X-ray absorption spectroscopic analysis of Fe(II) and Cu(II) forms of a herbicide-degrading alpha-ketoglutarate dioxygenase

The first step in the degradation of 2,4-dichlorophenoxyacetic acid (2, 4-D ) by Ralstonia eutropha JMP134 is catalyzed by the alpha-ketoglutarate (alp ha-KG)-dependent dioxygenase TfdA. Previously, EPR and ESEEM studies on ina ctive Cu(II)-substituted TfdA suggested a mixture of nitrogen/oxygen coordi nation 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 rear rangement of the g-tensor principal axis system. Herein, we report the use of X-ray absorption spectroscopy (XAS) to further characterize the metal co ordination environment of Cu TMA as well as that in the active, wild-type F e(II) enzyme. The EXAFS data are interpreted in terms of four NIO ligands ( two imidazole-like) in the Cu TfdA sample and six N/O ligands tone or two i midazole-like) in the Fe TfdA sample. Addition of alpha-KG results in no si gnificant structural change in coordination fur Cu or Fe TFdA. However, add ition 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 bath in the Fe and Cu EXAFS, loss of one histidine ligand upon 2,4-D addition best describes the phenom enon. These XAS data clearly demonstrate that changes occur in the atomic e nvironment of the metallocenter upon substrate binding.