CueO from Eschenchia coli is a multicopper oxidase (MCO) involved in copper tolerance under aerobic conditions It features four copper atoms that act as electron transfer (T1) and dioxygen reduction (T2, T3; trinuclear) sites. In addition, it displays a methionine-rich insert which includes a helix that blocks physical access to the T1 site and which provides an extra labile site T4 adjacent to the T1 center This T4 site is required for CueO function. Like many MCOs, CueO exhibits phenol oxidase activity with broad substrate specificity. Maximal activity with model substrate 2,6-dimethoxyphenol required stoichiometric occupation of T4 by Cu-II (notation. Cu-II-CueO). This was achieved in Mops buffer which has little affinity for Cu2+ However, pH buffers that bind or precipitate Cu2+ (Tris, BisTris, and KPi) generated enzyme with a vacant T4 site (notation square-CueO) which has no phenol oxidase activity Addition of excess Cu2+ effectively generated a Cu2+ buffer and recovered the activity partially or completely, depending upon the specific pH buffer This phenomenon allowed reliable estimation of the affinity of T4 for Cu-II center dot K-D = 5 5 x 10(-9) M. CueO is involved in copper tolerance and has been suggested to be a cuprous oxidase The anion [Cu-I(Bca)(2)](3-) (Bca = bicinchoninate) acted as a novel chromophoric substrate It is a robust reagent, being air-stable and having a Cu-I affinity comparable to those of periplasmic Cu-I binding proteins The influences of pH buffer composition and of excess Cu2+ on cuprous oxidation were diametrically opposite to those seen for phenol oxidation, suggesting that square-CueO, not Cu-II-CueO, is the resting form of the cuprous oxidase Steady-state kinetics demonstrated that the intact anion [Cu-I(Bca)(2)](3-), not "free" Cu+, is the substrate that donates Cu-I directly to T4 The data did not follow classical Michaelis-Menten kinetics but could be fitted satisfactorily by an extension that considered the effect of free ligand Bca. The K-m term consists of two components, allowing estimation of the transient affinity of T4 for Cu-I K-D = 1 3 X 10(-13) M It may be concluded that Cu-I carried by [Cu-I(Bca)(2)](3-) is oxidized only upon complete transfer of Cu-I to T4. The transfer is required to induce a negative shift in the copper reduction potential to allow oxidation and electron transfer to the T1 site. The results provide compelling evidence that CueO is a cuprous oxidase The new approach will have significant application to the study of metallo-oxidase enzymes