A visual inspection of SyrB2's active site reveals two hydrophobic Phe residues that were ~4 Å away from iron's ligands (Fig. ). Specifically, the F104 residue is 3.8 Å from a water ligand and F121 residue is 3.9 Å away from the chloride ligand. To assess if the hydrophobic nature of these residues was important, tyrosine mutants of both residues, i.e. F104Y and F121Y SyrB2, were constructed, expressed, and purified. Upon purification, WT SyrB2 and F104Y exhibited no signatures in the visible range of the UV-Vis spectrum, implying that both halogenases purify without iron bound to the active site (Fig. ). In contrast, SyrB2-F121Y purified as a bluegreen solution with a λmax at 650 nm (Fig ). This blue-green chromophore has been observed in a few NHFe proteins previously, and has been postulated to originate from a ligand-to-metal charge transfer band (LMCT) of a DOPA-Fe 3+ species. We hypothesize that such a species has formed from intra-cellular oxidation of the F121Y mutant and will refer to this SyrB2 variant as F121Y/DOPA. To further investigate the iron oxidation state in F121Y/DOPA, we attempted to oxidize the protein using hydrogen peroxide (Fig. ). Even after incubation with 100 equivalents of peroxide, no shift in the LMCT band was observed, which implies that the iron center is not in the ferrous state. To further characterize the electronic structure of the iron center, we investigated the F121Y/DOPA protein with EPR spectroscopy. The X-band EPR spectrum shows a distinct signal at g ~ 4.29 with additional features at g ~ 5.03 and 9.06 (Fig ). The signal at g ~ 4.29 is a hallmark of high-spin ferric iron species in a highly orthorhombic environment (E/D = 0.33). The broad shoulder at g ~ 5.03 and small peak at g ~ 9.06 could be originating from a different Fe 3+ -bound species in a more rhombic environment (E/D = 0.2).