Consequently, the much more computationally demanding M06-2X/6-31+G(d,p)/MM US method was applied only to the exploration of the mechanism rendering the E-I final product. The resulting free energy profile for the covalent inhibition of SARS CoV-2 M pro with B3 is depicted in Figure , while the evolution of the selected bond distances along the PMF is shown in Figure . pair complex, E (+/-) :I, a zwitterion species that according to previous studies is well described by the M06-2X functional here, is clearly less stable than the initial complex in which both residues of the C145/H41 dyad are in their neutral states (by ca. 8 kcal•mol - 1 ). This result agrees with our previous computational studies of the proteolysis reaction and the inhibition reaction with different inhibitors. Thus, despite the quantitative energetic difference between the ion pair and the neutral form appears to be dependent on the substrate, the neutral dyad must be considered as the starting state of the reaction catalyzed by M pro . As shown in Figure , the proton transfer from C145 to H41 is associated with a slight approach of the sulfur atom of the former to the nucleophilic atom of the substrate (from 3.5 to 2.8 Å). Then, the covalent bond formation between C145 and the Cb atom of the substrate takes place concertedly with the proton transfer from the protonated H41 to the Ca atom of the substrate to reach the final E-I covalent complex.