Copper homeostasis mechanisms are critical for bacterial resistance to copper-induced stress. The Escherichia coli multicopper oxidase copper efflux oxidase (CueO) is part of the copper detoxification system in aerobic conditions. CueO contains a methionine-rich (Met-rich) domain believed to interact with copper, but its exact function and the importance of related copper-binding sites remain unclear. This study investigates these open questions by employing a multimodal and multiscale approach. Through the design of various E. coli CueO (EcCueO) variants with altered copper-coordinating residues and domain deletions, we employ biological, biochemical, and physico-chemical approaches to unravel in vitro CueO catalytic properties and in vivo copper resistance. Strong correlation between the different methods enables evaluation of EcCueO variants’ activity as a function of Cu+ availability. Our findings demonstrate the Met-rich domain is not essential for cuprous oxidation, but it facilitates Cu+ recruitment from strongly chelated forms, acting as transient copper binding domain thanks to multiple methionines. They also indicate that the Cu6/7 copper-binding sites previously observed within the Met-rich domain play a negligible role. Meanwhile, Cu5, located at the interface with the Met-rich domain, emerges as the primary and sole substrate-binding active site for cuprous oxidation. The Cu5 coordination sphere strongly affects the enzyme activity and the in vivo copper resistance. This study provides insights into the nuanced role of CueO Met-rich domain, enabling the functions of copper-binding sites and the entire domain itself to be decoupled. This paves the way for a deeper understanding of Met-rich domains in the context of bacterial copper homeostasis.