Characterization of an O2 Adduct of an Active Cobalt-Substituted Extradiol-Cleaving Catechol Dioxygenase

The first example of an O2 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 O2 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 O2 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 O2 binding and activation.