Copper toxicity in Escherichia coli primarily targets iron-sulfur clusters in dehydratases. The study shows that copper inhibits the growth of both wild-type and mutant strains lacking copper homeostatic systems. Copper blocks branched-chain amino acid biosynthesis by inactivating iron-sulfur cluster enzymes, such as isopropylmalate dehydratase and fumarase A. These enzymes are sensitive to copper, which displaces iron atoms from the cluster, leading to inactivation. Copper toxicity is not dependent on oxygen, as shown by in vivo and in vitro experiments. Copper efflux, glutathione chelation, and cluster repair systems enhance resistance to copper. The study also demonstrates that copper directly damages iron-sulfur clusters in vitro, with Cu(I) being the primary culprit. Copper-damaged clusters can be repaired in vivo, but the process is hindered by residual copper. The structure of damaged clusters suggests that copper displaces iron atoms, leading to cluster degradation. Enzymes with buried iron-sulfur clusters are resistant to copper, while those with solvent-exposed clusters are vulnerable. Glutathione and the Suf iron-sulfur cluster assembly system contribute to copper resistance. Copper toxicity is focused on cell processes that rely on proteins with solvent-exposed clusters. The study highlights the importance of copper homeostasis in preventing damage to essential enzymes and the role of various cellular mechanisms in mitigating copper toxicity.Copper toxicity in Escherichia coli primarily targets iron-sulfur clusters in dehydratases. The study shows that copper inhibits the growth of both wild-type and mutant strains lacking copper homeostatic systems. Copper blocks branched-chain amino acid biosynthesis by inactivating iron-sulfur cluster enzymes, such as isopropylmalate dehydratase and fumarase A. These enzymes are sensitive to copper, which displaces iron atoms from the cluster, leading to inactivation. Copper toxicity is not dependent on oxygen, as shown by in vivo and in vitro experiments. Copper efflux, glutathione chelation, and cluster repair systems enhance resistance to copper. The study also demonstrates that copper directly damages iron-sulfur clusters in vitro, with Cu(I) being the primary culprit. Copper-damaged clusters can be repaired in vivo, but the process is hindered by residual copper. The structure of damaged clusters suggests that copper displaces iron atoms, leading to cluster degradation. Enzymes with buried iron-sulfur clusters are resistant to copper, while those with solvent-exposed clusters are vulnerable. Glutathione and the Suf iron-sulfur cluster assembly system contribute to copper resistance. Copper toxicity is focused on cell processes that rely on proteins with solvent-exposed clusters. The study highlights the importance of copper homeostasis in preventing damage to essential enzymes and the role of various cellular mechanisms in mitigating copper toxicity.