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 030.356 December 19, 2007
Cytochrome Pigment 450
•
N N
•
Fe
N S
III
N
•
Cys
Deoxy form of cytochrome p450 active site
•
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
Cytochrome P450 Catalytic Cycle
RH = Substrate
ROH = Oxidized Substrate
O-O bond cleavage!!
Image from Dinisov, I.G. Chem.Rev. 2005, 105, 22532277
Proposed Mechanisms for O-O Bond Cleavage
O O
FeV
OH O
+ FeIV
•
“Compound I”
A
S
S
Cys
Cys
FeIII
S O
Cys
B
FeIV
S
“Compound II”
Cys
• Pathway A : 2 e- push from metal, resulting in heterolytic cleavage • Pathway B : 1 e- push from metal, resulting in homolytic cleavage
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
Pan, Z; Newcomb, M. Inorg. Chem. 2007, 46, 6767-6774
Independent Proposal
The Kinetic Study of Oxidation Reactions of (TDFPP)Fe IVO complex, Model Compound of Heme Iron Center in Cytochrome P450
Cl Ar F O
O N FeIII N Ar OH
O Fe
III
OH
m-chloroperoxybenzoic acid
F
N OH N
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!
Ar O
hexanone
OH O
ROH
hexanol
+ IV • Fe
“Compound I”
Experimental Procedure
• Make 0.188 mM 5,10,15,20-tetrakis(2,6difluorophenyl)porphyrin iron(III)hydroxo complex, (TDFPP)FeIIIOH, stock solution in CH3CN • Dilute 532 μl in 4.468 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
Oxidation of (TDFPP)FeIIIOH
O
MCPBA
FeIII FeIV
Soret band
2.0
1.5
Absorbance (AU)
(TDFPP)Fe OH 1 eq MCPBA 2 eq MCPBA
III
1.0
0.5
Q band
0.0
• Room Temp • Soret band 406 → 412 nm Q band 566 → 550 nm • Successful Oxidation! • But no kinetic study due to non-continuous stirring
400
600
800
Wavelength (nm)
Oxidation of (TDFPP)FeIIIOH
-Low Temperature Kinetic Study1.0
406 412
(TDFPP)Fe OH 1 eq MCPBA 2 eq MCPBA 3 eq MCPBA 4 eq MCPBA
III
Change of [FeIIIOH] at 406 nm
-4.31
Log [Fe(III)OH] Log [Fe(III)OH]
Absorbance (AU)
-4.32 -4.33 -4.34 -4.35 -4.36 -4.37 0 20 40 Time (s) 60 80
0.5
550 566
0.0 400 600 800
Wavelength (nm)
• 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
Slope = -6.7 (± 0.8) x10-4 s-1 ∴Rate of Oxidation k=6.7 (± 0.8) x10-4 s-1
Oxidation of Hexanol
-Room Temperature Kinetic StudyOH O
1:1000
FeIVO : Hexanol
O FeIV FeIII
1.2
1.2
412
Absorbance (AU)
1.0
Absorbance (AU)
1.0 0.8 0.6 0.4 0.2 0.0 400 600
Decrease in absorbance at 412 nm! ∴Oxidation of substrate by (TDFPP)FeIVO observed
0.8
390
400
410
420
Wavelength (nm)
ε of FeIIIOH at 412 nm = 6.83 x 104 mol l-1 cm-1
800
Wavelength (nm)
ε of FeIVO at 412 nm = 9.63 x 104 mol l-1 cm-1
Oxidation of Hexanol
-Room Temp vs. Low Temp Change in [FeIVO] at 412 nm
Room Temperature
-4.345
-4.32 -4.33 -4.34 -4.35 -4.36 -4.37 -4.38 0 200 400 600 800 1000 Time (s)
Low Temperature (O oC)
-4.35
Log(Fe(IV)O) Log [Fe(IV)O]
Log [Fe(IV)O] Log []
-4.355 -4.36 -4.365 -4.37 -4.375 0 1000 2000 Time (s) 3000 4000
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
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
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