Understanding the Origin of Suicide Inactivation in the Extradiol Dioxygenases Timothy E. Machonkin, Department of Chemistry, Whitman College, Walla Walla, WA 99362
Our aim is to understand why most chlorinated substrates lead to suicide inactivation of non-heme Fe(II)-containing ring-cleaving dioxygenases, such as the well-characterized extradiol catechol dioxygenases. We are using a multifaceted approach: 1. Paramagnetic NMR studies of enzyme-bound substrate, in order to characterize its pKa and electronic structure.
2. Characterization of model complexes with ligands similar to those found in the enzyme active site (facialcapping triad of N, N, O).
Homology-based structural model of 2,6dichloro-F-hydroquinone dioxygenase:
Aqueous substrate (4,4'-difluoro-2,3-dihydroxybiphenyl): Structure of L2Fe complex: Distal Fluorine
3. Biochemical studies of unusual Fe(II)-containing ring-cleaving dioxygenases that can cleave chlorinated substrates.
Proximal Fluorine
pH = 6.9 pH = 7.4 pH = 7.9 H159
pH = 8.5 pH = 10.9
Enzyme-bound substrate:
NMR evidence for L2Fe/L2Fe(H2O)3 equilibrium:
linewidth = 420 Hz 1:3
1:2
Proximal Fluorine linewidth = 6700 Hz 1:1.5 LFe(H2O)3
L2Fe
L2Fe
LFe(H2O)3
1:1 51
50
49
15
14
13
12
H227 Y266
increasing L:Fe(II) ratio
Distal Fluorine
H11 E276
ppm
