DNA microarray experiments showed that the expression of the Escherichia coli ytfE gene is highly increased upon exposure to nitric oxide. We also reported that deletion of ytfE significantly alters the phenotype of E. coli, generating a strain with enhanced susceptibility to nitrosative stress and defective in the activity of several iron-sulfur-containing proteins. In this work, it is shown that the E. coli ytfE confers protection against oxidative stress. Furthermore, we found that the damage of the [4Fe-4S](2+) clusters of aconitase B and fumarase A caused by exposure to hydrogen peroxide and nitric oxide stress occurs at higher rates in the absence of ytfE. The ytfE null mutation also abolished the recovery of aconitase and fumarase activities, which is observed in wild type E. coli once the stress is scavenged. Notably, upon the addition of purified holo-YtfE protein to the mutant cell extracts, the enzymatic activities of fumarase and aconitase are fully recovered and at rates similar to the wild type strain. We concluded that YtfE is critical for the repair of iron-sulfur clusters damaged by oxidative and nitrosative stress conditions.