This step, which is the rate limiting step of the process with a free energy barrier of 13.5 kcal•mol -1 measured from the reactants E:I complex, appears to be a very asynchronous process (see Figure ). The transition state, TS2, defined as the maximum of the PMF but also confirmed by optimizing and characterizing a representative structure at M06-2X/6-31+G(d,p)/MM level (see Figure and Tables and), is characterized by Sg-Cb bond formation in a very advanced stage of the process (1.89 Å) but a proton transfer in an early stage of the reaction Hg-Ca distance of ca. 1.70 Å. This concerted character was also confirmed by tracing the IRC down to the ion pair intermediate and the product from the optimized TS2, which in fact was used to generate the free energy profile with the FEP method described above (see Supporting Information). From a technical point of view, it is important to note that both methods, US and FEP, render the same description of the process with just slightly quantitative energetic differences (see Figure vs Figure ). Finally, the analysis of the average interaction energies (electrostatic plus Lennard-Jones) between residues of M pro and each fragment of the inhibitor B3 computed at the TS2 shows that the pattern of interactions does not significantly change from the one obtained in the E:I complex (see Supporting Information vs Figure ).