NO binding to Mn-substituted homoprotocatechuate 2,3-dioxygenase

Relationship to O2 reactivity

Joshua A. Hayden, Erik R. Farquhar, Lawrence Que, John D. Lipscomb, Michael P. Hendrich

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Iron(II)-containing homoprotocatechuate 2,3-dioxygenase (FeHPCD) activates O2 to catalyze the aromatic ring opening of homoprotocatechuate (HPCA). The enzyme requires FeII for catalysis, but MnII can be substituted (MnHPCD) with essentially no change in the steady-state kinetic parameters. Near simultaneous O2 and HPCA activation has been proposed to occur through transfer of an electron or electrons from HPCA to O2 through the divalent metal. In O2 reactions with MnHPCD-HPCA and the 4-nitrocatechol (4NC) complex of the His200Asn (H200N) variant of FeHPCD, this transfer has resulted in the detection of a transient MIII-O2 ·- species that is not observed during turnover of the wild-type FeHPCD. The factors governing formation of the MIII-O2 ·- species are explored here by EPR spectroscopy using MnHPCD and nitric oxide (NO) as an O2 surrogate. Both the HPCA and the dihydroxymandelic substrate complexes of MnHPCD bind NO, thus representing the first reported stable MnNO complexes of a nonheme enzyme. In contrast, the free enzyme, the MnHPCD-4NC complex, and the MnH200N and MnH200Q variants with or without HPCA bound do not bind NO. The MnHPCD-ligand complexes that bind NO are also active in normal O 2-linked turnover, whereas the others are inactive. Past studies have shown that FeHPCD and the analogous variants and catecholic ligand complexes all bind NO, and are active in normal turnover. This contrasting behavior may stem from the ability of the enzyme to maintain the approximately 0.8-V difference in the solution redox potentials of FeII and Mn II. Owing to the higher potential of Mn, the formation of the NO adduct or the O2 adduct requires both strong charge donation from the bound catecholic ligand and additional stabilization by interaction with the active-site His200. The same nonoptimal electronic and structural forces that prevent NO and O2 binding in MnHPCD variants may lead to inefficient electron transfer from the catecholic substrate to the metal center in variants of FeHPCD during O2-linked turnover. Accordingly, past studies have shown that intermediate FeIII species are observed for these mutant enzymes.

Original languageEnglish (US)
Pages (from-to)717-728
Number of pages12
JournalJournal of Biological Inorganic Chemistry
Volume18
Issue number7
DOIs
StatePublished - Oct 1 2013

Fingerprint

3,4-dihydroxyphenylacetate 2,3-dioxygenase
Nitric Oxide
Enzymes
Electrons
Ligands
Metals
Substrates
Catalysis
Kinetic parameters
Oxidation-Reduction
Paramagnetic resonance

Keywords

  • Electron paramagnetic resonance
  • Homoprotocatechuate dioxygenase
  • Manganese(II)
  • Manganese(III)
  • Nitric oxide

Cite this

NO binding to Mn-substituted homoprotocatechuate 2,3-dioxygenase : Relationship to O2 reactivity. / Hayden, Joshua A.; Farquhar, Erik R.; Que, Lawrence; Lipscomb, John D.; Hendrich, Michael P.

In: Journal of Biological Inorganic Chemistry, Vol. 18, No. 7, 01.10.2013, p. 717-728.

Research output: Contribution to journalArticle

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abstract = "Iron(II)-containing homoprotocatechuate 2,3-dioxygenase (FeHPCD) activates O2 to catalyze the aromatic ring opening of homoprotocatechuate (HPCA). The enzyme requires FeII for catalysis, but MnII can be substituted (MnHPCD) with essentially no change in the steady-state kinetic parameters. Near simultaneous O2 and HPCA activation has been proposed to occur through transfer of an electron or electrons from HPCA to O2 through the divalent metal. In O2 reactions with MnHPCD-HPCA and the 4-nitrocatechol (4NC) complex of the His200Asn (H200N) variant of FeHPCD, this transfer has resulted in the detection of a transient MIII-O2 ·- species that is not observed during turnover of the wild-type FeHPCD. The factors governing formation of the MIII-O2 ·- species are explored here by EPR spectroscopy using MnHPCD and nitric oxide (NO) as an O2 surrogate. Both the HPCA and the dihydroxymandelic substrate complexes of MnHPCD bind NO, thus representing the first reported stable MnNO complexes of a nonheme enzyme. In contrast, the free enzyme, the MnHPCD-4NC complex, and the MnH200N and MnH200Q variants with or without HPCA bound do not bind NO. The MnHPCD-ligand complexes that bind NO are also active in normal O 2-linked turnover, whereas the others are inactive. Past studies have shown that FeHPCD and the analogous variants and catecholic ligand complexes all bind NO, and are active in normal turnover. This contrasting behavior may stem from the ability of the enzyme to maintain the approximately 0.8-V difference in the solution redox potentials of FeII and Mn II. Owing to the higher potential of Mn, the formation of the NO adduct or the O2 adduct requires both strong charge donation from the bound catecholic ligand and additional stabilization by interaction with the active-site His200. The same nonoptimal electronic and structural forces that prevent NO and O2 binding in MnHPCD variants may lead to inefficient electron transfer from the catecholic substrate to the metal center in variants of FeHPCD during O2-linked turnover. Accordingly, past studies have shown that intermediate FeIII species are observed for these mutant enzymes.",
keywords = "Electron paramagnetic resonance, Homoprotocatechuate dioxygenase, Manganese(II), Manganese(III), Nitric oxide",
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T1 - NO binding to Mn-substituted homoprotocatechuate 2,3-dioxygenase

T2 - Relationship to O2 reactivity

AU - Hayden, Joshua A.

