title: Herbicide-degrading alpha-keto acid-dependent enzyme TfdA: metal coordination environment and mechanistic insights.
abstract: TfdA is a non-heme iron enzyme which catalyzes the first step in the oxidative degradation of the widely used herbicide (2, 4-dichlorophenoxy)acetate (2,4-D). Like other alpha-keto acid-dependent enzymes, TfdA utilizes a mononuclear Fe(II) center to activate O(2) and oxidize substrate concomitant with the oxidative decarboxylation of alpha-ketoglutarate (alpha-KG). Spectroscopic analyses of various Cu(II)-substituted and Fe(II)-reconstituted TfdA complexes via electron paramagnetic resonance (EPR), electron spin-echo envelope modulation (ESEEM), and UV-vis spectroscopies have greatly expanded our knowledge of the enzyme's active site. The metal center is coordinated to two histidine residues as indicated by the presence of a five-line pattern in the Cu(II) EPR signal, for which superhyperfine splitting is attributed to two equivalent nitrogen donor atoms from two imidazoles. Furthermore, a comparison of the ESEEM spectra obtained in H(2)O and D(2)O demonstrates that the metal maintains several solvent-accessible sites, a conclusion corroborated by the increase in multiplicity in the EPR superhyperfine splitting observed in the presence of imidazole. Addition of alpha-KG to the Cu-containing enzyme leads to displacement of an equatorial water on copper, as determined by ESEEM analysis. Subsequent addition of 2,4-D leads to the loss of a second water molecule, with retention of a third, axially bound water. In contrast to these results, in Fe(II)-reconstituted TfdA, the cosubstrate alpha-KG chelates to the metal via a C-1 carboxylate oxygen and the alpha-keto oxygen as revealed by characteristic absorption features in the optical spectrum of Fe-TfdA. This binding mode is maintained in the presence of substrate, although the addition of 2,4-D does alter the metal coordination environment, perhaps by creating an O(2)-binding site via solvent displacement. Indeed, loss of solvent to generate an open binding site upon the addition of substrate has also been suggested for the alpha-keto acid-dependent enzyme clavaminate synthase 2 [Zhou et al. (1998) J. Am. Chem. Soc. 120, 13539-13540]. Nitrosyl adducts of various Fe-TfdA complexes have also been investigated by optical and EPR spectroscopy. Of special interest is the tightly bound NO complex of Fe-TfdA.(alpha-KG).(2,4-D), which may represent an accurate model of the initial oxygen-bound species.
authors.affiliation: Department of Chemistry, Center for Metals in Biocatalysis, University of Minnesota, Minneapolis 55455, USA.
authors.forename: E L
authors.initials: EL
authors.lastname: Hegg
authors.forename: A K
authors.initials: AK
authors.lastname: Whiting
authors.forename: R E
authors.initials: RE
authors.lastname: Saari
authors.forename: J
authors.initials: J
authors.lastname: McCracken
authors.forename: R P
authors.initials: RP
authors.lastname: Hausinger
authors.forename: L
authors.initials: L
authors.lastname: Que
mesh_terms: D015084:2,4-Dichlorophenoxyacetic Acid / Q000737:chemistry* / Q000378:metabolism; D000421:Alcaligenes; D001665:Binding Sites; D001673:Biodegradation, Environmental; D003300:Copper / Q000737:chemistry; D003903:Deuterium; D004578:Electron Spin Resonance Spectroscopy; D005290:Ferric Compounds / Q000737:chemistry / Q000378:metabolism; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D008670:Metals / Q000737:chemistry* / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism
chemical_list: D005290:Ferric Compounds; D008024:Ligands; D008670:Metals; D015084:2,4-Dichlorophenoxyacetic Acid; D009569:Nitric Oxide; D003300:Copper; D003903:Deuterium; D007501:Iron; D006899:Mixed Function Oxygenases; C084087:2,4-dichlorophenoxyacetate-alpha-ketoglutarate dioxygenase; D010100:Oxygen