The first mass at 37408 Da corresponds to the apo SyrB2-F121Y mutant. This mass is lower than expected for F121Y SyrB2 (theoretical mass: 37525 Da). This missing mass (117 Da) can be accounted for by the C-terminal residue, valine, which was cleaved during ionization in the MS experiments. A second less intense peak was found at 37585 Da, which is 177 Da greater than the apo F121Y species. Given the presence of iron and DOPA modification accounts for only 72 Da of mass, an extra 105 Da is left unaccounted for. Since the binding of native ligands 2OG (144 Da) and succinate (116 Da) cannot account for this mass differential, other ligands may be present bound to the ferric-DOPA active site. We propose that a non-native ligand, pyruvate (87 Da), is bound to ferric ion in the active site via its alpha-keto functional group. The active site is possibly quite perturbed with the formation of the ferric-DOPA species (F121 is located ~6 Å from the iron center in the WT crystal structure) and it's feasible that the protein's affinity for various ligands has changed. Given the ubiquity of pyruvate in the cellular environment (390 µM), the overexpressed protein would have an abundance of this ligand available to bind. The rest of the unaccounted mass (18 Da) is explained by an aqua ligand, which is present in the WT crystal structure and numerous other non-heme iron structures. Overall, using a combination of spectroscopic, mass-spectrometric, and biochemical studies, we were able to confirm the formation of DOPA post-translational modification in SyrB2 halogenase.