It is worth noting that the choice of buffer in this study was different in order to prevent any external ligand binding to the Fe cofactor. This buffer condition closely mimicked the experimental conditions for peroxidation, enabling us to gain a better understanding of the structural dynamics during the oxidation process. In contrast to the crystal structure of Biot2 ─ FePP • Sav S112A K121A, where the axial imidazole ligands from the crystallization buffer fixed the porphyrin plane in one conformation, the structure of Biot2 ─ FePP • Sav S112E K121H exhibited significant disorder around the iron center. This complexity made determining the orientation of the porphyrin challenging. The difficulties in determining the porphyrin orientation arise because the iron center occupies a special position on a crystallographic axis. While the presence and location of the Fe are unambiguous, any porphyrin disorder will be reflected to some extent in the crystallographically-related symmetry position within the same vestibule. This complexity adds to the intricacy of studying the structural dynamics of the system. Despite these challenges, the electron density maps unveiled the presence of at least three potential conformers for the porphyrin at different contour levels of the 2Fo-Fc maps (0.5 σ -1 σ) (Figure , three conformers are depicted in pink, yellow and cyan). After refinement, two conformations of the porphyrin were found to be canted relative to the surface of the biotin-binding vestibule and are symmetry related, while the third conformation sits parallel. However, upon comparison with the crystal structure of Biot2 ─ FePP • Sav S112A K121A, we were able to visualize a single conformer for the porphyrin plane in Biot2 ─ FePP • Sav S112E K121H, Figure . Additionally, the structure revealed no axial ligation originating from Sav, thus confirming our previous hypothesis that the residue at position 124 does not provide any axial ligation to the cofactor.