AbstractThe main protease (M^pro) of SARS-CoV-2 is critical for viral function and a key drug target. M^pro is only active when reduced; turnover ceases upon oxidation but is restored by re-reduction. This suggests the system has evolved to survive periods in an oxidative environment, but the mechanism of this protection has not been confirmed. Here, we report a crystal structure of oxidized M^pro showing a disulfide bond between the active site cysteine, C145, and a distal cysteine, C117. Previous work proposed this disulfide provides the mechanism of protection from irreversible oxidation. M^pro forms an obligate homodimer, and the C117-C145 structure shows disruption of interactions bridging the dimer interface, implying a correlation between oxidation and dimerization. We confirm dimer stability is weakened in solution upon oxidation. Finally, we observe the protein’s crystallization behavior is linked to its redox state. Oxidized M^pro spontaneously forms a distinct, more loosely packed lattice. Seeding with crystals of this lattice yields a structure with an oxidation pattern incorporating one cysteine-lysine-cysteine (SONOS) and two lysine-cysteine (NOS) bridges. These structures further our understanding of the oxidative regulation of M^pro and the crystallization conditions necessary to study this structurally.