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

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Page 1: Investigating the mechanism of naphthalene 1,2-dioxygenase ...pei300w.princeton.edu/media/meet-our-interns/interns-2009/Zozula_Alex_sol.pdf · Investigating the mechanism of naphthalene

Investigating the mechanism of naphthalene 1,2-dioxygenase in whole

cells

Alex Zozula Groves Lab

September 25, 2009

Page 2: Investigating the mechanism of naphthalene 1,2-dioxygenase ...pei300w.princeton.edu/media/meet-our-interns/interns-2009/Zozula_Alex_sol.pdf · Investigating the mechanism of naphthalene

Mycobacterium tuberculosis The alveolar macrophage detects M. tb.

Page 3: Investigating the mechanism of naphthalene 1,2-dioxygenase ...pei300w.princeton.edu/media/meet-our-interns/interns-2009/Zozula_Alex_sol.pdf · Investigating the mechanism of naphthalene

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

Page 4: Investigating the mechanism of naphthalene 1,2-dioxygenase ...pei300w.princeton.edu/media/meet-our-interns/interns-2009/Zozula_Alex_sol.pdf · Investigating the mechanism of naphthalene

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

Page 5: Investigating the mechanism of naphthalene 1,2-dioxygenase ...pei300w.princeton.edu/media/meet-our-interns/interns-2009/Zozula_Alex_sol.pdf · Investigating the mechanism of naphthalene

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 

Page 6: Investigating the mechanism of naphthalene 1,2-dioxygenase ...pei300w.princeton.edu/media/meet-our-interns/interns-2009/Zozula_Alex_sol.pdf · Investigating the mechanism of naphthalene

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  

Page 7: Investigating the mechanism of naphthalene 1,2-dioxygenase ...pei300w.princeton.edu/media/meet-our-interns/interns-2009/Zozula_Alex_sol.pdf · Investigating the mechanism of naphthalene

Bicyclo[4.1.0]heptane (norcarane) 

krear = 2x108 s‐1 → radical 

life=me ≈ 8.1 ns 

Page 8: Investigating the mechanism of naphthalene 1,2-dioxygenase ...pei300w.princeton.edu/media/meet-our-interns/interns-2009/Zozula_Alex_sol.pdf · Investigating the mechanism of naphthalene

trans‐1‐methyl‐2‐phenylcyclopropane (PMCP) 

krear ≈ 1011 s‐1 → radical 

life=me ≈ 15 ps 

Page 9: Investigating the mechanism of naphthalene 1,2-dioxygenase ...pei300w.princeton.edu/media/meet-our-interns/interns-2009/Zozula_Alex_sol.pdf · Investigating the mechanism of naphthalene

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 

Page 10: Investigating the mechanism of naphthalene 1,2-dioxygenase ...pei300w.princeton.edu/media/meet-our-interns/interns-2009/Zozula_Alex_sol.pdf · Investigating the mechanism of naphthalene

The Groves Group (2008)