Date of Award

Spring 2021

Degree Type



Chemistry and Biochemistry

Director of Thesis

Dr. Thomas Makris

First Reader

Dr. Maksymilian Chruszcz

Second Reader

Dr. Maksymilian Chruszcz


OleT, a member of the CYP152 family of cytochrome P450s (CYPs), decarboxylates fatty acids using hydrogen peroxide as an oxidant. The resultant products are a terminal alkene and carbon dioxide. This C–C cleavage reaction is highly atypical for CYPs, which prototypically oxygenate substrates, and provides a potential means to enzymatically produce drop-in fuels. OleT contains a heme-iron cofactor that facilitates decarboxylation through the activation of hydrogen peroxide. The catalytic cycle, as determined by transient kinetics, includes two ferryl intermediates known as Compound I (Ole-I) and Compound II (Ole-II). Ole-I performs substrate hydrogen abstraction and subsequent single electron transfer to Ole-II induces C–C bond cleavage. Previous studies of OleT orthologs have demonstrated that chemoselectivity of the enzyme is reinforced, in part, from exquisite binding of fatty acids within the distal substrate-binding pocket. However, it has remained unclear how ground-state thermodynamics may also influence OleT ferryl reactivity. This research probes the impact of heme-electronics on the intermediates of OleT catalysis through the substitution of native iron-protoporphyrin IX with an iron-mesoporphyrin and iron-deuteroporphyrin IX, both of which alter the redox potential of the iron. Transient kinetic studies at variable temperatures have been used to measure the direct impact of this alteration on C–H abstraction barriers. These results suggest a strong linkage between heme electronics and the metabolic efficiency of OleT and offer a strategy for modulating the reactivity of this ubiquitously distributed superfamily of enzymes.

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© 2021, Alexis J Holwerda