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.

Direct Hemin Transfer from IsdA to IsdC in the Iron-regulated Surface Determinant (Isd) Heme Acquisition System of Staphylococcus aureus

“…1A). These absorp-tion features are consistent with the previous observation that the heme iron in IsdA is pentacoordinate (19,24).…”

“…IsdA and the B. anthracis homologue of IsdC are also important for hemin uptake (12,19). IsdB, IsdA, and IsdC bind heme (11,19,20,23,24), and structural studies show that IsdA and IsdC bind hemin in a pentacoordinate complex with a tyrosine residue as the only axial ligand (19,20), in contrast to the hexacoordination of the heme iron in Shp and HtsA (14,15). It has been proposed that IsdH and IsdB capture haptoglobin-hemoglobin and hemoglobin, respectively, and heme is transferred from bound hemoglobin to IsdA, then IsdC, and finally to ABC transporters IsdDEF and/or HtsABC (25,26).…”

The Near-iron Transporter (NEAT) Domains of the Anthrax Hemophore IsdX2 Require a Critical Glutamine to Extract Heme from Methemoglobin

“…To confirm that the mutations did not affect heme binding, recombinant NEAT domains expressed overnight were assayed for their ability to co-purify with endogenous heme during expression in E. coli. Each NEAT domain was purified as stated under "Experimental Procedures," and bound heme was detected by measuring the Soret absorbance at ϳ400 nm, a well documented spectroscopic assay used to detect the presence of bound heme iron (7,12,20,24,(35)(36)(37)(38)(39)(40). As indicated in Fig.…”

Crystal Structure of the Heme-IsdC Complex, the Central Conduit of the Isd Iron/Heme Uptake System in Staphylococcus aureus

“…Although biochemical and spectroscopic studies on some components of the system have been carried out (for example, Refs. [17][18][19][20][21], the structural basis of heme binding and the molecular mechanisms of heme transfer are still unexplored. Molecular data on the NEAT domain-containing proteins could also help in the design of better vaccines.…”