To gain insight on the biophysics of this fascinating protein, here we attempt to establish the true nature of the enzyme's cooperativity by applying an arsenal of biophysical methods. First, we ask ourselves whether the doubly occupancy might arise by the fact that an excess of ligand is used. We address this by using MX and binding assays. Next, we investigate the impact of solvation, which leads to a loss of symmetry of the apoprotein on passing from the solid state to aqueous solution . Anticipating our results, we show that in co-crystals obtained in conditions of 1:1 ligand/protomer stoichiometry, the protein features NC with only one ligand bound in one active site, possibly because binding of one ligand in site distorts the other one. This contrasts with what was found in the 500 MX ligand-bound structures solved so far, which might have been obtained in excess of ligands. Ribbon representation of Mpro's subunit "A", shown in shades of red, and "B", in the foreground, represented with gray low-opacity ribbons (PDBid 7PHZ). Each subunit consists of three domains. The first two are the chymotrypsin-like β-barrel domains I and II (residues 10 to 99 and 100 to 182, respectively) with six-stranded antiparallel β barrels that harbor the substratebinding site between them. The catalytic center is a CYS-HIS dyad. The last domain (residues 198-303) is a globular cluster of five helices, involved in dimerization of the enzyme. The insets show details of the catalytic dyad and of the interactions that stabilize the reactive geometry and that were previously reported to be fundamental for site activation/deactivation , namely the hydrophobic interactions between PHE140 and HIS163, and the proximity of GLU166 to the SER1 of the adjacent protomer, which allows for the formation of inter-protomer H-bonds. .