The Kinetic Study of Oxidation Reactions of (TDFPP)FeIVO, Model Compound of Heme Iron Center in Cytochrome P450 - PowerPoint PPT Presentation

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The Kinetic Study of Oxidation Reactions of (TDFPP)FeIVO, Model Compound of Heme Iron Center in Cytochrome P450

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Title: The Kinetic Study of Oxidation Reactions of (TDFPP)FeIVO, Model Compound of Heme Iron Center in Cytochrome P450


1
The Kinetic Study of Oxidation Reactions of
(TDFPP)FeIVO,Model Compound of Heme Iron Center
in Cytochrome P450
  • Se Ryeon Lee

Department of Chemistry Johns Hopkins University
Independent Project for Advanced Inorganic Lab
030.356December 19, 2007
2
Cytochrome Pigment 450
  • Monooxygenase with heme center
  • Catalyze the oxidation of organic substrates by
    dioxygen
  • Important role in biosynthesis, metabolism, and
    detoxification of harmful substances
  • Found in all organisms

Deoxy form of cytochrome p450 active site
3
Cytochrome P450 Catalytic Cycle
RH Substrate ROH Oxidized Substrate
O-O bond cleavage!!
Image from Dinisov, I.G. Chem.Rev. 2005, 105,
2253-2277
4
Proposed Mechanisms for O-O Bond Cleavage
Compound I

A
B
Compound II
  • Pathway A 2 e- push from metal, resulting in
    heterolytic cleavage
  • Pathway B 1 e- push from metal, resulting in
  • homolytic cleavage

5
Research Results from the Newcomb Group
  • Kinetic study of Iron(IV)oxo complex with three
    different aryl groups
  • a. 2,6-Cl2C6H3 b. 2,6-F2C6H3 c. C6F3
  • Theory
  • - Increase in electron-withdrawing effects
  • Electron demand a lt b lt c
  • - ? e- demand, ? reactive metal-oxo complex
  • - ?Kinetic Rate ? a lt b lt c
  • Was this true ? NO!!

6
Research Results from the Newcomb Group
Disproportionation Equilibrium
  • Less favorable disproportionation equilibrium
    with increase of e- demand of macrocycle
  • ? decrease in reactive species
  • ?Kinetic rate ? a gt b gt c

Pan, Z Newcomb, M. Inorg. Chem. 2007, 46,
6767-6774
7
Independent Proposal
The Kinetic Study of Oxidation Reactions of
(TDFPP)FeIVO complex, Model Compound of Heme Iron
Center in Cytochrome P450
Originally, planned to use 5,10,15,20-tetrakis
(pentafluorophenyl)-porphyrin High electron
demand ? less favorable disproportionation
equilibrium ? less reactive species ? Slow
oxidation rate Feasible to perform in inorganic
lab!

Compound I

8
Experimental Procedure
  • Make 0.188 mM 5,10,15,20-tetrakis(2,6-difluorophen
    yl)porphyrin iron(III)hydroxo complex,
    (TDFPP)FeIIIOH, stock solution in CH3CN
  • Dilute 532 µl in 4.468 ml CH3CN gt 20 µM in 5 ml
  • Add 1 eq m-chloroperoxybenzoic acid, MCPBA, to
    oxidize
  • Add more MCPBA (1 eq at a time) until
    (TDFPP)FeIVO is observed using UV/Vis kinetic
    study
  • Add 1000 eq substrate (hexanol) and observe any
    change using UV/Vis kinetic study
  • Analyze change in peak to calculate the rate
    constant

9
Oxidation of (TDFPP)FeIIIOH
Soret band
  • Room Temp
  • Soret band
  • 406 ? 412 nm
  • Q band
  • 566 ? 550 nm
  • Successful Oxidation!
  • But no kinetic study due to non-continuous
    stirring

Q band
10
Oxidation of (TDFPP)FeIIIOH -Low Temperature
Kinetic Study-
412
406
Change of FeIIIOH at 406 nm
Log Fe(III)OH
550
566
Slope -6.7 ( 0.8) x10-4 s-1 ?Rate of Oxidation

k6.7 ( 0.8) x10-4 s-1
  • UV/Vis taken at 0 oC under constant stirring

e of FeIIIOH at 406 nm 7.32 x 104 mol l-1
cm-1 e of FeIVO at 406 nm 8.62 x 104 mol l-1
cm-1
11
Oxidation of Hexanol-Room Temperature Kinetic
Study-
11000 FeIVO Hexanol
Decrease in absorbance at 412 nm! ?Oxidation of
substrate by (TDFPP)FeIVO observed
412
e of FeIIIOH at 412 nm 6.83 x 104 mol l-1
cm-1 e of FeIVO at 412 nm 9.63 x 104 mol l-1
cm-1
12
Oxidation of Hexanol-Room Temp vs. Low Temp -
Change in FeIVO at 412 nm
Low Temperature (O oC)
Room Temperature
Log Fe(IV)O
Log Fe(IV)O
Slope -5.0 (0.3) x10-5 s-1 ?Rate of oxidation
of hexanol k5.0 (0.3) x10-5 s-1
Slope -5.1 (0.4) x10-6 s-1 ?Rate of oxidation
of hexanol k5.1 (0.4) x10-6 s-1
13
Conclusion Shortcomings
  • Conclusion
  • Successful oxidation reaction of porphyrins and
    substrates under both room temperature (RT) and
    low temperature (0 oC) (LT)
  • Was able to calculate the rate and compare RT and
    LT
  • Shortcomings
  • Using (TPFPP)FeIIIOH instead of (TDFPP)FeIIIOH
    may have been easier to study
  • Not enough data due to many unsuccessful
    experiments
  • e.g. using CH3Cl as solvent ? no oxidation
  • Only one substrate and one porphyrin used for
    oxidation reaction
  • ? need more various substrates and porphyrins to
    compare the rate
  • Not able to identify the oxidized substrates
  • ? need GC analysis

14
Applications
  • The experiment shows a promising oxidation
    reaction that is slow enough to be detected in
    room temperature which suggests
  • - Comparing the oxidation of different substrates
    by various porphyrins may help to understand the
    mechanistic details of oxidation reactions
  • - It can be performed in class with no
    sophisticated instruments to understand the
    cytochrome p450 mechanism
  • Acknowledgements
  • Mark Schopfer (Karlin Lab at JHU)
  • Jun Wang (Karlin Lab at JHU)
  • References
  • Denisov, I.G. Makris, T.M. Sligar, S.G.
    Schlichting, I. Chem. Rev. 2005, 105, 2253-2277
  • Dolphin, D. Traylor, T.G. Xxie, L.Y. Acc. Chem.
    Res. 1997, 30, 251-259
  • Lee, W.A. Calderwood, T.S. Bruice, T.C. Proc.
    Natl. Acad. Sci. U.S.A. 1985, 82, 4301-4305
  • Lim, M.H. Lee, Y.J. Goh, Y.M. Nam, W. Kim, C.
    Bull. Chem. Soc. Jpn. 1999, 72, 707-713
  • Lippard, S.J. Berg, J.M. Principles of
    Bioinorganic Chemistry. University Science Books
    California, 1994.
  • Pan, Z Newcomb, M. Inorg. Chem. 2007, 46,
    6767-6774
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