Understanding the Origin of Suicide Inactivation in the Extradiol Dioxygenases Timothy E. Machonkin, Department of Chemistry, Whitman College, Walla Walla, WA 99362
We have re-focussed our efforts to study poorly characterized non-catechol ring-cleaving dioxygenases, especially the hydroquinone dioxygenases that actually prefer chlorinated (and even brominated) substrates, PcpA and LinE. We are using a two-pronged approach of studying both the actual enzymes and synthesis of novel model complexes. 1. Biochemical studies of unusual Fe(II)-containing ring-cleaving dioxygenases that cleave chlorinated substrates. Substrate-specificity of PcpA: only 2,6-disubstituted hydroquinones are substrates, monosubstituted hydroquinones are inihibitors/inactivators. Substrates:
OH Cl
OH Cl Br
OH
Inhibitors/Inactivators:
His159
OH Cl
His227
His11
Glu276
Tyr266
OH
OH
Br
No evidence of binding:
OH
H3 C
OH
OH
OH Cl Cl
OH
CH 3
OH
OH
OH
OH
1
OH Br H3 C
Cl
Relative initial rate
Structural model of 2,6-dichlorohydroquinone dioxygenase (PcpA) validated by site-directed mutagenesis.
0.8
HO
OH Cl
0.6
Br HO Br
0.4
H3C HO
0.2 0 0
OH
H3C
50
100
150
200
250
Substrate concentration (μM)
2. Fe(II)-containing model complexes that contain ortho-chlorophenolates with Fe-Cl secondary bonds. Paramagnetic NMR spectra of (TACH-o-tol) Fe(II) complexes upon addition of substituted phenols.
OH
300
Crystal structure of [(TACH-o-tol)FeII(2-dichloro-phenolate)]OTf (left) and [(TACH-o-tol)FeII(2,6-dichlorophenolate)]OTf (right).
