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

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.