Date of Award


Document Type

Open Access Dissertation


Chemistry and Biochemistry


College of Arts and Sciences

First Advisor

Thomas Makris


A recently discovered cytochrome P450 has garnered much interest for its ability to oxidatively decarboxylate n length fatty acids to n-1 terminal olefins using hydrogen peroxide as an oxidant. This enzyme (P450 OleT) is mechanistically peculiar, as it seemingly abrogates the oxygen insertion chemistry that typifies the P450 superfamily. In this work we explore the origin of this deviant catalysis.

Using stopped-flow and transient kinetic methodologies the ferryl-oxo pi-cation radical and ferryl-hydroxo intermediates, known as compound-I (Cpd-I) and compound-II (Cpd-II) respectively, were isolated in high yield. This is the first time either species has been observed in a substrate bound P450 using a native oxidant. The accumulation of these intermediates has enabled direct observation of the decarboxylation mechanism of P450 OleT. We have found that decarboxylation is initiated with substrate hydrogen atom abstraction by Cpd-I, similar to oxygen insertion chemistries. The branch point in the reaction coordinate occurs at Cpd-II where, through a combination of substrate positioning and stabilization of ferryl-hydroxo by active site hydrogen bonding, oxygen rebound is inhibited allowing for a proton coupled one electron oxidation of the substrate to occur. This one electron oxidation liberates the carboxylate as CO2 through either a carbocation or biradical mechanism. Additionally, the isolation of Cpd-I in the absence of a bound substrate has allowed for an examination of how the peptide backbone is protected from a highly oxidizing intermediate, crucial for preventing non-productive self-oxidation.

Included in

Chemistry Commons