SOD1 Catalyses Thiol Oxidation to Thiosulfinates

Abstract Cu/Zn superoxide dismutase (SOD1) is canonically regarded as a superoxide scavenging antioxidant yet is paradoxically associated with multiple diseases. Here, we show that SOD1 catalyzes thiol oxidation to thiosulfinates (RS(O)SR), revealing a previously unrecognized copper mediated oxidant forming reaction in biology. Steady state kinetics demonstrate robust, O 2 dependent thiol consumption, and ATR FTIR spectra of the SOD1–cysteine reaction display S–O bands (≈1042/1169 cm −1 ) identical to cysteine thiosulfinate, including matching pseudo–first order decay in excess cysteine. SOD1 generated thiosulfinates are potent electrophiles and oxidants, supported by dimedone trapping and O-atom transfer to TCEP and horseradish peroxidase, with alkaline lability consistent with thiosulfinate hydrolysis rather than H 2 O 2 . Exogenous thiosulfinates phenocopy SOD1 mediated thiol oxidation, including GSH depletion, protein sulfenylation and cytotoxicity. SOD1 inhibition strongly suppresses cysteine and homocysteine induced toxicity, demonstrating that thiol driven oxidative stress requires SOD1 activity. SOD1 overexpression in human cells triggers oxidative stress and reduces proliferation, an effect that is absent in a copper-deficient mutant. N-acetylcysteine treatment further amplified this SOD1-dependent oxidative stress. At lower levels, nanomolar thiosulfinates elicit a hormetic proliferative response and rescue SOD1 deficient growth, identifying a pro-growth signaling function mediated by basal thiosulfinate formation. Kinetic modelling indicates that thiol–thiosulfinate turnover can match basal superoxide dismutation, indicating that thiosulfinate synthesis is a major catalytic output of SOD1. These findings identify SOD1 as a thiol oxidizing enzyme that generates thiosulfinates, establishing a core sulfur-based oxidation pathway and revealing that two classical “antioxidants”, SOD1 and thiols, together generate potent oxidants that link thiol metabolism to both cytotoxic and growth promoting outcomes. Significance Statement Although SOD1 is classically defined as an antioxidant, its association with diverse oxidative-stress–driven diseases suggests additional chemistry at work. Here we identify thiosulfinate synthesis as a major catalytic output of SOD1, revealing that the enzyme is not merely a superoxide detoxifier but a thiol-oxidizing catalyst. This activity provides a unifying mechanism for two long-standing biological paradoxes: the unexplained toxicity of elevated thiols and the pro-growth, pro-survival signaling linked to basal SOD1 activity. By establishing thiosulfinates as a central oxidative currency in cells, this work reframes SOD1 as a bifunctional oxidase that shapes both stress responses and proliferative programs.