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

Andrew J. Fielding, John D. Lipscomb, Lawrence Que

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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
Issue number2
StatePublished - Jan 18 2012


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