Next, we tested the impact of DOPA post-translational modification on the halogenation catalysis of SyrB2. To that end, we developed halogenation assays for SyrB2 with its native substrate, Thr, which is delivered to SyrB2's active site while covalently bound to the ppt group of SyrB1. Chlorinated, hydroxylated (minor product), or unreacted threonine were liberated from SyrB1 after incubation with TycF thioesterase. These products were first derivatized with 6aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) and then analyzed by reversed-phase UPLC/Multiple-Reaction-Monitoring-MS/MS for detection. WT-SyrB2, when subjected to halogenation assay, reveals AQC-Cl-Thr as the major product, eluting slightly after the unreacted AQC-Thr (Fig ). Isotopic ratios of AQC- Cl-Thr to AQC- Cl-Thr were shown to be 3:1, consistent with the natural abundance of chlorine, and providing validation to the identity of chlorinated Thr (Fig ). The AQC-OH-Thr minor product was also observed, eluting prior to the unreacted AQC-Thr, though with much less intensity than AQC-Cl-Thr. SyrB2-F121Y/DOPA, on the other hand, when subjected to similar halogenation assays (with or without the reductant ascorbate), showed no chlorinated or hydroxylated products (Fig 3B ). The only analyte observed corresponds to the unreacted Thr substrate (Fig ). While a low-intensity peak is present close to the noise at 2.52 min. in the AQC- Cl-Thr channel, no such peak is seen at the same elution time in the AQC- Cl-Thr channel suggesting no chlorinated product was formed. Even in the presence of ten equivalents of ascorbate, no products were observed (Fig ). With these observations, we postulate that the ferric-DOPA SyrB2 species is a catalytically inactive, off-cycle species. We hypothesize that the modification of tyrosine to DOPA itself isn't necessarily the reason why F121Y/DOPA is catalytically inactive. It's the binding of ferric iron by the catecholate group of DOPA that results in an enzymatic species that's not part of the overall catalytic cycle. The DOPA121 is possibly bound to the ferric iron in a bidentate fashion which would result in large distortions in the active site, since F121 is located ~6 Å away from iron in the WT active site. To test this hypothesis, we performed UV-Vis titration of chloride into SyrB2-F121Y/DOPA and observed no perturbation in the LMCT band eluding to the fact that DOPA121 residue is binding in a position where chloride would bind or that distortion of the active site prevents chloride binding (Fig ).