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Item Spectroscopic Identification of Heme Axial Ligands in HtsA That Are Involved in Heme Acquisition by Streptococcus pyogenes(2010-04) Ran, Yanchao; Liu, Mengyao; Zhu, Hui; Nygaard, Tyler K.; Brown, Doreen E.; Fabian, Marian; Dooley, David M.; Lei, BenfangThe heme-binding proteins Shp and HtsA of Streptococcus pyogenes are part of the heme acquisition machinery in which Shp directly transfers its heme to HtsA. Mutagenesis and spectroscopic analyses were performed to identify the heme axial ligands in HtsA and to characterize axial mutants of HtsA. Replacements of the M79 and H229 residues, not the other methionine and histidine residues, with alanine convert UV−vis spectra of HtsA with a low-spin, hexacoordinate heme iron into spectra of high-spin heme complexes. Ferrous M79A and H229A HtsA mutants possess magnetic circular dichroism (MCD) spectra that are similar with those of proteins with pentacoordinate heme iron. Ferric M79A HtsA displays UV−vis, MCD, and resonance Raman (RR) spectra that are typical of a hexacoordinate heme iron with histidine and water ligands. In contrast, ferric H229A HtsA has UV−vis, MCD, and RR spectra that represent a pentacoordinate heme iron complex with a methionine axial ligand. Imidazole readily forms a low-spin hexacoordinate adduct with M79A HtsA with a Kd of 40.9 μM but not with H229A HtsA, and cyanide binds to M79A and H229A with Kd of 0.5 and 19.1 μM, respectively. The ferrous mutants rapidly bind CO and form simple CO complexes. These results establish the H229 and M79 residues as the axial ligands of the HtsA heme iron, indicate that the M79 side is more accessible to the solvent than the H229 side of the bound heme in HtsA, and provide unique spectral features for a protein with pentacoordinate, methionine-ligated heme iron. These findings will facilitate elucidation of the molecular mechanism and structural basis for rapid and direct heme transfer from Shp to HtsA.Item Axial Ligand Replacement Mechanism in Heme Transfer from Streptococcal Heme-Binding Protein Shp to HtsA of the HtsABC Transporter(2013-09) Ran, Yanchao; Malmirchegini, G. Reza; Clubb, Robert T.; Lei, BenfangThe heme-binding protein Shp of Group A Streptococcus rapidly transfers its heme to HtsA, the lipoprotein component of the HtsABC transporter, in a concerted two-step process with one kinetic phase. Heme axial residue-to-alanine replacement mutant proteins of Shp and HtsA (ShpM66A, ShpM153A, HtsAM79A, and HtsAH229A) were used to probe the axial displacement mechanism of this heme transfer reaction. Ferric ShpM66A at high pH and ShpM153A have a pentacoordinate heme iron complex with a methionine axial ligand. ApoHtsAM79A efficiently acquires heme from ferric Shp but alters the reaction mechanism to two kinetic phases from a single phase in the wild-type protein reactions. In contrast, apoHtsAH229A cannot assimilate heme from ferric Shp. The conversion of pentacoordinate holoShpM66A into pentacoordinate holoHtsAH229A involves an intermediate, whereas holoHtsAH229A is directly formed from pentacoordinate holoShpM153A. Conversely, apoHtsAM79A reacts with holoShpM66A and holoShpM153A in mechanisms with one and two kinetic phases, respectively. These results imply that the Met79 and His229 residues of HtsA displace the Met66 and Met153 residues of Shp, respectively. Structural docking analysis supports this mechanism of the specific axial residue displacement. Furthermore, the rates of the cleavage of the axial bond in Shp in the presence of a replacing HtsA axial residue are greater than that in the absence of a replacing HtsA axial residue. These findings reveal a novel heme transfer mechanism of the specific displacement of the Shp axial residues with the HtsA axial residues and the involvement of the HtsA axial residues in the displacement.Item The mechanism of heme transfer from the strepcoccal cell surface protein Shp to HtsA of the HtsABC transporter(Montana State University - Bozeman, College of Agriculture, 2010) Ran, Yanchao; Chairperson, Graduate Committee: Benfang LeiGroup 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.