title: Characterization of an O2 adduct of an active cobalt-substituted extradiol-cleaving catechol dioxygenase.
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 (59)Co 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.
authors.affiliation: Department of Chemistry and Center for Metals in Biocatalysis, University of Minnesota, Minneapolis, Minnesota 55455, USA.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Fielding
authors.forename: John D
authors.initials: JD
authors.lastname: Lipscomb
authors.forename: Lawrence
authors.initials: L
authors.lastname: Que
mesh_terms: D002396:Catechols / Q000737:chemistry / Q000378:metabolism; D003035:Cobalt / Q000737:chemistry*; D049308:Dioxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D015394:Molecular Structure; D009154:Mutation; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*
chemical_list: D002396:Catechols; D003035:Cobalt; D007501:Iron; D049308:Dioxygenases; C020055:3,4-dihydroxyphenylacetate 2,3-dioxygenase; D010100:Oxygen