As shown in Figure and schematically depicted in Scheme 3, the inhibition process with B4 is equivalent to that obtained with B3. Thus, the generation of a transient ion pair intermediate E (+/-) :I by a proton transfer from C145 to H41 precedes the formation of the covalent complex between C145 and the carbonyl carbon atom of B4. The first step is virtually the same as in the case of the inhibition with B3, confirming that once again, the neutral reactant complex is favored with respect to the ion pair dyad. Evolution of the key distances with the progression of the reaction shows that the proton transfer from C145 to H41 is also associated with an approach of the former to the carbonyl carbon atom, from 3.0 to 2.2 Å thus generating a more reactive conformation (Figure ). Then, the second step, which as in the case of B3, represents the rate-limiting step of the full inhibition process, involves the acylation of the protein together with the proton transfer from the protonated H41 to the carbonyl oxygen atom of the inhibitor with an energy barrier, 15.2 kcal•mol -1 . This barrier is slightly higher than that obtained for B3, 13.5 kcal•mol -1 . This difference of ~2 kcal•mol -1 is also observed in the reaction energies, being the reaction with B3 slightly more exergonic (-12.5 kcal•mol -1 ), than the reaction with B4 (-10.5 kcal•mol -1 ). A recent computational study of the inhibition mechanisms of rhodesain cysteine protease by a dipeptidyl enoate carried out in our laboratory confirmed that the inhibition process can take place through the active site cysteine attack on either the Cβ atom or to the carbonyl carbon atom of the inhibitor in an exergonic process with a low activation energy barrier. Interestingly, and as revealed by the evolution of the interatomic distances monitored in Figure , the proton transfer from the positively charged H41 to the carbonyl oxygen atom does not take place directly but through the hydroxyl group of the substrate. Thus, the proton Hg is transferred from the Ne of H41 to the oxygen atom of the hydroxyl group, O*, simultaneous with the proton transfer, H*, from this hydroxyl oxygen atom to the carbonyl oxygen atom, O, of the substrate. A similar mechanism has been found for the inhibition reaction of M pro with PF-00835231 using similar QM/MM methods but with the B3LYP functional. Our activation free energy is 4.5 kcal•mol -1 lower than the one obtained in that study, which could be due to chemical and/or methodological differences. In this regard, it has been previously noted that limitations of B3LYP for describing thio-Michael additions. It is also worth mentioning that our ion pair intermediate is clearly a stable minimum in the free energy surface, while a metastable ion pair catalytic dyad is formed by the proton transfer from Cys145 to His41 in that work. The transition state of this second step, defined as the maximum of the PMF but also confirmed by optimizing a representative structure at M06-2X/6-31+G(d,p)/MM level (see Figure and Tables and) and tracing the IRC down to the ion pair and the final product complex, support the proposed mechanism. The role played by the terminal hydroxyl group of B4 in the proton transfer from H41 to the carbonyl oxygen atom of the inhibitor agrees with the results of Hoffman and co-workers who measured a drop of potency observed in different HMKs when the terminal hydroxyl group was substituted by other groups.