NC semiquinone radical species. Our results demonstrate facile electron transfer between FeII, O2, and the organic ligand, thereby supporting the proposed wild-type enzyme mechanism.

Original languageEnglish (US)
Pages (from-to)16788-16793
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume107
Issue number39
DOIs
StatePublished - Sep 28 2010

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3,4-dihydroxyphenylacetate 2,3-dioxygenase
Iron
Enzymes
Heme
Catalytic Domain
Spectrum Analysis
Electrons
Ligands
Mutation
4-nitrocatechol
semiquinone radicals

Keywords

  • Oxygen activation
  • Oxygenase
  • Spectroscopy
  • Superoxide

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Trapping and spectroscopic characterization of an FeIII-superoxo intermediate from a nonheme mononuclear iron-containing enzyme. / Mbughuni, Michael M.; Chakrabarti, Mrinmoy; Hayden, Joshua A.; Bominaar, Emile L.; Hendrich, Michael P.; Münck, Eckard; Lipscomb, John D.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 107, No. 39, 28.09.2010, p. 16788-16793.

Research output: Contribution to journalArticle

Mbughuni, Michael M. ; Chakrabarti, Mrinmoy ; Hayden, Joshua A. ; Bominaar, Emile L. ; Hendrich, Michael P. ; Münck, Eckard ; Lipscomb, John D. / Trapping and spectroscopic characterization of an FeIII-superoxo intermediate from a nonheme mononuclear iron-containing enzyme. In: Proceedings of the National Academy of Sciences of the United States of America. 2010 ; Vol. 107, No. 39. pp. 16788-16793.
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abstract = "FeIII-O2 •- intermediates are well known in heme enzymes, but none have been characterized in the nonheme mononuclear FeII enzyme family. Many steps in the O2 activation and reaction cycle of FeII-containing homoprotocatechuate 2,3-dioxygenase are made detectable by using the alternative substrate 4-nitrocatechol (4NC) and mutation of the active site His200 to Asn (H200N). Here, the first intermediate (Int-1) observed after adding O2 to the H200N-4NC complex is trapped and characterized using EPR and M{\"o}ssbauer (MB) spectroscopies. Int-1 is a high-spin (S1 = 5/2) FeIII antiferromagnetically (AF) coupled to an S2 = 1/2 radical (J ≈ 6 cm-1 in H = JS1•S2). It exhibits parallel-mode EPR signals at g = 8.17 from the S = 2 multiplet, and g = 8.8 and 11.6 from the S = 3 multiplet. These signals are broadened significantly by 17O2 hyperfine interactions (A17O ≈ 180 MHz). Thus, Int-1 is an AF-coupled FeIII-O2 •- species. The experimental observations are supported by density functional theory calculations that show nearly complete transfer of spin density to the bound O2. Int-1 decays to form a second intermediate (Int-2). MB spectra show that it is also an AF-coupled Fe III-radical complex. Int-2 exhibits an EPR signal at g = 8.05 arising from an S = 2 state. The signal is only slightly broadened by 17O2 (<3{\%} spin delocalization), suggesting that Int-2 is a peroxo-FeIII-4NC semiquinone radical species. Our results demonstrate facile electron transfer between FeII, O2, and the organic ligand, thereby supporting the proposed wild-type enzyme mechanism.",
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T1 - Trapping and spectroscopic characterization of an FeIII-superoxo intermediate from a nonheme mononuclear iron-containing enzyme

AU - Mbughuni, Michael M.

AU - Chakrabarti, Mrinmoy

AU - Hayden, Joshua A.

AU - Bominaar, Emile L.

AU - Hendrich, Michael P.

AU - Münck, Eckard

AU - Lipscomb, John D.

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N2 - FeIII-O2 •- intermediates are well known in heme enzymes, but none have been characterized in the nonheme mononuclear FeII enzyme family. Many steps in the O2 activation and reaction cycle of FeII-containing homoprotocatechuate 2,3-dioxygenase are made detectable by using the alternative substrate 4-nitrocatechol (4NC) and mutation of the active site His200 to Asn (H200N). Here, the first intermediate (Int-1) observed after adding O2 to the H200N-4NC complex is trapped and characterized using EPR and Mössbauer (MB) spectroscopies. Int-1 is a high-spin (S1 = 5/2) FeIII antiferromagnetically (AF) coupled to an S2 = 1/2 radical (J ≈ 6 cm-1 in H = JS1•S2). It exhibits parallel-mode EPR signals at g = 8.17 from the S = 2 multiplet, and g = 8.8 and 11.6 from the S = 3 multiplet. These signals are broadened significantly by 17O2 hyperfine interactions (A17O ≈ 180 MHz). Thus, Int-1 is an AF-coupled FeIII-O2 •- species. The experimental observations are supported by density functional theory calculations that show nearly complete transfer of spin density to the bound O2. Int-1 decays to form a second intermediate (Int-2). MB spectra show that it is also an AF-coupled Fe III-radical complex. Int-2 exhibits an EPR signal at g = 8.05 arising from an S = 2 state. The signal is only slightly broadened by 17O2 (<3% spin delocalization), suggesting that Int-2 is a peroxo-FeIII-4NC semiquinone radical species. Our results demonstrate facile electron transfer between FeII, O2, and the organic ligand, thereby supporting the proposed wild-type enzyme mechanism.

AB - FeIII-O2 •- intermediates are well known in heme enzymes, but none have been characterized in the nonheme mononuclear FeII enzyme family. Many steps in the O2 activation and reaction cycle of FeII-containing homoprotocatechuate 2,3-dioxygenase are made detectable by using the alternative substrate 4-nitrocatechol (4NC) and mutation of the active site His200 to Asn (H200N). Here, the first intermediate (Int-1) observed after adding O2 to the H200N-4NC complex is trapped and characterized using EPR and Mössbauer (MB) spectroscopies. Int-1 is a high-spin (S1 = 5/2) FeIII antiferromagnetically (AF) coupled to an S2 = 1/2 radical (J ≈ 6 cm-1 in H = JS1•S2). It exhibits parallel-mode EPR signals at g = 8.17 from the S = 2 multiplet, and g = 8.8 and 11.6 from the S = 3 multiplet. These signals are broadened significantly by 17O2 hyperfine interactions (A17O ≈ 180 MHz). Thus, Int-1 is an AF-coupled FeIII-O2 •- species. The experimental observations are supported by density functional theory calculations that show nearly complete transfer of spin density to the bound O2. Int-1 decays to form a second intermediate (Int-2). MB spectra show that it is also an AF-coupled Fe III-radical complex. Int-2 exhibits an EPR signal at g = 8.05 arising from an S = 2 state. The signal is only slightly broadened by 17O2 (<3% spin delocalization), suggesting that Int-2 is a peroxo-FeIII-4NC semiquinone radical species. Our results demonstrate facile electron transfer between FeII, O2, and the organic ligand, thereby supporting the proposed wild-type enzyme mechanism.

KW - Oxygen activation

KW - Oxygenase

KW - Spectroscopy

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