Intrigued by how the central aromatic residue in motif 1 enhances HheC activity towards the epoxide-ring opening reaction, we decided to computationally evaluate HheC wild-type and mutant F12Y by means of Quantum Mechanics (QM) and Molecular Dynamics (MD) simulations. Considering the proximity of position F12 to the catalytic residues (Figure ) and the big impact on reaction rate (Table ), we hypothesized that the additional hydroxyl group could potentially establish hydrogen bonds with either the catalytic and binding residues, the nucleophile binding pocket and/or the substrates epichlorohydrin (1) and azide to promote catalysis. To elucidate whether mutation F12Y directly impacts the activation barrier for the epoxide ring-opening reaction, we generated a cluster model of the active site and used DFT as done previously by the group of Himo (Figure , 5B). We observed that both in the reactant complex (RC) (Figure ) and the transition state (TS1) the additional hydroxyl group thanks to mutation F12Y can establish a hydrogen bond with azide, which helps to retain the nucleophile in the nucleophile binding pocket and more importantly positions azide in a good orientation for epoxidering opening (Figure , left panel). The terminal nitrogen of azide establishes a hydrogen bond with the backbone of L178 and the hydroxyl group of Y12, whereas the nitrogen involved in the nucleophilic attack is hydrogen-bonded to a crystallographic water molecule (Figure , left panel). This hydrogen bond network impacts the charge distribution at the azide and favors the accumulation of more negative partial charge at the nitrogen responsible for the nucleophilic attack (Table ). The activation barrier at this conformation towards the epoxide ring opening 1 is ca. 9 kcal/mol at the M06-2X/Def2-TZVPP level of theory. We located another TS2 that does not present the F12Y-azide interaction, instead the terminal nitrogen of azide makes a hydrogen bond with both the crystallographic water molecule and L178 (Figure , right panel). This difference in the hydrogen bond network with respect to TS1 slightly modifies the negative charge on the nucleophilic nitrogen of the azide, thus leading to an activation barrier for TS2 ca. 6 kcal/mol higher than for TS1 (the activation barrier for TS2 is ca. 16 kcal/mol). This additional TS2 is extremely similar to the one found for HheC wild type (Figure ). In the wild type, the activation barrier for the ring opening of 1 is ca. 14 kcal/mol as found for TS2 in the case of F12Y. The computed barriers are in line with the previously reported barriers for HheC with azide and other epoxides reported by Himo and coworkers. It should be mentioned that the large differences in the activation barriers found for HheC F12Y and wild type are overestimated as compared to the experimental kinetic constants, but in line with the observed big impact of mutation F12Y on the catalytic turnover (minimum 5-fold increase in kobs,max). We observed in the DFT-optimized RC as well as TS2 that F12Y makes a hydrogen bond with the carbonyl backbone of P175 (the distance between the hydrogen of the hydroxyl group of Y12 and the oxygen of the backbone of P175 is ca. 2 Å in all cases, see Figure and). The carbonyl group of P175 was found to provide electrostatic stabilization to C, which favors the attack at this position. The hydrogen bond established between the hydroxyl group of F12Y and P175 induces a slight bend of the carbonyl backbone. HheC mutant F12Y therefore also favors the proper positioning of the P175 backbone close to the epoxide substrate and favors the attack at the less substituted carbon.