While we've shown that F121 residue plays a significant role in preventing unproductive oxidation in SyrB2, we wanted to assess the impact of this residue across similar proteins. Using the SyrB2 sequence, we performed a BLAST analysis and sorted hits into hydroxylases (HXD/E motif) or halogenases (HXA/G motif). After removing redundant hits, 635 protein sequences were aligned, and LOGOS plots were generated (Fig ). For hydroxylases, the prominent amino acid in position 121 is a redox-active tyrosine. In halogenases, on the other hand, the redox-inactive phenylalanine is strongly conserved at position 121. Since mutation of phenylalanine to tyrosine at this position in SyrB2 has led to an off-cycle species, it's intriguing that the hydroxylases have evolved to have a redox-active tyrosine proximal to the reactive iron center. We believe the differential residue conservation at position 121 for hydroxylases and halogenases is due to the isomeric flexibility of the reactive ferryl intermediate (Fig ). Only one isomer of ferryl intermediate is plausible for hydroxylases: a trigonal bipyramidal structure with the oxo ligand and a histidine ligand defining the axial plane. Due to the presence of a third amino acid residue (Asp or Glu) coordinating to the iron center, the ferryl intermediate in hydroxylases is "locked" into one geometry. Even if a tyrosine ligand were present in the active site, so long as it's not proximal to the oxo ligand, unproductive oxidation to DOPA would not occur. Note that while all structures are drawn with succinate in monodentate coordination, bidentate coordination is also plausible. In halogenases, on the other hand, the Glu or Asp in the facial triad are replaced by Ala or Gly. This replacement sterically and electrostatically accommodates halide binding to the iron center. As a consequence, the protein's ability to "lock" the reactive intermediate into a single isomer is lost. Three different isomers for the haloferryl intermediate have been proposed for halogenase systems (Fig ). One is analogous to hydroxylases and exists as trigonal bipyramidal structure with the oxo ligand and a histidine ligand defining the axial plane (1). Another isomer is structurally similar to 1 but swaps the positions of the oxo and halide ligands (2). The last isomer is best described as a heavily distorted trigonal bipyramidal structure, with the halide ligand and the coordinating oxygen of the succinate ligand defining the axial plane (3). Given this flexibility, any one of these intermediates could be reactive across the 2OG-dependent halogenase enzymes. Since reactive haloferryl intermediates exist in the timescales of hundreds of seconds, halogenases must prevent a non-productive isomer from damaging the protein or leading to an unreactive enzyme intermediate. Evolution of the redox-inactive phenylalanine residue at position 121, therefore, prevents an unproductive oxidation from occurring, maintains catalytic activity, and allows the halogenase to align a productive ferryl isomer for its halogenation function.