Heme acquisition system A (HasA), secreted by various gram-negative pathogens, uses a unique histidine iron tyrosine (His-Fe-Tyr) coordination to scavenge heme (i.e., protoporphyrin IX-Fe complex) as an iron source. In order to study heme uptake by HasA, we utilized axial ligand mutants and protoporphyrin IX. Our results suggest that the binding of heme in apoprotein is initiated by interactions between heme and hydrophobic residues in the heme pocket. Subsequently, the sequential coordination of Tyr-75 and His-32 with the iron appears to complete holoprotein formation.