The redox potential of type I copper in the Escherichia coli multicopper oxidase CueO was shifted in the positive or negative direction as a result of the single, double, and triple mutations in the first and second coordination spheres: the formation of the NH center dot center dot center dot S-(Cys500 ligand) hydrogen bond, the breakdown of the NH(His443 ligand)center dot center dot center dot O-(Asp439) hydrogen bond, and the substitution of the Met510 ligand for the non-coordinating Leu or coordinating Gin. Laccase activities of CueO were maximally enhanced 140-fold by virtue of the synergistic effect of mild mutations at and at around the ligand groups to type I copper. (C) 2013 Elsevier Inc. All rights reserved.