AU - Farquhar, Erik R.

AU - Que, Lawrence

AU - Lipscomb, John D.

AU - Hendrich, Michael P.

PY - 2013/10/1

Y1 - 2013/10/1

N2 - Iron(II)-containing homoprotocatechuate 2,3-dioxygenase (FeHPCD) activates O2 to catalyze the aromatic ring opening of homoprotocatechuate (HPCA). The enzyme requires FeII for catalysis, but MnII can be substituted (MnHPCD) with essentially no change in the steady-state kinetic parameters. Near simultaneous O2 and HPCA activation has been proposed to occur through transfer of an electron or electrons from HPCA to O2 through the divalent metal. In O2 reactions with MnHPCD-HPCA and the 4-nitrocatechol (4NC) complex of the His200Asn (H200N) variant of FeHPCD, this transfer has resulted in the detection of a transient MIII-O2 ·- species that is not observed during turnover of the wild-type FeHPCD. The factors governing formation of the MIII-O2 ·- species are explored here by EPR spectroscopy using MnHPCD and nitric oxide (NO) as an O2 surrogate. Both the HPCA and the dihydroxymandelic substrate complexes of MnHPCD bind NO, thus representing the first reported stable MnNO complexes of a nonheme enzyme. In contrast, the free enzyme, the MnHPCD-4NC complex, and the MnH200N and MnH200Q variants with or without HPCA bound do not bind NO. The MnHPCD-ligand complexes that bind NO are also active in normal O 2-linked turnover, whereas the others are inactive. Past studies have shown that FeHPCD and the analogous variants and catecholic ligand complexes all bind NO, and are active in normal turnover. This contrasting behavior may stem from the ability of the enzyme to maintain the approximately 0.8-V difference in the solution redox potentials of FeII and Mn II. Owing to the higher potential of Mn, the formation of the NO adduct or the O2 adduct requires both strong charge donation from the bound catecholic ligand and additional stabilization by interaction with the active-site His200. The same nonoptimal electronic and structural forces that prevent NO and O2 binding in MnHPCD variants may lead to inefficient electron transfer from the catecholic substrate to the metal center in variants of FeHPCD during O2-linked turnover. Accordingly, past studies have shown that intermediate FeIII species are observed for these mutant enzymes.

AB - Iron(II)-containing homoprotocatechuate 2,3-dioxygenase (FeHPCD) activates O2 to catalyze the aromatic ring opening of homoprotocatechuate (HPCA). The enzyme requires FeII for catalysis, but MnII can be substituted (MnHPCD) with essentially no change in the steady-state kinetic parameters. Near simultaneous O2 and HPCA activation has been proposed to occur through transfer of an electron or electrons from HPCA to O2 through the divalent metal. In O2 reactions with MnHPCD-HPCA and the 4-nitrocatechol (4NC) complex of the His200Asn (H200N) variant of FeHPCD, this transfer has resulted in the detection of a transient MIII-O2 ·- species that is not observed during turnover of the wild-type FeHPCD. The factors governing formation of the MIII-O2 ·- species are explored here by EPR spectroscopy using MnHPCD and nitric oxide (NO) as an O2 surrogate. Both the HPCA and the dihydroxymandelic substrate complexes of MnHPCD bind NO, thus representing the first reported stable MnNO complexes of a nonheme enzyme. In contrast, the free enzyme, the MnHPCD-4NC complex, and the MnH200N and MnH200Q variants with or without HPCA bound do not bind NO. The MnHPCD-ligand complexes that bind NO are also active in normal O 2-linked turnover, whereas the others are inactive. Past studies have shown that FeHPCD and the analogous variants and catecholic ligand complexes all bind NO, and are active in normal turnover. This contrasting behavior may stem from the ability of the enzyme to maintain the approximately 0.8-V difference in the solution redox potentials of FeII and Mn II. Owing to the higher potential of Mn, the formation of the NO adduct or the O2 adduct requires both strong charge donation from the bound catecholic ligand and additional stabilization by interaction with the active-site His200. The same nonoptimal electronic and structural forces that prevent NO and O2 binding in MnHPCD variants may lead to inefficient electron transfer from the catecholic substrate to the metal center in variants of FeHPCD during O2-linked turnover. Accordingly, past studies have shown that intermediate FeIII species are observed for these mutant enzymes.

KW - Electron paramagnetic resonance

KW - Homoprotocatechuate dioxygenase

KW - Manganese(II)

KW - Manganese(III)

KW - Nitric oxide

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