Chairperson, Graduate Committee: Benfang LeiRan, Yanchao2013-06-252013-06-252010https://scholarworks.montana.edu/handle/1/2102Group A Streptococcus relies on heme as a source of iron. The proteins Shp and HtsA are part of the heme acquisition machinery of Group A Streptococcus. Shp rapidly transfers its heme to HtsA; however, the mechanism of the Shp/HtsA reaction is unknown. This project was conducted to elucidate the structural basis and molecular mechanism of this rapid heme transfer reaction. Site-directed mutagenesis was used to identify the axial ligands of the heme iron in Shp and HtsA, and kinetic and spectroscopic analyses and animal infection were used to assess the effects of the elimination of the axial ligands on coordination and spin state of the heme iron, kinetic mechanism of the heme transfer, autoxidation of the Shp heme iron, and GAS virulence. The axial ligands of the heme iron in Shp and HtsA were found to be Met66/Met153 and Met79/His229, respectively. The Met153 Shp and His229 HtsA residues are critical for the affinity of the proteins for heme, and the other axial side of the heme irons is more accessible to solvent. The Met66Ala and Met153Ala replacements of Shp alter the kinetic mechanism of Shpto- HtsA heme transfer and unexpectedly slow down heme transfer, which allows detection of transfer intermediates. The HtsA Met79Ala and HtsA His229Ala mutant proteins cannot acquire heme from ferrous Shp but induce rapid autoxidation of ferrous Shp. The significance of these findings is three-fold. Firstly, the structural basis of the heme binding in Shp and HtsA and the spectral properties of their axial ligand mutants enable the interpretation of the kinetics and spectral changes of the heme transfer reactions. Secondly, HtsA axial mutant-induced autoxidation of ferrous Shp provides evidence for the activated heme transfer mechanism and the formation of a Shp-HtsA complex that weakens the heme binding in Shp. Thirdly, the findings allow us to propose a reaction model in which the side chains of the axial residues from HtsA are inserted into the axial positions of the heme in Shp to extract it from the surface protein and pull it into the transporter active site. The project significantly advances the understanding of how heme is rapidly transferred from one protein to another in heme acquisition.enStreptococcusHemoproteinsDissertationCopyright 2010 by Yanchao Ran