Influence of unique structural features of multicopper oxidase CueO on halide sensitivity

Multicopper oxidases are attractive biocatalysts for oxygen reduction, but their use is frequently limited by halide inhibition. The cuproxidases CueO are bacterial multicopper oxidases characterized by a domain rich in histidine and methionine residues (His/Met-rich) of variable size that occludes the T1 copper substrate-access site. Here, we compared chloride and fluoride effects on two CueO homologues, from Escherichia coli and Hafnia alvei , alongside variants lacking the His/Met-rich domain (ΔHis/Met). Using both electrochemistry and in-solution assays, we show that fluoride is a strong inhibitor with IC₅₀ values near 1 mM and little dependence on applied potential or enzyme identity. In agreement with other MCOs’ behavior, chloride inhibition of CueOs is weaker and strongly potential- and pH-dependent. However, a striking feature of CueOs is their apparent resistance to inhibition with evidence of activation at low potentials in the presence of chloride. This effect is attributed to the electrochemical metalation of the enzyme, promoted by electrogenerated Cu(I) species stabilized through chloride complexation, highlighting the complex and variable nature of chloride responses in CueOs. In situ electrochemically metalated enzymes enable the inhibition process to be examined independently. Notably, pre-metalated WT enzymes showed greater chloride resistance than their ΔHis/Met counterparts, suggesting that the His/Met-rich domain provides steric protection against chloride inhibition. In contrast, inhibition by fluoride is as strong with pre-metalated WT enzymes as with ΔHis/Met variants. Our findings, consistent across both electrochemical and in-solution methods, support the hypothesis that fluoride targets the trinuclear copper cluster, whereas chloride acts closer to the T1 region of CueOs.