We were pleased to observe that Biot2 ─ FePP binds to both biotin-binding sites flanking the vestibule. Notably, due to the longer chain length of the linkers, we anticipated multiple conformations of the porphyrin core. To mitigate this, Sav AA crystals were grown in conditions containing imidazole that would provide axial ligation to the Fe-porphyrin, fixing it to a specific conformation. Consequently, in the electron density map of Biot2 ─ FePP • Sav AA, we found porphyrin core in a single conformation with residual density that could be clearly attributed to the binding of at least one external imidazole ligands. Due to crystallographic symmetry constraints, it is not possible to determine whether both axial ligands are bound simultaneously. Coordination of an axial imidazole pointing towards the biotin-binding vestibule, suggests that there is room enough between the Fe-porphyrin and the protein to accommodate a substrate, tightly embedded within the vestibule. Inspection of second coordination sphere interactions between the cofactor and close-lying residues revealed a hydrogen bond between the sulfonate group of the cofactor and Sav N49. The amino acid residues closest to this imidazole include Sav S112A, K121A, and L124, Figure for Biot2 ─ FePP is displayed as grey mesh and red mesh for anomalous scattering for Fe (modelled at 80 % occupancy). The external imidazole ligands (originating from the crystallisation buffer) are displayed in blue. The H-bonding interaction of the SO3 ─ group with the Sav N49 is depicted in yellow. Close-lying residues, including S112A, K121A, and L124, are highlighted color-coded sticks (C in magenta).