Redirecting electron flows in glutamate oxidases by selective anchoring of osmium complexes

Abstract

L-Glutamate is the most abundant and essential excitatory neurotransmitter in the nervous system. However, its direct electrochemical detection is challenging due to its inherently non-electroactive nature. In this study, we redesigned L-glutamate oxidase (GlutOx) by covalently attaching osmium polypyridyl complexes as electron mediators at selected sites. Most engineered enzymes retained their native catalytic activity, while exhibiting significantly altered catalytic currents during L-glutamate oxidation, depending on the proximity, orientation, and microenvironments of the osmium complexes relative to the FAD cofactors. Notably, two mutants significantly enhanced catalytic currents, revealing selectively and efficiently rerouted electron transfer pathways from the enzyme active site to Os complexes. These findings provide an effective strategy for designing redox-active enzymes for electrochemical biosensors.