Role of the Multicopper Oxidase CueO in Copper Homeostasis Under Anaerobic Conditions in Enterobacteria

The periplasmic multicopper oxidase CueO plays a crucial role in copper detoxification in enterobacteria. CueO contains a catalytic site, the Cu-T1 center, and a methionine-rich (Met-rich) domain capable of binding copper. This enzyme oxidizes cuprous ions (Cu⁺) to the less toxic cupric ions (Cu²⁺), coupled with oxygen reduction. This oxygen dependence has established CueO's role in alleviating copper stress under aerobic conditions, but its function in anaerobic environments remains uncertain. In this study, we demonstrated that under anaerobic conditions and copper stress in E. coli and S. Typhimurium, CueO is produced and contributes to copper homeostasis through its catalytic activity. Using CueO variants with either a mutated catalytic site or a deleted Met-rich domain, we showed that CueO's catalytic activity, rather than its copper-binding capacity, is essential for copper resistance. Additionally, we found that deleting other copper homeostasis systems in E. coli, the inner membrane transporter CopA and the efflux pump CusCBA, leads to the overproduction of CueO under anaerobic conditions. This overproduction enhances the copper resistance of the ∆copA ∆cusB strain. Overall, our findings provide evidence for CueO's role in copper detoxification under anaerobic conditions, highlighting its importance in such environments, that is, host-pathogen interactions or biofilm formation.