Iron is an essential nutrient that is actively acquired by bacterial pathogens during infections. Clinically important Staphylococcus aureus obtains iron by extracting heme from hemoglobin using the IsdH surface receptor; extraction is done by a tri-domain unit that contains its second (N2) and third (N3) NEAT domains, joined by a helical linker. Previous studies found these three domains are necessary for heme transfer and resolved the IsdH:Hb complex, but the underlying mechanism remains unknown. In this work, we probed the mechanism of heme capture by using steered and conventional molecular dynamic simulations to explain NMR and stopped-flow transfer kinetics measurements. An alanine mutagenesis analysis reveals that two subsites trigger heme with the transiently forming receptor-Hb interface by contacting Hb's F-helix. The subsites are located within the N3 and linker domains and appear to play distinct roles stabilizing the heme transfer transition state. Using this information, we present a model of the transfer reaction that provides new insight into the mechanism underpinning microbial heme transfer.