Date of Award


Degree Type


Degree Name

Master of Science




Dr. Martin J. Stillman

Second Advisor

Dr. Melvyn C. Usselman

Third Advisor

Dr. Ronald R. Martin


Staphylococcus aureus infection is becoming a widespread threat in both hospitals and communities worldwide, resulting in substantial treatment costs and numerous patient deaths. Bacterial survival is largely dependant on the organism’s ability to scavenge iron from a host via specialized pathways. The recently identified cell walland membrane-associated Iron regulated surface determinant (Isd) proteins are one such pathway. They serve to transport iron-containing heme, from hemoglobin, from the environment into the bacterial cell for nourishment. To date very little is known about the heme binding properties of these proteins and the overall Isd heme scavenging mechanism. Through the use of UV-visible absorption and magnetic circular dichroism spectroscopies, mass spectrometry, and mutational analysis, the heme-binding properties of IsdA, IsdC, and IsdE have been characterized. Recombinant IsdA (rIsdA) was found to bind a single high-spin ferric heme, coordinated by tyrosine 166 of the IsdA near abc transporter (NEAT) domain. Recombinant IsdC (rIsdC) was purified bound primarily to protoporphyrin IX, with traces of high-spin ferric heme and an unidentified ligand of approximately 320 Da. Recombinant IsdE (rIsdE) was purified bound to two heme molecules and an unidentified ligand of approximately 320 Da. One heme is ferric and is bound near the edge of a heme binding pocket through the imidazole group of histidine 229. The other is low-spin ferrous and is inaccessible to small ligands. The ferrous heme binding amino acid residue(s) remain unknown but it appears to be bound deep within the heme binding pocket of rIsdE. iii



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