During oxidative stress in Escherichia coli, the SufABCDSE stress response pathway mediates iron-sulfur (Fe-S) cluster biogenesis rather than the Isc pathway. To determine why the Suf pathway is favored under stress conditions, the stress response SufS-SufE sulfur transfer pathway and the basal housekeeping IscS-IscU pathway were directly compared. We found that SufS-SufE cysteine desulfurase activity is significantly higher than IscS-IscU at physiological cysteine concentrations and after exposure to H(2)O(2). Mass spectrometry analysis demonstrated that IscS-IscU is more susceptible than SufS-SufE to oxidative modification by H(2)O(2). These important results provide biochemical insight into the stress resistance of the Suf pathway.