Multiprotein Heme Shuttle Pathway in Staphylococcus aureus: Iron-Regulated Surface Determinant Cog-Wheel Kinetics

Iron is a critically important nutrient for all species. Bacteria have evolved specialist survival systems to chelate and transport iron across the wall and membrane into the cytoplasm. One such system in the human pathogenic bacteria Staphylococcus aureus involves extracting heme from hemoglobin and then transporting the intact heme across the wall and membrane. The iron-regulated surface determinant (Isd) proteins act in concert to carry out the heme scavenging and subsequent transport. While details of the static heme-binding reaction are currently quite well known, little mechanistic data are available. In this paper, we describe detailed time-resolved mass spectral and magnetic circular dichroism spectral data recorded as heme is transferred unidirectionally from holo-IsdA to apo-IsdE via IsdC. The electrospray mass spectral data simultaneously monitor the concentrations of six protein species involved in the trans-wall transport of the extracted heme and show for the first time the mechanistic details of heme transfer that is key to the Staphylococcus aureus Isd heme-scavenging system. Bimolecular kinetic analysis of the ESI-mass spectral data shows that heme transfer from IsdA to IsdC is very fast, whereas the subsequent heme transfer from IsdC to IsdE is slower. Under limiting IsdC conditions, the IsdC intermediary cycles between heme-free and heme-containing forms until either all heme has been transferred from holo-IsdA or no further apo-IsdE is available. The data show that a unique role for IsdC is acting as the central cog-wheel that facilitates heme transfer from IsdA to IsdE.