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 (Cu 2+ ), coupled with the reduction of O 2 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 Ec CueO crystal structures, located in close proximity to TNC. One conserved ligand of the Cu8-site is the amino acid H 145 , in both Cu + and Cu 2+ redox states. By designing and characterizing the H 145 S variant, this work demonstrates for the first time the pivotal role of H 145 in the functional maturation/metalation of Ec CueO active sites under conditions of low Cu 2+ /Cu + availability. Moreover, we show that H 145 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. In environments where the availability of Cu + /Cu 2+ is limited, the presence of H 145 residue is crucial for the recruitment of cofactors to the Cu8-site and for ensuring the rapid metalation of APO CueO to HOLO CueO. • Cu8-site is putative transient copper-binding site of multicopper oxidases (MCOs). • H 145 , a ligand of Cu8-site in E. coli ( Ec )CueO, was mutated to non-coordinating serine. • H 145 S demonstrates a delayed metalation of binuclear Cu-T3 site. • H 145 is pivotal for Ec CueO metalation under low environmental Cu + /Cu 2+ availability • H 145 belongs to a HxHx(H/C/M) motif conserved across bacterial MCOs.