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 December 19, 2007

2. Deoxy form of cytochrome p450
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,

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 < b < c
- ↑ e- demand, ↑ reactive metal-oxo complex
- ∴Kinetic Rate ⇒ a < b < 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 > b > c
Pan, Z; Newcomb, M. Inorg. Chem. 2007, 46,

7. Feasible to perform in inorganic lab!
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 mM 5,10,15,20-tetrakis(2,6-difluorophenyl)porphyrin iron(III)hydroxo complex, (TDFPP)FeIIIOH, stock solution in CH3CN
Dilute 532 μl in ml CH3CN => 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-
406
412
Change of [FeIIIOH] at 406 nm
Log [Fe(III)OH]
550
566
Slope = -6.7 (± 0.8) x10-4 s-1
∴Rate of Oxidation k=6.7 (± 0.8) x10-4 s-1
UV/Vis taken at 0 oC under constant stirring
ε of FeIIIOH at 406 nm = 7.32 x 104 mol l-1 cm-1
ε of FeIVO at 406 nm = 8.62 x 104 mol l-1 cm-1

11. Oxidation of Hexanol -Room Temperature Kinetic Study-
1:1000
FeIVO : Hexanol
Decrease in absorbance at 412 nm!
∴Oxidation of substrate by (TDFPP)FeIVO observed
412
ε of FeIIIOH at 412 nm = 6.83 x 104 mol l-1 cm-1
ε 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
Room Temperature
Low Temperature (O oC)
Log [Fe(IV)O]
Log [Fe(IV)O]
Slope = -5.0 (±0.3) x10-5 s-1
∴Rate of oxidation of hexanol
k=5.0 (±0.3) x10-5 s-1
Slope = -5.1 (±0.4) x10-6 s-1
∴Rate of oxidation of hexanol
k=5.1 (±0.4) x10-6 s-1

13. Conclusion & Shortcomings
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,
Dolphin, D.; Traylor, T.G.; Xxie, L.Y. Acc. Chem. Res. 1997, 30,
Lee, W.A.; Calderwood, T.S.; Bruice, T.C. Proc. Natl. Acad. Sci. U.S.A. 1985, 82,
Lim, M.H.; Lee, Y.J.; Goh, Y.M.; Nam, W.; Kim, C. Bull. Chem. Soc. Jpn. 1999, 72,
Lippard, S.J.; Berg, J.M. Principles of Bioinorganic Chemistry. University Science Books; California, 1994.
Pan, Z; Newcomb, M. Inorg. Chem. 2007, 46,