Graduate Program

Chemistry

Degree Name

Master of Science (MS)

Semester of Degree Completion

2009

Thesis Director

Scott Tremain

Thesis Committee Member

Barbara Lawrence

Thesis Committee Member

Mark McGuire

Thesis Committee Member

Doug Klarup

Abstract

A heme protein is a metalloprotein containing the heme prosthetic group. This prosthetic group is responsible for the wide range of biological functions found in heme proteins, from oxygen transport to electron transport. Moreover, heme proteins show a fascinating range of folding architectures, leading to their interest in research on structure-function relationships. The objective of this research in bioinorganic chemistry is to study the structural stability of two heme proteins, iron cytochrome c (Fecyt) and horseradish peroxidase (HRP). Absorbance spectroscopy is used to monitor the structural changes in the protein environment around the heme prosthetic group.

With just a change in the oxidation state of iron, differences in the structural stability of Fecyt can be distinguished by monitoring changes in the heme Soret absorbance band over time. In this work, urea denaturation studies are performed on iron(II) cytochrome c (FeIIcyt) and iron(III) cytochrome c (FeIIIcyt) after various times of incubation. Structural stability is quantified by determining the Gibbs free energy of unfolding in the absence of denaturant, ~Gu(H2O). The ~Gu(H2O) value for FeIIcyt was determined to be 16.52 +/- 0.75 KJ/mol, whereas it was determined to be 6.3 +/- 1. 1 KJ/mol for FeIIIcyt. Based on these results, FeIIcyt is more stable towards urea denaturation than FeIIIcyt.

Differences in the structural stability ofHRP in the presence of various acids can also be distinguished by monitoring changes in the heme Soret absorbance band over time. The three acids used are acetic acid, phosphoric acid and sulfuric acid. Since acetic acid is a weak acid, changes in the heme Soret absorbance band of HRP are only observed at high concentrations of acetic acid. HRP remains folded until the concentration of acetic acid reaches 500 rnM. In the presence of phosphoric acid, HRP is more easily unfolded, requiring only 50 rnM phosphoric acid to initiate the unfolding process. Since sulfuric acid is a strong acid, only 5 mM sulfuric acid is required to initiate the unfolding process in HRP. Unexpectedly, higher concentrations of sulfuric acid cause spectroscopic changes in the heme Soret absorbance band that resemble refolded HRP.

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