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

Abstract

The iron-regulated surface determinants (Isd) of Staphylococcus aureus, including surface proteins IsdA, IsdB, IsdC, and IsdH and ATP-binding cassette transporter IsdDEF, constitute the machinery for acquiring heme as a preferred iron source. Here we report hemin transfer from hemin-containing IsdA (holo-IsdA) to hemin-free IsdC (apo-IsdC). The reaction has an equilibrium constant of 10 ؎ 5 at 22°C in favor of holo-IsdC formation. During the reaction, holo-IsdA binds to apo-IsdC and then transfers the cofactor to apo-IsdC with a rate constant of 54.3 ؎ 1.8 s ؊1 at 25°C. The transfer rate is >70,000 times greater than the rate of simple hemin dissociation from holoIsdA into solvent (k transfer ‫؍‬ 54.3 s ؊1 versus k ؊hemin ‫؍‬ 0.00076 s ؊1 ). The standard free energy change, ⌬G 0 , is ؊27 kJ/mol for the formation of the holo-IsdA-apo-IsdC complex. IsdC has a higher affinity for hemin than IsdA. These results indicate that the IsdA-to-IsdC hemin transfer is through the activated holoIsdA-apo-IsdC complex and is driven by the higher affinity of apo-IsdC for the cofactor. These findings demonstrate for the first time in the Isd system that heme transfer is rapid, direct, and affinity-driven from IsdA to IsdC. These results also provide the first example of heme transfer from one surface protein to another surface protein in Gram-positive bacteria and, perhaps most importantly, indicate that the mechanism of activated heme transfer, which we previously demonstrated between the streptococcal proteins Shp and HtsA, may apply in general to all bacterial heme transport systems.

HtaA Is an Iron-Regulated Hemin Binding Protein Involved in the Utilization of Heme Iron in Corynebacterium diphtheriae

“…It is predicted that hemin bound to HtaA is transferred to HtaB, which may function in an intermediate step in the movement of hemin from HtaA to HmuT. This mechanism of action for HtaB would be similar to the function proposed for some of the Isd proteins from S. aureus, which are involved in the movement of hemin through the cell wall (22,46,50). HmuT is proposed to deliver hemin to the HmuU permease, a component of the ABC transporter (HmuU and HmuV), which facilitates the uptake of hemin into the cytosol, where it is thought to be degraded by the heme oxygenase enzyme HmuO, releasing iron for cellular metabolism.…”

“…S. aureus binds hemin or hemoglobin to its cell envelope through various surface-anchored proteins, termed iron-regulated surface determinants (Isd), which contain sortase recognition signals at their C termini that are essential for the covalent linkage of these proteins to the peptidoglycan (24,46). It is believed that hemin is transported through the cell envelope via a cascade mechanism in which hemin is transferred between various Isd receptors (22,50). In S. pyogenes, hemin is proposed to bind initially to surface-exposed proteins, designated Shp and Shr, which appear to be anchored to the membrane by a putative transmembrane region in their C termini (21,49).…”

Overcoming the Heme Paradox: Heme Toxicity and Tolerance in Bacterial Pathogens

“…IsdB and IsdH are responsible for binding hemoglobin and hemoglobinhaptoglobin, respectively (24). IsdA extracts heme from IsdB or IsdH for passage to IsdC (64,73,94,131). This efficient heme-scavenging system brings heme across the membrane into the cytoplasm through the IsdDEF ABC transporter, where it can either be degraded by the heme oxygenases IsdG and IsdI to release free iron or be trafficked intact to the cell membrane (68,94).…”

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.…”

“…The pocket is bordered by a 3 10 -helix that is thought to be critical for NEAT function, including heme and/or hemoprotein binding (9,12,19,20). NEAT-NEAT heme transfer can be facilitated by the conserved iron-coordinating tyrosine (21)(22)(23)(24). However, the exact structural and molecular mechanisms mediating NEAT domain heme acquisition from methemoglobin (metHb) remains to be identified.…”