In enterics, the transcription factor SoxR triggers a global stress response by sensing a broad spectrum of redox-cycling compounds. In the non-enteric bacteria Pseudomonas aeruginosa and Streptomyces coelicolor, SoxR is activated by endogenous redox-active small molecules and only regulates a small set of genes. We investigated if the more general response in enterics is reflected in the ability of SoxR to sense a wider range of redox-cycling compounds. Indeed, while Escherichia coli SoxR is tuned to structurally diverse compounds that span a redox range of −450 to +80 mV, P. aeruginosa and S. coelicolor SoxR are less sensitive to viologens, which have redox potentials below −350 mV. Using a mutagenic approach, we pinpointed three amino acids that contribute to the reduced sensitivity of P. aeruginosa and S. coelicolor SoxR. Notably these residues are not conserved in homologues of the Enterobacteriaceae. We further identified a motif within the sensor domain that tunes the activity of SoxR from enterics – inhibiting constitutive activity while allowing sensitivity to drugs with low redox potentials. Our findings highlight how small alterations in structure can lead to the evolution of proteins with distinct specificities for redox-active small molecules.