Andress, Samuel2025-08-152025-08-152025-08-152025-08-11https://hdl.handle.net/10012/22182The main protease (Mpro) of SARS-CoV-2 is essential for viral replication. Its proteolytic mechanism relies on a catalytic dyad formed by the C145 and H41 residues. Recent studies have focused on understanding how Mpro defends itself against oxidative stress as the C145 side chain is susceptible to oxidative damage, which can irreversibly inactivate the enzyme. Oxidative conditions, such as those induced by the immune response, are known to trigger structural changes in Mpro, including the formation of a disulfide bond between C145 and the proximal C117, as well as a shift toward an inactive, monomeric state. It has been proposed that non-catalytic cysteines may act as redox-sensitive switches, or as oxidative sinks that reduce harmful oxidants before reaching the catalytic C145. In this study, functional assays (kinetic analysis and thermal shift assays) combined with structural methods (small-angle X-ray scattering and X-ray crystallography) reveal that mimicking oxidation at C117 via a C117D point mutation drives Mpro toward an inactive monomeric conformation. This transition involves changes in the substrate-binding pocket, rigidification of the dimerization domain, and increased disorder at the N-and C-termini. In contrast, a C145D mutation, designed to mimic oxidation at the catalytic residue, had no impact on the enzyme’s conformation or oligomeric state. These findings present the first solved structure of the monomeric form of Mpro and reveal a novel role for C117 as a redox sensor that mediates oxidative regulation of the protease’s structure and function.enMaster Thesis