Date of Award

Fall 12-14-2017

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Professor Aimin Liu

Second Advisor

Professor Donald Hamelberg

Third Advisor

Professor Jenny Yang

Abstract

Metalloenzymes catalyze a wide range of chemical transformations. Their remarkable versatility is imparted to them by their metal centers that are redox active or function as Lewis acids. They utilize transition metals such as iron that has accessibility to a variety of redox states, allowing them to efficiently activate and insert molecular oxygen into a wide range of unactivated organic substrates. The work described in this dissertation is the structural and mechanistic characterization of both heme and non-heme iron-dependent metalloenzymes. Precisely, the focus is placed on three enzymes, a cytochrome P450, CYP121, a tyrosine hydroxylase, LmbB2, and an extradiol dioxygenase, 3-hydroxyanthranilate 3,4-dioxygenase (HAO). The first two enzymes expand the repertoire of activities performed by hemoproteins. CYP121 catalyzes an unusual C-C crosslinking reaction that is distinct from traditional oxygenase chemistry performed by this family. LmbB2 is one of the first enzymes that mediate aromatic amino acid hydroxylation without using a pterin cofactor. Finally, HAO serves as the prototype for type III extradiol dioxygenase chemistry. A broad spectrum of biochemical and spectroscopic techniques was used to investigate the oxygen activation techniques by both LmbB2 and CYP121 and the role of second sphere ligands in the HAO active site. Specifically, rapid-kinetic methods, EPR spectroscopy, and X-ray crystallography allowed the elucidation of enzyme structure and function properties. In CYP121, using peracetic acid as an oxidant, similarities with canonical P450 enzymes through the use of a catalytic shunt pathway were demonstrated. We also identify the first intermediate in this pathway, an alkyperoxo species with an unusual 5/2 spin signal observed by EPR spectroscopy. In the LmbB2 study, a compound ES-like species was observed by UV-Vis and EPR spectroscopy. This intermediate was shown to be catalytically competent and able to react specifically to the substrate L-tyrosine. Finally, the role of a second sphere Pro97 residue in the active site of HAO was investigated using enzymatic assays. Long-range remote structural influences were observed using protein crystallography techniques between the active site and the conformation of the surface residues.

Available for download on Tuesday, December 10, 2019

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