Immobilization of high-potential multicopper oxidases like laccase from Trametes versicolor (TvLc) on cathode surfaces in an orientation that enables direct electron transfer (DET) to the active center allows to profit from the enzyme's low overpotential towards the oxygen reduction reaction (ORR). In this work, TvLc is immobilized on mixed self-assembled monolayers (SAMs) of thiolates on Au(111). The SAMs consist of two species, a linker molecule, thiolated veratric acid (tVA) with a moiety similar to the enzyme's natural substrates, and a diluent molecule, 3-mercaptopropionic acid (MPA), which increases the lateral distance between neighboring tVA molecules when incorporated between the linkers. In this work we study the effect of linker separation on enzyme activity. Electrochemical scanning tunneling microscopy (EC-STM) is used to determine the molecular arrangement of the two species in two different SAMs. After enzyme immobilization, negligible enzymatic activity is found on a monolayer, where a separation between tVA and MPA phases is observed. This is explained by the close packing of tVA linkers, which prevents access to the enzyme's active center. Maximum bioelectrocatalytic currents at low overpotentials are measured on a SAM, where tVA and MPA homogeneously mix in a single phase. The increased distance between isolated linkers allows for induced-fit binding of TvLc to the linker moiety, which leads to a proper orientation of the enzyme and allows for DET from the electrode through the linker molecule to the enzyme's active center. (C) 2016 Elsevier Ltd. All rights reserved.