Herbicide-degrading alpha-keto acid-dependent enzyme TfdA: Metal coordination environment and mechanistic insights

TfdA is a non-heme iron enzyme which catalyzes the first step in the oxidat ive degradation of the widely used herbicide (2,4-dichlorophenoxy)acetate ( 2,4-D). Like other alpha-keto acid-dependent enzymes, TfdA utilizes a monon uclear Fe(II) center to activate O-2 and oxidize substrate concomitant with the oxidative decarboxylation of alpha-ketoglutarate (alpha-KG). Spectrosc opic analyses of various Cu(II)-substituted and Fe(II)-reconstituted TfdA c omplexes via electron paramagnetic resonance (EPR), electron spin-echo enve lope modulation (ESEEM), and UV-vis spectroscopies have greatly expanded ou r 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 patter n in the Cu(II) EPR signal, for which superhyperfine splitting is attribute d to two equivalent nitrogen donor atoms from two imidazoles,Furthermore, a comparison of the ESEEM spectra obtained in H2O and D2O demonstrates that the metal maintains several solvent-accessible sites, a conclusion corrobor ated by the increase in multiplicity in the EPR superhyperfine splitting ob served in the presence of imidazole. Addition of alpha-KG to the Cu-contain ing enzyme leads to displacement of an equatorial water on copper, as deter mined 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 a lpha-KG chelates to the metal via a C-l carboxylate oxygen and the alpha-ke to oxygen as revealed by characteristic absorption features in the optical spectrum of Fe-TfdA. This binding mode is maintained in the presence of sub strate, although the addition of 2,4-D does alter the metal coordination en vironment, 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 enz yme clavaminate synthase 2 [Zhou et al. (1998) J. Am. Chem. Sec. 120, 13539 -13540]. Nitrosyl adducts of various Fe-TfdA complexes have also been inves tigated 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 ac curate model of the initial oxygen-bound species.