Volume 278, May 2026, 113257Author links open overlay panel, , , , , , , , Highlights•

Cu8-site is putative transient copper-binding site of multicopper oxidases (MCOs).

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H145, a ligand of Cu8-site in E. coli (Ec)CueO, was mutated to non-coordinating serine.

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H145S demonstrates a delayed metalation of binuclear Cu-T3 site.

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H145 is pivotal for EcCueO metalation under low environmental Cu+/Cu2+ availability

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H145 belongs to a HxHx(H/C/M) motif conserved across bacterial MCOs.

Abstract

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.

Graphical abstractIn environments where the availability of Cu+/Cu2+ is limited, the presence of H145 residue is crucial for the recruitment of cofactors to the Cu8-site and for ensuring the rapid metalation of APO CueO to HOLO CueO.Download: Download high-res image (270KB)Download: Download full-size imageKeywords

Cuproxidase CueO

Metalation

Maturation

Copper homeostasis

Electrochemistry

Multicopper oxidase

Abbreviations(MCOs)

Multicopper oxidases

(ORR)

Oxygen Reduction Reaction

(DET)

Direct electron transfer

(PCR)

Polymerase reaction chain

(PBS)

Phosphate buffer saline

(EPR)

Electron Paramagnetic Resonance

(ABTS)

2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)

(BCA)

Bicinchoninic acid disodium salt hydrate

(NHE)

Normal Hydrogen Electrode

(LAB)

Lactic acid bacteria

(Vmax/appKm)

Catalytic efficiency

© 2026 The Authors. Published by Elsevier Inc.