MESSAI.iO

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CueO, a copper efflux oxidase in E. coli, undergoes reductive inactivation in the presence of Cu2+ due to the coordination of labile copper by histidine residues. Deletion of the putative Cu8 ligands His145 and His406 significantly suppressed this inactivation, indicating their roles as main and auxiliary ligands. The results provide insights into the structural and biochemical mechanisms underlying CueO's bioelectrocatalytic activity.

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Abstract

Copper efflux oxidase (CueO) is involved in copper homeostasis in Escherichia coli by catalyzing the oxidation of cuprous ion (Cu + ) to cupric ion (Cu 2+ ). CueO has been studied as a direct electron transfer (DET)-type bioelectrocatalyst owing to its high dioxygen-reducing activity. Our previous study demonstrated reductive inactivation in the DET-type bioelectrocatalysis of CueO in the presence of Cu 2+ , which was known to facilitate substrate oxidation in solution. Considering the structural data, we hypothesized that eighth bound copper (Cu8) induced reductive inactivation. CueO variants deleting putative Cu8 ligands (His145, His406, and Met417) were characterized bioelectrochemically. As expected, the inactivation was significantly suppressed in H145A while being slightly suppressed in the H406A variant. In contrast, Cu 2+ tolerance was slightly decreased in the M417A variant. These results indicate that His145 and His406 are the main and auxiliary ligands, respectively, of Cu8 that induce reductive inactivation, whereas Met417 might contribute to stabilizing the Cu8 coordination sphere. Furthermore, kinetic analysis revealed that the deletion of Cu8 ligands affected both the binding and redox kinetics of the enzyme-substrate-copper complex. Additionally, Cu8-induced inactivation was observed using an enzymatic assay in solution. Therefore, reductive inactivation likely occurs in concert with biological Cu + oxidation, which may contribute to regulating the Cu 2+ /Cu + ratio.

Keywords

CopperRedoxEffluxKineticsOxidase testSubstrate (aquarium)

Identifiers

DOI
10.1021/acs.inorgchem.5c03674
PubMed
41381400
Journal
Inorganic chemistry
Year
2026