Date of Award

1-1-2012

Document Type

Campus Access Dissertation

Department

Chemistry and Biochemistry

Sub-Department

Chemistry

First Advisor

John H Dawson

Abstract

Heme proteins are highly versatile with important roles in biochemistry including oxygen transport, storage, and catalysis. Work on heme proteins has often led the way for studying biological systems, and while different heme protein families have unique gene sequences, protein folding and varied roles in biochemistry, some characteristics are similar throughout all heme proteins and a variety of spectroscopic methods have been first applied to the study of heme proteins over the years. Using the two defining features of all heme proteins, the iron metal center and the tetrapyrrole macrocycle, UV-visible (UV-Vis) electronic absorption and rapid-scan, stopped-flow spectroscopies were utilized to determine reaction mechanisms and kinetics data. Due to the chromophore properties of heme-containing proteins, magnetic circular dichroism (MCD) spectrophotometry was used to determine structural aspects of different heme-containing proteins.

In part one of this dissertation, two projects highlight the kinetics of reacting thiolate-ligated heme proteins with different oxo-donors. The reaction between ferric Caldariomyces fumago chloroperoxidase (CCPO) and meta-chloroperoxybenzoic acid (mCPBA) has been examined, but unlike most CCPO reactions, Compound I (Cpd I) and Compound II (Cpd II) are formed using the same reactant. Thus, the peracid is used as an oxo donor to produce Cpd I and then as a reductant to reduce Cpd I to Cpd II, and finally, Cpd II to the ferric state. In the second project, the reaction of ferric cytochrome P450cam (Cyp101) with substituted (Cl, CH3, OCH3) perbenzoic acids was studied using rapid scanning stopped flow spectroscopy. An absorption appears en route to the formation of the high-valent moiety known as Compound I that is thought to be an ferric acylperoxo heme adduct similar to the known Compound 0 of other peroxidases. Further support for this conclusion derive from additional linear Hammett correlation plots for both the rate of formation of the intermediate as well as for its conversion to Compound I vs. the substituent Hammett ρ constant.

In the second part, MCD studies were completed on a variety of heme-containing proteins in order to determine the axial ligands critical for binding the heme cofactor. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an enzyme recently implicated in a new role for eukaryotic systems: heme transport protein. Different spectroscopic techniques (UV-Vis and MCD) were employed to identify the axial binding site for heme to GAPDH, which was determined to be a bis-histidine ligand set. Another group of proteins studied for their heme-uptake properties were the gram-positive bacteria Streptococcus pyogenes Shr NEAT1 and NEAT 2 domains and Corynebactrium diphtheriae HmuT. Both NEAT1 and NEAT2 were determined to contain two thioether donors, presumably bis-methionine in the various oxidation states. For HmuT, the native protein was determined to be ligated to a nitrogen donor and an oxo-donor. It was concluded that wild-type HmuT adopts the His/Tyr active site similar to other known heme-uptake systems. During the course of mutation studies, His136 and Tyr235 were determined to be the axial ligands of wild-type HmuT.

Finally, the last project completed was spectroscopic studies completed on Saccharomyces cervisiae iso-1-cytochrome c and its F82H, M80C, F82C, and T78C/K79G mutants. UV-vis absorption and MCD spectroscopy were utilized to compare these mutants to the wild-type yeast cytochrome c and its His/Met active site ligand set, as well as to the F82H bis-His mutant. Spectroscopic changes in these mutants upon heme reduction suggest a new Cys-to-Met ligand switch, pointing to the flexibility of the heme environment in these systems.

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