Rational Tuning of Superoxide Sensitivity in SoxR, the [2Fe-2S] Transcription Factor: Implications of Species-Specific Lysine Residues

In Escherichia coli, the [2Fe-2S] transcriptional factor, SoxR, functions as a sensor of oxidative stress. The transcriptional activity in SoxR is regulated by the reversible oxidation and reduction of [2Fe-2S] clusters. We previously proposed that superoxide (O2•–) has a direct role as a signal for E. coli SoxR and that the sensitivity of the E. coli SoxR response to O2•– is 10-fold higher than that of Pseudomonas aeruginosa SoxR. The difference between the two homologues reflects interspecies differences in the regulatory role of O2•– activation. To investigate the determinants of SoxR’s sensitivity to O2•–, we substituted several amino acids that are not conserved among enteric bacteria SoxR homologues and investigated the interaction of SoxR with O2•– using pulse radiolysis. The substitution of E. coli SoxR Lys residues 89 and 92 with Ala residues (K89AK92A), located close to [2Fe-2S] clusters, dramatically affected this protein’s reaction with O2•–. The second-order rate constant of the reaction was ...