title: X-ray absorption spectroscopic analysis of Fe(II) and Cu(II) forms of a herbicide-degrading alpha-ketoglutarate dioxygenase.
abstract: The first step in the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) by Ralstonia eutropha JMP134 is catalyzed by the alpha-ketoglutarate (alpha-KG)-dependent dioxygenase TfdA. Previously, EPR and ESEEM studies on inactive Cu(II)-substituted TfdA suggested a mixture of nitrogen/oxygen coordination with two imidazole-like ligands. Differences between the spectra for Cu TfdA and alpha-KG- and 2,4-D-treated samples were interpreted as a rearrangement of the g-tensor principal axis system. Herein, we report the use of X-ray absorption spectroscopy (XAS) to further characterize the metal coordination environment of Cu TfdA as well as that in the active, wild-type Fe(II) enzyme. The EXAFS data are interpreted in terms of four N/O ligands (two imidazole-like) in the Cu TfdA sample and six N/O ligands (one or two imidazole-like) in the Fe TfdA sample. Addition of alpha-KG results in no significant structural change in coordination for Cu or Fe TfdA. However, addition of 2,4-D results in a decrease in the number of imidazole ligands in both Cu and Fe TfdA. Since this change is seen both in the Fe and Cu EXAFS, loss of one histidine ligand upon 2,4-D addition best describes the phenomenon. These XAS data clearly demonstrate that changes occur in the atomic environment of the metallocenter upon substrate binding.
authors.affiliation: Center for Metalloenzyme Studies, University of Georgia, Athens 30602-2556, USA.
authors.forename: N J
authors.initials: NJ
authors.lastname: Cosper
authors.forename: C M
authors.initials: CM
authors.lastname: Stålhandske
authors.forename: R E
authors.initials: RE
authors.lastname: Saari
authors.forename: R P
authors.initials: RP
authors.lastname: Hausinger
authors.forename: R A
authors.initials: RA
authors.lastname: Scott
mesh_terms: D015084:2,4-Dichlorophenoxyacetic Acid / Q000737:chemistry / Q000378:metabolism; D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D006540:Herbicides / Q000378:metabolism*; D007501:Iron / Q000737:chemistry / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000737:chemistry / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D013057:Spectrum Analysis / Q000379:methods; D014965:X-Rays
chemical_list: D006540:Herbicides; D007656:Ketoglutaric Acids; D015084:2,4-Dichlorophenoxyacetic Acid; D003300:Copper; D007501:Iron; D006899:Mixed Function Oxygenases; C084087:2,4-dichlorophenoxyacetate-alpha-ketoglutarate dioxygenase