On the acetophenone side, 2,4,6-trifluoro substituted 1b showed improved conversions with both EREDs, and boosted ee in the case of GluER T36A. Yet, an electron-withdrawing para-nitro group (2c) resulted in very low conversions with both EREDs. An electron-donating para-methoxy group (2d) reduced conversion only for GluER T36A. This suggests both EREDs are affected to different extents by both steric and electronic effects. reaction over competing dehalogenation. A gating mechanism has also been suggested for a non-light dependent reaction, but has not been directly shown. To investigate this hypothesis (Figure ), we carried out pre-steady state kinetics by stopped-flow with a diode array detector, to follow the reoxidation of flavin hydroquinone (FMNhq) by 1a in the presence or absence of 2a. We observed FMNhq and FMNox, but not the FMN semiquinone (FMNsq, Figure ), indicating that the initial single electron transfer (SET) is much faster than the subsequent radical reactions and the final hydrogen atom transfer (HAT). Thus, the stopped-flow data was most appropriately modelled as a single step from FMNhq to FMNox (Figure ). ), implying that the SET is the rate determining step. The reaction was thus modelled as a single step (rate constant kox). An acceleration of the reaction was observed in the presence of the vinyl co-substrate (2a). The affinity for 2a was one order of magnitude higher compared to 1a, explaining the exquisite selectivity of the enzyme for the formation of 3aa. C: Deuterium incorporation experiments, showing that the hydrogen at the benzylic position originates from the FMN cofactor.