Characterization of an O 2 adduct of an active cobalt-substituted extradiol-cleaving catechol dioxygenase

Andrew J. Fielding, John D Lipscomb, Larry Que

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

The first example of an O 2 adduct of an active Co-substituted oxygenase has been observed in the extradiol ring cleavage of the electron-poor substrate 4-nitrocatechol (4NC) by Co(II)-homoprotocatechuate 2,3-dioxygenase (Co-HPCD). Upon O 2 binding to the high-spin Co(II) (S = 3/ 2) enzyme-substrate complex, an S = 1/ 2 EPR signal exhibiting 59Co hyperfine splitting (A = 24 G) typical of a low-spin Co(III)-superoxide complex was observed. Both the formation and decay of the new intermediate are very slow in comparison to the analogous steps for turnover of 4NC by native high-spin Fe(II)-HPCD, which is likely to remain high-spin upon O 2 binding. A similar but effectively stable S = 1/ 2 intermediate was formed by the inactive [H200N-Co-HPCD(4NC)] variant. The observations presented shed light on the key roles played by the substrate, the second-sphere His200 residue, and the spin state of the metal center in facilitating O 2 binding and activation.

Original languageEnglish (US)
Pages (from-to)796-799
Number of pages4
JournalJournal of the American Chemical Society
Volume134
Issue number2
DOIs
StatePublished - Jan 18 2012

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3,4-dihydroxyphenylacetate 2,3-dioxygenase
Dioxygenases
Cobalt
Substrates
Oxygenases
Superoxides
Paramagnetic resonance
Enzymes
Metals
Chemical activation
Electrons
4-nitrocatechol
catechol

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Characterization of an O 2 adduct of an active cobalt-substituted extradiol-cleaving catechol dioxygenase. / Fielding, Andrew J.; Lipscomb, John D; Que, Larry.

In: Journal of the American Chemical Society, Vol. 134, No. 2, 18.01.2012, p. 796-799.

Research output: Contribution to journalArticle

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N2 - The first example of an O 2 adduct of an active Co-substituted oxygenase has been observed in the extradiol ring cleavage of the electron-poor substrate 4-nitrocatechol (4NC) by Co(II)-homoprotocatechuate 2,3-dioxygenase (Co-HPCD). Upon O 2 binding to the high-spin Co(II) (S = 3/ 2) enzyme-substrate complex, an S = 1/ 2 EPR signal exhibiting 59Co hyperfine splitting (A = 24 G) typical of a low-spin Co(III)-superoxide complex was observed. Both the formation and decay of the new intermediate are very slow in comparison to the analogous steps for turnover of 4NC by native high-spin Fe(II)-HPCD, which is likely to remain high-spin upon O 2 binding. A similar but effectively stable S = 1/ 2 intermediate was formed by the inactive [H200N-Co-HPCD(4NC)] variant. The observations presented shed light on the key roles played by the substrate, the second-sphere His200 residue, and the spin state of the metal center in facilitating O 2 binding and activation.

AB - The first example of an O 2 adduct of an active Co-substituted oxygenase has been observed in the extradiol ring cleavage of the electron-poor substrate 4-nitrocatechol (4NC) by Co(II)-homoprotocatechuate 2,3-dioxygenase (Co-HPCD). Upon O 2 binding to the high-spin Co(II) (S = 3/ 2) enzyme-substrate complex, an S = 1/ 2 EPR signal exhibiting 59Co hyperfine splitting (A = 24 G) typical of a low-spin Co(III)-superoxide complex was observed. Both the formation and decay of the new intermediate are very slow in comparison to the analogous steps for turnover of 4NC by native high-spin Fe(II)-HPCD, which is likely to remain high-spin upon O 2 binding. A similar but effectively stable S = 1/ 2 intermediate was formed by the inactive [H200N-Co-HPCD(4NC)] variant. The observations presented shed light on the key roles played by the substrate, the second-sphere His200 residue, and the spin state of the metal center in facilitating O 2 binding and activation.

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