HasA_SM, a hemophore secreted by the Gram-negative bacteria Serratia marcescens, extracts heme from host hemoproteins and shuttles it to HasR_SM, a specific hemophore outer membrane receptor. Heme iron in HasA_SM is in a six-coordinate ferric state. It is linked to the protein by the heretofore uncommon axial ligand set, His32 and Tyr75. A third residue of the heme pocket, His83, plays a crucial role in heme ligation through hydrogen bonding to Tyr75. The vibrational frequencies of coordinated carbon monoxide constitute a sensitive probe of trans ligand field, FeCO structure, and electrostatic landscape of the distal heme pockets of heme proteins. In this study, carbonyl complexes of wild-type (WT) HasA_SM and its heme pocket mutants His32Ala, Tyr75Ala, and His83Ala were characterized by resonance Raman spectroscopy. The CO complexes of WT HasA_SM, HasA_SM(His32Ala), and HasA_SM(His83Ala) exhibit similar spectral features and fall above the line that correlates ν_Fe-CO and ν_C-O for proteins having a proximal imidazole ligand. This suggests that the proximal ligand field in these CO adducts is weaker than that for heme-CO proteins bearing a histidine axial ligand. In contrast, the CO complex of HasA_SM(Tyr75Ala) has resonance Raman signatures consistent with ImH-Fe-CO ligation. These results reveal that in WT HasA_SM, the axial ImH side chain of His32 is displaced by CO. This is in contrast to other heme proteins known to have the His/Tyr axial ligand set, wherein the phenolic side chain of the Tyr ligand dissociates upon CO addition. The displacement of His32 and its stabilization in an unbound state is postulated to be relevant to heme uptake and/or release.