Roles of Sixth Bound Copper in Reductive Inactivation of Copper Efflux Oxidase

Copper efflux oxidase (CueO) is involved in copper homeostasis in Escherichia coli by catalyzing the oxidation of cuprous ion (Cu+) to cupric ion (Cu2+). 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 Cu2+, which was known to facilitate substrate oxidation in solution. Considering the structural data, we hypothesized that sixth-bound copper (Cu6) induced reductive inactivation. CueO variants deleting putative Cu6 ligands (His145, His406, and Met417) were characterized bioelectrochemically. As expected, the reductive inactivation was significantly suppressed in H145A while slightly suppressed in H406A variants. In contrast, Cu2+ tolerance was slightly decreased in the M417A variant. These results indicate that His145 and His406 are the major and minor ligands of Cu6 that induce reductive inactivation, respectively, whereas Met417 might contribute to stabilizing the Cu6 coordination sphere. Furthermore, kinetic analysis of chronoamperograms representing the time-dependent Cu2+ response revealed that the deletion of Cu6 ligands affected both the binding and redox kinetics of the enzyme-substrate-copper complex. Additionally, Cu6-induced inactivation was observed using an enzymatic assay in solution for the oxidation of 1,1′-ferrocenedimethanol. Therefore, reductive inactivation likely occurs in concert with biological Cu+ oxidation, which may contribute to the regulation of the Cu2+/Cu+ ratio.