Copper-Mediated Antimicrobial Activity : Targeting Cytochrome c Complexes Across Bacterial Species

Copper (Cu) exerts potent antibacterial activity and contributes to mammalian immune defense. Although solvent exposed 4Fe-4S clusters in cytoplasmic dehydrogenases are major intracellular targets of Cu, its chemical reactivity and subcellular distribution suggest that additional cellular processes may be vulnerable to Cu toxicity. Here, we identify c-type cytochrome biogenesis as a conserved target of Cu excess across diverse pathogenic and environmental bacteria. Analysis of respiratory membrane complexes from pathogens, including Vibrio cholerae and Pseudomonas aeruginosa, revealed pronounced and selective sensitivity of c-type cytochrome-containing complexes to excess Cu. In P. aeruginosa, elevated Cu impaired the cbb3-type cytochrome c oxidase and NapBC-containing cytochrome c complexes while largely sparing complexes independent of c-type cytochromes, including the aa3-type cytochrome c oxidase and succinate dehydrogenase. In contrast, the caa3-type cytochrome c oxidase of Bacillus subtilis, which contains a covalently bound cytochrome c subunit, was also severely affected. Cu likewise depleted c-type cytochromes in plant-associated and photosynthetic bacteria, indicating that this vulnerability is broadly conserved. Complementary analyses in B. subtilis and Escherichia coli showed that Cu primarily disrupts c-type cytochrome assembly rather than promoting the degradation of preassembled complexes. These findings establish c-type cytochrome-containing respiratory complexes as conserved targets of Cu toxicity. Given their central role in electron transport and adaptation to oxygen-limited environments, disruption of cytochrome c biogenesis may contribute to Cu antimicrobial activity and expose a potential vulnerability in pathogens that rely on c-type cytochromes for microaerobic respiration and colonization of hypoxic host tissues.