Table 1 Redox-dependent enzymatic activities of SARS-CoV-2 M^pro wild-type and variants

M^pro Variants	Reduced ^a Relative activity (%)	Oxidized ^b Relative activity (%)	Reactivation (after 200s) ^c
			Relative activity (%)	Change (%)
Wild-type	100.0 ± 2.6^d	30.5 ± 1.0	34.0 ± 1.0	+3.5%
			Fully reversible overnight^e
Cys-Ser variants
C16S	75.3 ± 1.1	25.4 ± 0.1	27.1 ± 0.2	+1.7%
C22S	84.4 ± 1.2	26.3 ± 0.2	31.2 ± 0.8	+4.9%
C38S	92.1 ± 0.5	31.8 ± 0.3	36.4 ± 0.4	+4.6%
C44S	16.4 ± 0.1	7.3 ± 0.3	7.6 ± 0.4	+0.3%
C85S	71.2 ± 1.7	26.4 ± 0.8	29.5 ± 0.1	+3.1%
C117S	41.9 ± 0.6	16.7 ± 0.1	14.2 ± 1.1	–2.5%
			irreversible overnight^e
C128S	74.3 ± 0.6	22.5 ± 0.6	28.4 ± 1.1	+5.9%
C145S	n. a.^f	n. a.	n. a.
C156S	77.5 ± 1.8	28.7 ± 0.7	35.0 ± 0.9	+6.3%
C160S	92.7 ± 3.0	28.2 ± 0.5	30.1 ± 0.1	+1.9%
C265S	85.1 ± 2.3	30.7 ± 0.4	34.4 ± 1.9	+3.7%
C300S	92.7 ± 0.3	32.1 ± 0.4	35.9 ± 2.0	+3.8%
Additional SONOS variants
C44A	29.4 ± 0.4	12.4 ± 0.5	11.1 ± 0.4	–1.3
C22S_C44S	10.4 ± 0.4	4.0 ± 0.0	2.3 ± 0.2	–1.7
K61A	55.2 ± 2.0	20.2 ± 2.4	18.7 ± 2.1	–1.5
K61A_C22S	42.4 ± 1.9	16.6 ± 2.2	17.2 ± 0.7	+0.6
K61A_C44S	10.2 ± 0.3	3.2 ± 0.0	3.5 ± 0.1	+0.3
K61A_C22S_C44S	7.0 ± 1.4	3.0 ± 0.1	2.0 ± 0.3	–1.0
			Partially reversible overnight^e
Y54F	19.4 ± 0.0	7.1 ± 1.8	8.2 ± 0.5	+1.1

^ain presence of 1 mM DTT.
^bafter oxidation with 1 mM H₂O₂ for 2 h on ice.
^cre-activation was initiated by addition of 20 mM DTT to the oxidized enzyme and kinetically analyzed 200 s after reduction.
^d100% activity refers to the activity of wild-type M^pro under reducing conditions.
^esee Supplementary Fig. 5 (wild-type), Supplementary Fig. 11 (variant C117S) and Supplementary Fig. 12 (variant K61A_C22S_C44S).
^fno measurable activity.
Experimental details are provided in the Supplementary Methods.

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