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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• 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