title: Characterization of the heme binding properties of Staphylococcus aureus IsdA.
abstract: We report the first characterization of the physical and spectroscopic properties of the Staphylococcus aureus heme-binding protein IsdA. In this study, a combination of gel filtration chromatography and analytical centrifugation experiments demonstrate that IsdA, in solution, is a monomer and adopts an extended conformation that would suggest that it has the ability to protrude from the staphylococcal cell wall and interact with the extracellular environment. IsdA efficiently scavenged intracellular heme within Escherichia coli. Gel filtration chromatography and electrospray mass spectrometry together showed that rIsdA in solution is a monomer, and each monomer binds a single heme. Magnetic circular dichroism analyses demonstrate that the heme in rIsdA is a five-coordinate high-spin ferric heme molecule, proximally coordinated by a tyrosyl residue in a cavity that restricts access to small ligands. The heme binding is unlike that in a typical heme protein, for example, myoglobin, because we report that no additional axial ligation is possible in the high-spin ferric state of IsdA. However, reduction to ferrous heme is possible which then allows CO to axially ligate to the ferrous iron. Reoxidation forms the ferric heme, which is once again isolated from exogenous ligands. In summary, rIsdA binds a five-coordinate, high-spin ferric heme which is proximally coordinated by tyrosine. Reduction results in formation of five-coordinate, high-spin ferrous heme with a neutral axial ligand, most likely a histidine. Subsequent addition of CO results in a six-coordinate low-spin ferrous heme also with histidine likely bound proximally. Reoxidation returns the tyrosine as the proximal ligand.
authors.affiliation: Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada N6A 5C1.
authors.forename: Christie L
authors.initials: CL
authors.lastname: Vermeiren
authors.forename: Mark
authors.initials: M
authors.lastname: Pluym
authors.forename: John
authors.initials: J
authors.lastname: Mack
authors.forename: David E
authors.initials: DE
authors.lastname: Heinrichs
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Stillman
mesh_terms: D000942:Antigens, Bacterial / Q000737:chemistry* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D006418:Heme / Q000378:metabolism*; D013058:Mass Spectrometry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013211:Staphylococcus aureus / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C512791:IsdA protein, Staphylococcus aureus; D011994:Recombinant Proteins; D006418:Heme