The Kinetic Study of Oxidation Reactions of (TDFPP)Fe IV O, 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
Jan 12, 2016
The Kinetic Study of Oxidation Reactions of
(TDFPP)FeIVO,Model Compound of Heme Iron Center in
Cytochrome P450
Se Ryeon Lee
Department of ChemistryJohns Hopkins University
Independent Project for Advanced Inorganic Lab 030.356
December 19, 2007
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 organismsDeoxy form of cytochrome p450
active site
N
NN
N
FeIII
S
Cys
Cytochrome P450 Catalytic Cycle
RH = Substrate
ROH = Oxidized Substrate
O-O bond cleavage!!
Image from Dinisov, I.G. Chem.Rev. 2005, 105, 2253-2277
Proposed Mechanisms for O-O Bond Cleavage
• Pathway A : 2 e- push from metal, resulting in heterolytic cleavage
• Pathway B : 1 e- push from metal, resulting in homolytic cleavage
A
B
“Compound I”
FeIII
O
OH
S
Cys
FeV
O
S
Cys
FeIV
O
S
Cys
FeIV
O
S
Cys
+
“Compound II”
•
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!!
Research Results from the Newcomb Group
• 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, 6767-6774
Disproportionation Equilibrium
Independent ProposalThe 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”•
N
NN
N
FeIII
OH
Ar
Ar
Ar
F
F
O
O
OH
Cl
OH
O
FeIV
O
ROH
FeIII
O OH
hexanone
hexanol
m-chloroperoxybenzoic acid
+
Experimental Procedure
• Make 0.188 mM 5,10,15,20-tetrakis(2,6-difluorophenyl)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
• Room Temp• Soret band
406 → 412 nm
Q band
566 → 550 nm• Successful Oxidation!• But no kinetic study due to
non-continuous stirring400 600 800
0.0
0.5
1.0
1.5
2.0
Abs
orba
nce
(AU
)
Wavelength (nm)
(TDFPP)FeIIIOH 1 eq MCPBA 2 eq MCPBA
Soret band
Q band
MCPBA
FeIII FeIV
O
400 600 800
0.0
0.5
1.0
Abs
orba
nce
(AU
)
Wavelength (nm)
(TDFPP)FeIIIOH 1 eq MCPBA 2 eq MCPBA 3 eq MCPBA 4 eq MCPBA
Oxidation of (TDFPP)FeIIIOH -Low Temperature Kinetic Study-
• UV/Vis taken at 0 oC under constant stirring
412
550 566
406
-4.37
-4.36
-4.35
-4.34
-4.33
-4.32
-4.31
0 20 40 60 80
Time (s)Lo
g [F
e(III
)OH
]Lo
g [
Fe(I
II)O
H]
Slope = -6.7 (± 0.8) x10-4 s-1
∴Rate of Oxidation k=6.7 (± 0.8) x10-4 s-1
Change of [FeIIIOH] at 406 nm
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
390 400 410 420
0.8
1.0
1.2A
bso
rba
nce
(A
U)
Wavelength (nm)
400 600 800
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Ab
sorb
an
ce (
AU
)
Wavelength (nm)
Oxidation of Hexanol-Room Temperature Kinetic Study-
FeIV
O
FeIII
OH O
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
1:1000
FeIVO : Hexanol
Oxidation of Hexanol-Room Temp vs. Low Temp -
-4.38
-4.37
-4.36
-4.35
-4.34
-4.33
-4.32
0 200 400 600 800 1000
Time (s)
Log(
Fe(
IV)O
)
Change in [FeIVO] at 412 nm
Log
[F
e(I
V)O
]
Slope = -5.0 (±0.3) x10-5 s-1
∴Rate of oxidation of hexanol
k=5.0 (±0.3) x10-5 s-1
-4.375
-4.37
-4.365
-4.36
-4.355
-4.35
-4.345
0 1000 2000 3000 4000
Time (s)Lo
g []
Log
[F
e(I
V)O
]
Low Temperature (O oC)Room Temperature
Slope = -5.1 (±0.4) x10-6 s-1
∴Rate of oxidation of hexanol
k=5.1 (±0.4) x10-6 s-1
Conclusion & ShortcomingsConclusion• 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
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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