Abstract
CueO is a multicopper oxidase involved in Cu-homeostasis of E. coli. CueO has the four catalytic copper binding sites, a type 1 Cu, a type 2 Cu and a pair of type 3 Cu's, in a single chain protein molecule consisting of 484 amino acids. CueO has the fifth Cu-binding site constructed by Met355, Asp360, Met439, Met439 as the substrate-binding site, which is isolated from bulk waters by the Met-rich segment comprised of amino acids 355-400. The high cuprous oxidose activity of CueO is realized by the presence of this fifth Cu-binding site as revealed by the Km value and the point mutations for the ligating amino acids. CueO showed enhanced oxidizing activities for organic substrates in the presence of Cu(II) ion at the fifth Cu-binding site because this extra Cu(II) ion functioned as the mediator of electron-transfer between exogenous substrate and type 1 Cu. Further, mutations at Asp112 adjacent to the trinuclear Cu center formed by type 2 Cu and type 3 Cu's indicated that this acidic amino acid functioned not only as a proton donor for dioxygen but also as a modulator for the binding of dioxygen.
