A hyperactive cobalt-substituted extradiol-cleaving catechol dioxygenase

Andrew J. Fielding, Elena G. Kovaleva, Erik R. Farquhar, John D Lipscomb, Larry Que

Research output: Contribution to journalArticle

57 Citations (Scopus)

Abstract

Homoprotocatechuate 2,3-dioxygenase from Brevibacterium fuscum (HPCD) has an Fe(II) center in its active site that can be replaced with Mn(II) or Co(II). Whereas Mn-HPCD exhibits steady-state kinetic parameters comparable to those of Fe-HPCD, Co-HPCD behaves somewhat differently, exhibiting significantly higher K?MO2 and kcat. The high activity of Co-HPCD is surprising, given that cobalt has the highest standard M(III/II) redox potential of the three metals. Comparison of the X-ray crystal structures of the resting and substrate-bound forms of Fe-HPCD, Mn-HPCD, and Co-HPCD shows that metal substitution has no effect on the local ligand environment, the conformational integrity of the active site, or the overall protein structure, suggesting that the protein structure does not differentially tune the potential of the metal center. Analysis of the steady-state kinetics of Co-HPCD suggests that the Co(II) center alters the relative rate constants for the interconversion of intermediates in the catalytic cycle but still allows the dioxygenase reaction to proceed efficiently. When compared with the kinetic data for Fe-HPCD and Mn-HPCD, these results show that dioxygenase catalysis can proceed at high rates over a wide range of metal redox potentials. This is consistent with the proposed mechanism in which the metal mediates electron transfer between the catechol substrate and O2 to form the postulated [M(II)(semiquinone)superoxo] reactive species. These kinetic differences and the spectroscopic properties of Co-HPCD provide new tools with which to explore the unique O2 activation mechanism associated with the extradiol dioxygenase family.

Original languageEnglish (US)
Pages (from-to)341-355
Number of pages15
JournalJournal of Biological Inorganic Chemistry
Volume16
Issue number2
DOIs
StatePublished - Feb 1 2011

Fingerprint

Dioxygenases
Cobalt
Metals
3,4-dihydroxyphenylacetate 2,3-dioxygenase
Kinetics
Oxidation-Reduction
Catalytic Domain
Brevibacterium
Substrates
Catalysis
Kinetic parameters
Rate constants
Proteins
Substitution reactions
Crystal structure
Chemical activation
X-Rays
catechol
Electrons
Ligands

Keywords

  • Bioinorganic chemistry
  • Cobalt(II)
  • Extradiol-cleaving catechol dioxygenase

Cite this

A hyperactive cobalt-substituted extradiol-cleaving catechol dioxygenase. / Fielding, Andrew J.; Kovaleva, Elena G.; Farquhar, Erik R.; Lipscomb, John D; Que, Larry.

In: Journal of Biological Inorganic Chemistry, Vol. 16, No. 2, 01.02.2011, p. 341-355.

Research output: Contribution to journalArticle

Fielding, Andrew J. ; Kovaleva, Elena G. ; Farquhar, Erik R. ; Lipscomb, John D ; Que, Larry. / A hyperactive cobalt-substituted extradiol-cleaving catechol dioxygenase. In: Journal of Biological Inorganic Chemistry. 2011 ; Vol. 16, No. 2. pp. 341-355.
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AB - Homoprotocatechuate 2,3-dioxygenase from Brevibacterium fuscum (HPCD) has an Fe(II) center in its active site that can be replaced with Mn(II) or Co(II). Whereas Mn-HPCD exhibits steady-state kinetic parameters comparable to those of Fe-HPCD, Co-HPCD behaves somewhat differently, exhibiting significantly higher K?MO2 and kcat. The high activity of Co-HPCD is surprising, given that cobalt has the highest standard M(III/II) redox potential of the three metals. Comparison of the X-ray crystal structures of the resting and substrate-bound forms of Fe-HPCD, Mn-HPCD, and Co-HPCD shows that metal substitution has no effect on the local ligand environment, the conformational integrity of the active site, or the overall protein structure, suggesting that the protein structure does not differentially tune the potential of the metal center. Analysis of the steady-state kinetics of Co-HPCD suggests that the Co(II) center alters the relative rate constants for the interconversion of intermediates in the catalytic cycle but still allows the dioxygenase reaction to proceed efficiently. When compared with the kinetic data for Fe-HPCD and Mn-HPCD, these results show that dioxygenase catalysis can proceed at high rates over a wide range of metal redox potentials. This is consistent with the proposed mechanism in which the metal mediates electron transfer between the catechol substrate and O2 to form the postulated [M(II)(semiquinone)superoxo] reactive species. These kinetic differences and the spectroscopic properties of Co-HPCD provide new tools with which to explore the unique O2 activation mechanism associated with the extradiol dioxygenase family.

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