Glucose oxidase [GOx-(PT-PEONH2)] hybrids are synthesized by attaching phenothiazine (PT) groups to aspartic and glutamic acid residues on the enzyme surface via poly(ethylene oxide) (PEO) spacers of different molecular weights. A fast oxidation of FADH(2)/FADH by PT+ with the aid of the local motion of a hydrophilic, long, flexible PEO spacer is achieved for the GOx-(PT-PEONH2) hybrids and yields greater electron-transfer (ET) rates than that for GOx-(PTNH2) hybrids, in which the PT groups are directly bonded to the GOx surface. The ET rate of GOx-(PT-PEONH2) hybrids depends on the molecular weight of PT-PEONH2, and the maximum is obtained at a molecular weight of 3000. The ET rates of GOx hybrids are compared in terms of the location of the PT modification and the length and structure of the spacer chain connection of the PT mediator to a surface amino acid residue. Greater ET rates are obtained for the modification at aspartic and glutamic acid residues than for the lysine modification when the PT groups are bonded directly or via a short PEO spacer chain. In contrast, no advantage of aspartic and glutamic acid residues over lysine residues in generating a fast oxidation of FADH(2)/FADH by PT+ is observed for GOx hybrids in which the PT groups are attached via longer PEO spacers. The long PEO spacer is able to compensate the disadvantage of lysine residues locating far from the FAD center in GOx hybrids whose mediation reactions are based on the so-called wipe mechanism.