The study investigates the mechanisms of copper toxicity in *Escherichia coli* by focusing on the inactivation of iron-sulfur cluster enzymes, particularly dehydratases. Copper, a universally toxic metal, can cause severe health issues in humans and animals. The researchers used *copA cueO cus* mutants, which lack copper homeostatic systems, to identify intracellular targets of copper toxicity and to test the hypothesis that reactive oxygen species play a role. They found that low micromolar levels of copper were sufficient to inhibit the growth of both wild-type and mutant strains, and that branched-chain amino acids were required for growth, indicating that copper blocks their biosynthesis. Copper treatment rapidly inactivated isopropylmalate dehydratase, an iron-sulfur cluster enzyme, and other enzymes in the same family. This inactivation did not require oxygen and was associated with the displacement of iron atoms from the solvent-exposed cluster, suggesting that Cu(I) damages these proteins by liganding to the coordinating sulfur atoms. The study also explored the role of copper efflux systems, glutathione chelation, and cluster repair in enhancing cellular resistance to copper toxicity. Overall, the findings highlight the vulnerability of iron-sulfur cluster enzymes to copper and the importance of copper homeostasis in cellular defense mechanisms.The study investigates the mechanisms of copper toxicity in *Escherichia coli* by focusing on the inactivation of iron-sulfur cluster enzymes, particularly dehydratases. Copper, a universally toxic metal, can cause severe health issues in humans and animals. The researchers used *copA cueO cus* mutants, which lack copper homeostatic systems, to identify intracellular targets of copper toxicity and to test the hypothesis that reactive oxygen species play a role. They found that low micromolar levels of copper were sufficient to inhibit the growth of both wild-type and mutant strains, and that branched-chain amino acids were required for growth, indicating that copper blocks their biosynthesis. Copper treatment rapidly inactivated isopropylmalate dehydratase, an iron-sulfur cluster enzyme, and other enzymes in the same family. This inactivation did not require oxygen and was associated with the displacement of iron atoms from the solvent-exposed cluster, suggesting that Cu(I) damages these proteins by liganding to the coordinating sulfur atoms. The study also explored the role of copper efflux systems, glutathione chelation, and cluster repair in enhancing cellular resistance to copper toxicity. Overall, the findings highlight the vulnerability of iron-sulfur cluster enzymes to copper and the importance of copper homeostasis in cellular defense mechanisms.