Characterization of an O2 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(11)-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 Co-59 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.