In this study, we report experimental results that provide the first complete challenge of a proposed model for heme acquisition by Staphylococcus aureus via the Isd pathway first put forth by Mazmanian, S. K., Skaar, E. P., Gaspar, A. H., Humayun, M., Gornicki, P., Jelenska, J.

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.

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Staphylococcus aureus is the source of a large number of hospital-acquired infections, of which many are serious and can lead to death. Iron is critically important to the survival and growth of the bacterium, and complex, multistep mechanisms are present to fulfill the necessary iron requirement. Isd proteins located on the wall and membrane of S. aureus have been proposed to function in heme acquisition. We report characterization of the S. aureus heme-binding protein IsdE, the lipoprotein component of a membrane-localized ABC transporter that is believed key to receiving heme from cell wall-anchored Isd proteins. Magnetic circular dichroism (MCD) data, which greatly extend the results from our initial study of IsdE in bacterial cell lysates (Mack, J., Vermeiren, C., Heinrichs, D. E., and Stillman, M. J. (2004) Biochem. Biophys. Res. Commun. 320, 781-788), probe the ligand and redox properties of the bound heme. The MCD data show that IsdE, when overexpressed in E. coli, binds either ferric or ferrous heme but that the largest fraction is low spin ferrous heme. Studies of mutants allowed identification and characterization of the ligands in the fifth and sixth position on the heme iron as histidine, proximally, and methionine, distally. This histidine-methionine heme-iron ligation is unique to heme transport proteins. The smaller fraction of ferric heme in the protein is not bound by methionine, allowing for access by strong field ligands, such as cyanide. Electrospray ionization mass spectral data are reported for the first time and show that only one heme ligand binds per IsdE protein molecule. These data also show there is little change in the conformation of the protein between the heme-bound and heme-free species, indicating that the heme-free IsdE adopts a structure essentially independent of the heme. The mass spectral data clearly show that IsdE reversibly unwinds under denaturing conditions to form at least two distinct, heme-free conformations.

Staphylococcus aureus is a human pathogen that results in numerous infections in hospital settings and recently also in the wider community. Its antibiotic resistant forms are causing considerable alarm. A series of surface-anchored proteins that have heme uptake and transport properties have been reported. Through the use of absorption and magnetic circular dichroism spectroscopies and mass spectrometry, the iron-free, protoporphyrin IX and the iron-containing, heme-binding characteristics of bacterial rIsdC have been obtained. Mass spectrometry showed that following isolation and purification, the rIsdC is bound predominantly to protoporphyrin IX and to a lesser extent heme, unlike the case of rIsdA, which binds predominantly heme. Magnetic circular dichroism analysis provided further information regarding porphyrin binding because the characteristic magnetic circular dichroism band envelopes for the iron-free protoporphyrin IX and the iron-containing heme can be clearly distinguished in the spectrum of the rIsdC. Analysis of these spectral data showed that the minor heme component exists as a high-intermediate spin state ferric heme when bound to rIsdC, similar to the high-spin ferric heme reported for the rIsdA protein.