A conserved copper-binding site in multicopper oxidases regulates the metalation of CueO from Escherichia coli

CueOs are multicopper oxidases (MCOs) involved in key biological processes related to copper homeostasis. Their physiological function is the catalytic oxidation of toxic cuprous ions (Cu⁺) to cupric ions (Cu²⁺), coupled with the reduction of O₂ to water. In addition to the copper sites belonging to the classical electron transfer chain of MCOs, from Cu-T1 to the trinuclear cluster (TNC), a Cu8-site was previously identified in EcCueO crystal structures, located in close proximity to TNC. One conserved ligand of the Cu8-site is the amino acid H₁₄₅, in both Cu⁺ and Cu²⁺ redox states. By designing and characterizing the H₁₄₅S variant, this work demonstrates for the first time the pivotal role of H₁₄₅ in the functional maturation/metalation of EcCueO active sites under conditions of low Cu²⁺/Cu⁺ availability. Moreover, we show that H₁₄₅ is part of a conserved HxHxH motif in CueOs, and more generally in bacterial MCOs, suggesting a common copper-binding Cu8-site for metalation in vivo. The absence of this conserved motif in certain MCOs, or the presence of additional His/Met-rich or His-rich insertions, appears to be linked to cellular copper availability and highlights the adaptability of MCOs. Beyond this fundamental understanding of MCO metalation mechanism, this works paves the way for application in medicine and environmental copper detection.