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
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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
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Cytochrome P450 Catalytic Cycle
RH Substrate ROH Oxidized Substrate
O-O bond cleavage!!
Image from Dinisov, I.G. Chem.Rev. 2005, 105,
2253-2277
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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

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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!!

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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
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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

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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

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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
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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
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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
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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
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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

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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.
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• 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