Title: The Kinetic Study of Oxidation Reactions of (TDFPP)FeIVO, Model Compound of Heme Iron Center in Cytochrome P450
1The Kinetic Study of Oxidation Reactions of
(TDFPP)FeIVO,Model Compound of Heme Iron Center
in Cytochrome P450
Department of Chemistry Johns Hopkins University
Independent Project for Advanced Inorganic Lab
030.356December 19, 2007
2Cytochrome 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
3Cytochrome P450 Catalytic Cycle
RH Substrate ROH Oxidized Substrate
O-O bond cleavage!!
Image from Dinisov, I.G. Chem.Rev. 2005, 105,
2253-2277
4Proposed 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
5Research 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!!
6Research 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
7Independent 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
8Experimental 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
9Oxidation 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
10Oxidation 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
11Oxidation 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
12Oxidation 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
13Conclusion 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
14Applications
- 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