Date of Award
4-22-2008
Degree Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Chemistry
First Advisor
Dabney W. Dixon - Chair
Second Advisor
Kathryn B. Grant
Third Advisor
Jerry Smith
Abstract
Sulfonated phthalocyanines were characterized using capillary electrophoresis and mass spectrometry. Derivatives investigated included the copper, cobalt, zinc and metal-free sulfonated phthalocyanines. The electropherograms of commercially available copper phthalocyanine-3,4',4'',4'''-tetrasulfonic acid and 4,4',4'',4'''-tetrasulfonic acid were very different, consistent with the latter compound having a structure that is not fully sulfonated. Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) were used to characterize the sulfonated phthalocyanines. Mass spectral evidence was obtained for a pentasulfonated species of both the metal-free phthalocyanine and zinc phthalocyanine when these species were made by sulfonation of the metal-free phthalocyanine (followed by zinc insertion in the latter case). Many pathogenic bacteria require heme and obtain it from their environment. Heme transverses the cytoplasmic membrane via an ATP binding cassette (ABC) pathway. Although a number of heme ABC transport systems have been described in pathogenic bacteria, there is as yet little biophysical characterization of the proteins in these systems. The sia (hts) gene cluster encodes a heme ABC transporter in the Gram positive Streptococcus pyogenes. The heme binding protein (HBP) of this transporter is SiaA (HtsA). Several biophysical techniques were used to determine the coordination state, and spin state of both the ferric and ferrous forms of this protein. Identifiers from these techniques suggested that the heme is six-coordinate and low spin in both oxidation states of the protein, with methionine and histidine as axial ligands. The pKa of SiaA was determined, as were the reductive and oxidative midpoint potentials. Guanidinium titration studies of wild-type SiaA showed that the ferric state is less stable than the ferrous state. Free energy of unfolding values [ÄG(H2O)] for the oxidized and reduced proteins were 7.3 ± 0.8 and 16.0 ± 3.6 kcal mol−1, respectively. Denaturation of the histidine mutant H229A was not able to be followed via absorbance spectrometry, possibly due to the large amount of apoprotein present or to non-specific binding of the heme in the binding pocket. The biophysical characterization described herein will significantly advance our understanding of structure-function relationships in HBP.
DOI
https://doi.org/10.57709/1059262
Recommended Citation
Sook, Brian R., "I. Characterization of Sulfonated Phthalocyanines by Mass Spectrometry. II. Characterization of SIAA, a Streptococcal Heme-Binding Protein Associated with a Heme ABC Transport System." Dissertation, Georgia State University, 2008.
doi: https://doi.org/10.57709/1059262