Date of Award


Document Type

Open Access Dissertation


Chemistry and Biochemistry

First Advisor

John H. Dawson


Cytochrome P450 (P450 or CYP) catalysis involves the oxygenation of organic compounds via a series of catalytic intermediates, namely, the ferric-peroxo, ferric-hydroperoxo, Compound I (Cpd I), and FeIII(H2O2) intermediates. The general consensus is that the Cpd I intermediate is the most reactive species in the reaction cycle, especially when the reaction involves hydrocarbon hydroxylation. Other than Cpd I, there is a multitude of evidence, both experimental as well as theoretical, supporting the involvement of other intermediates in various types of oxidation reactions. In part I of this work, the multiple oxidant hypothesis of P450 catalysis has been probed using P450-CAM from Pseudomonas putida, a prototypical P450 enzyme. P450-CAM is a versatile catalyst that has been shown to catalyze many typical P450 reactions in camphor analog substrates. The active site threonine-252 to alanine (T252A) mutant of P450-CAM on reaction with camphor yields H2O2 and minimal oxidized camphor, presumably because it makes very little Compound I while still generating the ferric-peroxo and ferric-hydroperoxo species. This makes T252A P450-CAM an ideal catalyst to probe the multiple oxidant hypothesis. Using heteroatom-containing substrates, including camphor analog substrates modified at the fifth position, we have compared the quantitative product formation between WT and T252A P450-CAM to gain an insight in the multiple oxidant hypothesis.

Magnetic circular dichroism (MCD) spectroscopy also known as the Faraday effect, is an excellent fingerprinting tool of various heme systems.

It can be used for assigning axial ligand identity, coordination numbers as well as spin state determination of the heme iron, which can lead to important information about their structures and functions. In part two of this work, we present results from the application of MCD spectroscopy in the axial ligand(s) identification analysis of three novel heme proteins, sGAF2, Z-ISO and Phu_R, and in the characterization of the dioxygen complex of an engineered P450BM3 protein.