We hypothesized that mutation F12Y could also help in the preorganization of the active site pocket, thus impacting and favoring the productive binding of the azide (and epoxide) in place for the ring-opening reaction to occur. To that end, we ran nanosecond timescale MD simulations for both HheC wild type and mutant F12Y in the absence of any substrate, and in the presence of epoxide 1 and azide in the active site. The analysis of the MD simulations in the absence of any ligand indicated that F12Y establishes a hydrogen bond with the backbone of T131 that is adjacent to the catalytic S132 (Figure ). This interaction established between the hydroxyl group of Y12 and T131, which is obviously not possible in HheC wild type, has some important implications for the active site preorganization. Thanks to this interaction, the loop containing F12Y is slightly more rigid (Figure ), which helps in retaining azide in place for the epoxide-ring opening reaction (Figure , right panel). In HheC wild type, such an interaction is not possible, thus the side chain of F12 is substantially more flexible and clearly affects the binding of the azide in the nucleophile binding pocket (Figure , left panel). Moreover, this newly established hydrogen bond between Y12 and T131, and the resulting loop rigidification likely contribute to the observed higher thermal stability of HheC mutant F12Y as well, as suggested previously. Although the kinetic constant (kobs,max) for mutant F12Y is substantially improved, the mutation at the same time affects the binding of azide and induces stronger cooperativity (Table ). The higher K50 value found for azide in mutant F12Y can be explained by the distance between azide and position F12Y (Figure ). In F12Y, azide can adopt two different binding modes: the catalytically productive pose as shown in Figure , and an additional one in which azide displaces the epoxide and interacts with both the catalytic S132 and F12Y. This additional, catalytically non-productive binding mode likely causes the higher K50 value found experimentally for azide in mutant F12Y (Table ). We additionally applied our correlation-based tool Shortest Path Map (SPM) to investigate how the communication network between subunits might be altered through mutation F12Y (Figure ). Interestingly, we observed a much more interconnected network in mutant F12Y, thus suggesting that the establishment of the Y12-T131 interaction enhances the intramolecular interactions and the allosteric communication between subunits. This is in line with the higher Hill coefficient of HheC F12Y found experimentally (Table ).