Structural and mechanistic characterization of heme and non -heme iron metalloenzymes
Abstract
Iron is essential in many biological functions such as oxygen transport, storage and redox processes. Two different iron containing enzymes are the subject of this dissertation. One is chlorite dismutase (Cld), a heme b, O-O bond forming enzyme that transforms toxic chlorite into innocuous chloride and dioxygen. Cld is potentially important in the bioremediation of chlorite contamination. The mechanism of Cld is not known. As such, my research is focused on elucidating the chlorite degradation pathway by first showing that chlorite is the sole source of dioxygen as determined by 18O labeling studies. The reaction was monitored by rapid mixing UV/vis spectroscopy and freeze-quench electron paramagnetic resonance (EPR). Transient reaction intermediates were detected and characterized. The EPR spectrum of the enzyme under turnover conditions resembles the tryptophan radical of Cytochrome c peroxidase (Ccp) Compound I. The position of this radical was probed using site-directed mutagenesis Three iron porphyrin complexes were investigated as functional mimics of the enzyme. Based on these results, a mechanism for Cld was put forth. The second enzyme is phenylalanine hydroxylase (PAH), a non-heme oxygenase that is capable of converting phenylalanine to tyrosine using oxygen and tetrahydrobiopterin. Defects in PAH lead to the metabolic disorder phenylketonuria, which is characterized by mental developmental problems. I am interested in deciphering the structural basis of this disease using recombinant human PAH. I report herein the progress on the expression, purification and crystallization of the full length wild type human PAH. The structure of the full length human PAH has not been reported so far.
Degree
Ph.D.
Advisors
Abu-Omar, Purdue University.
Subject Area
Biochemistry
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