investigating the mechanism of naphthalene 1,2-dioxygenase...
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Investigating the mechanism of naphthalene 1,2-dioxygenase in whole
cells
Alex Zozula Groves Lab
September 25, 2009
Mycobacterium tuberculosis The alveolar macrophage detects M. tb.
FeII
S-Cys
R-H
R-H
FeIII
S-Cys
R-H
O2
R-OHS-Cys
H2O2
S-Cys
R-H
H+
H+
H2O
S-Cys
OHS-Cys
R-H
R-H
e-
e-
3
4
56
7
8
O
FeIII
FeIVO
FeIIIO
FeIIO2
HR
FeIIIO O H
FeVO
OHNDO
Related non-heme systems
DBHPHM C uII
O-OH
FeIVOFeIV
O
AlkBMMO FeIII
OFeIII
O-O
FeIV
OFeIV
HO
O
Rieske protein [Fe2S2]
Fe2+OO
N N
NN
H
H
Naphthalene Dioxygenase (NDO) A non-heme mono-Fe system
a remarkably simple, “heme-like” active site
NDO
Useful for bioremediation and enantioselective catalysis
Fe3+O
O H
Fe5+O
OH
Fe3+OH
R
Fe2+e-
O2
R
Fe3+O
O H
R
R
O
R
Fe5+O
OH
R
Fe5+O
OH
R
Fe3+OH
R
O
Fe4+O
R
Fe3+OH
R
O
OH
directaddition
radicalpathway
cationpathway
How does naphthalene dioxygenase work? There have been many proposals
In vivo technique
Allows proteins to react in their na=ve environments and avoids difficult isola=on
Used in the past with other enzyme systems (e.g. AlkB, P450) and has yielded results in accord with in vitro data
Can easily demonstrate cells are releasing representa=ve samples by lysing cells
Can demonstrate products aren't reac=ng further by feeding product mixtures to cells
Bicyclo[4.1.0]heptane (norcarane)
krear = 2x108 s‐1 → radical
life=me ≈ 8.1 ns
trans‐1‐methyl‐2‐phenylcyclopropane (PMCP)
krear ≈ 1011 s‐1 → radical
life=me ≈ 15 ps
Conclusions
Variety of diagnos=c probes hydroxylated in mechanis=cally informa=ve posi=ons with no over‐oxida=on or ca=onic products
Norcarane, bicyclohexane, and DECP intermediate life=mes agree well; TMCP and PMCP intermediate life=mes agree well → 1º vs. 2º carbon effect?
Various amounts of 18O in products → Fe(V)‐oxo‐hydroxo species formed but its reac=vity is substrate dependent
The Groves Group (2008)