## 3 Results and Discussion  
The EbSe (Figure 1) and EbSeS binding poses were obtained from the molecular docking, as described in the Computational Methods, and the adduct between EbSe and EbSeS with the model catalytic pocket was reoptimized via DFT. The reactant complex (RC) formed by the four amino acids cluster with docked EbSe was used as the starting point of our mechanistic investigation. From our calculations, (Figure 2) the overall inhibition mechanism closely resembles the acylation step of the fully functional MP\({}^{\text{pro}}\) shown in Scheme 1a.b. The presence of EbSe does not impair the activation of Cys145, which is effectively deprotonated by His41 with an activation energy of about 15.16 kcal mol\({}^{-1}\) (TS1), a value close to that previously reported for this step in a computational study on the functional enzyme (19.9 kcal mol\({}^{-1}\)) [5]. Notably, an Ion Pair (IP) between deprotonated cysteine (Cys') and protonated Histidine (HIP) was not located on the potential energy surface (PES); in addition, after the proton transfer, the cysteine residue efficiently attacks the Se atom of EbSe, leading to an adduct with an almost linear N-Se-S bond (three centers intermedi  
Figure 1: (**A**) Docking simulations between MP\({}^{\text{pro}}\) and EbSe. For better visualization, only the side chain of the main residues is shown. (**B**) Optimized MP\({}^{\text{pro}}\) cluster, with EbSe docked inside. For clarity, only C\({}^{\alpha}\), H\({}^{\alpha}\) and amino acids side chains are displayed, and it is oriented for optimal view. Level of theory: B3 LYP-D3(BJ)/6-311G(d,p), cc-pVTZ. mational freedom of the backbone might help in stabilizing an Ion Pair, which was located in another MP\({}^{\text{pro}}\) mechanistic investigation [50], our results suggest that the cysteine attack to the Se-N bond occurs with a very low, if any, activation energy. The TCI is situated 3.41 kcal mol\({}^{-1}\) above the RC; thus, the step is weakly endergonic. After the formation of the TCI, full covalent inhibition is reached with the back proton transfer from HIP to the N atom of EbSe, leading to the N-Se bond cleavage and to the complete formation of the S-Se bond between Cys145 and EbSe. This step is exergonic; the inhibited product (Pinhb) is almost 7 kcal mol\({}^{-1}\) more stable than the RC. While the back-proton transfer is predicted to have an activation energy of about 26 kcal mol\({}^{-1}\), thus being rate-determining for the whole process, the geometrical features of the transition state (TS2) appear slightly distorted, due to the constraints imposed to His41 backbone. Therefore, the activation energy of this process is likely overestimated in our model because of the limited flexibility of the catalytic pocket. Indeed, in the acylation step of the functional MP\({}^{\text{pro}}\), the analogous step occurs with a definitively lower activation energy [5]. However, previous investigations on enzymatic clusters [41; 42] and on oversimplified models [51] based on reactive Cys/Sec showed that while the barriers might be magnified in these last ones, the relevant features of the mechanism can be correctly reproduced even in very reduced systems, given that the relevant reactive residues are considered, as in our case. The Pinhb obtained here presented a similar binding pose when compared with the EbSe from the covalent docking, previously published [52]. Using the same approach, (Figure 3) the inhibition mechanism by EbSeS has been explored (energies are reported in Table S3 (Supplementary Materials)). As seen for EbSe, in this case, the inhibitor does not impair Cys145 activation, which occurs with an even lower activation energy of 8.83 kcal mol\({}^{-1}\) (TS1). However, this is almost certainly related to the closer proximity of Cys145 and His41 residues in the cluster (the distance between S and N atoms is 3.46 A; conversely, in the cluster docked with EbSe, the corresponding distance is 4.61 A). After TS1, the mechanism of EbSeS displays important differences with respect to that of EbSe.