While EPR spectroscopic studies confirm the presence of a high-spin ferric center in F121Y/DOPA sample, similar signals have been previously observed in a ferric-phenolate complex (i.e. a tyrosinate residue binding to Fe 3+ ) as well. However, high-spin ferric-phenolate species exhibit UV signatures blue-shifted (550 nm) relative to the F121Y/DOPA species (Fig To exclude the possibility of ferric-tyrosinate and confirm the presence of DOPA posttranslational modification in F121Y/DOPA sample, we executed a reaction that specifically targets DOPA amino acids. Nitration/oxidation of the DOPA amino acid results in the formation of a di-nitro-DOPA-quinone species with a distinct UV-Vis signature at 500 nm (Fig. ). We performed this reaction on different concentrations of L-DOPA free amino acid to develop a standard curve, which would quantify the extent of DOPA modification in F121Y/DOPA (Fig. ). After denaturation of F121Y/DOPA in acidic buffer, the nitration/oxidation reaction was performed, and the UV-Vis spectrum was recorded (Fig . A peak at 510 nm was present in the product, consistent with other DOPA-proteins treated with this method, supporting DOPA modification in F121Y/DOPA SyrB2. Based on the absorbance values (0.04, ε500 nm, L-DOPA = 9,800 M -1 cm -1 ), ~20% of the F121Y/DOPA protein mixture contains the DOPA modification. Thus, F121Y is not fully oxidized to F121DOPA during protein expression analogous to iron-substituted catechol dioxygenase and F208Y ribonucleotide reductase which also purified as a mixture of tyrosine and DOPA containing variants. Oxidation of tyrosine to DOPA likely occurs through the formation of the ferryl intermediate.