Article,
Herbicide-degrading alpha-keto acid-dependent enzyme TfdA: metal coordination environment and mechanistic insights.
Biochemistry, 38 (50): 16714--16726 (December 1999)
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.
