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

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

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 • “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

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 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, 6767-6774

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”

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 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

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

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

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

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

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