The stopped-flow experiments confirmed a rate enhancement of the SET in the presence of cosubstrate 2a. Furthermore, the affinity for 2a is an order of magnitude higher than for substrate 1a. This suggests the highly reactive α-acyl radical is predominantly formed in the presence of 2a, demonstrating a gating mechanism, where the α-acyl radical is predisposed for C-C bond formation. Deuterium labelling experiments confirm that the benzylic hydrogen in 3aa comes from the FMN cofactor, as expected (Figure ). : A: Outcome of biotransformations with GluER T36A with 2a only, 1a only, or both 1a and 2a. Concentrations determined by HPLC using a calibration curve. It is apparent that in the absence of 2a, the fate of the α-acyl intermediate is not predominantly 4a. Also shown is the appearance of biotransformations with GluER T36A after 24 h with no substrate, 2a only, 1a only, or both 1a and 2a. The yellow colour in the reaction with 1a is indicative of oxidized flavin, implying enzyme inactivation. B: UV-vis spectra of reactions containing 1a, as well as controls without substrate, GluER T36A, or GDH. The spectrum of FMN after a reaction with 1a only matches that of enzyme bound oxidized flavin (no substrate, no GDH control). The control containing 1a, but no GDH showed extensive precipitation of GluER T36A resulting in a lower flavin peak. In the absence of 1a, FMN remains fully reduced, with some slight turbidity. C: Coomassie-stained SDS-PAGE gel of biotransformations with GluER T36A after 24 h with no substrate, 2a only, 1a only, or both 1a and 2a. The orange box shows GluER T36A. D: Difference in peptide abundance after tryptic digest of the band of GluER T36A in the reaction with 1a only and without substrate. For the difference in peptide abundance of 2a only and 1a and 2a, see Figures S5&6. E: Active site of GluER T36A (PDB: 6MYW) with acetate bound, showing peptides with decreased abundance by colour, as well as putative amino acids in the active site that may become modified by radical reactions.