Direct bioelectrocatalysis applied in biosensors, biofuel cells, and bioelectrosynthesis is based on an efficient electron transfer between enzymes and electrodes in the absence of redox mediators. Some oxidoreductases are capable of direct electron transfer (DET), while others achieve the enzyme to electrode electron transfer (ET) by employing an electron-transferring domain. Cellobiose dehydrogenase (CDH) is the most-studied multidomain bioelectrocatalyst and features a catalytic flavodehydrogenase domain and a mobile, electron-transferring cytochrome domain connected by a flexible linker. The ET to the physiological redox partner lytic polysaccharide monooxygenase or, ex vivo, electrodes depends on the flexibility of the electron transferring domain and its connecting linker, but the regulatory mechanism is little understood. Studying the linker sequences of currently characterized CDH classes we observed that the inner, mobile linker sequence is flanked by two outer linker regions that are in close contact with the adjacent domain. A function-based definition of the linker region in CDH is proposed and has been verified by rationally designed variants of Neurospora crassa CDH. The effect of linker length and its domain attachment on electron transfer rates has been determined by biochemical and electrochemical methods, while distances between the domains of CDH variants were computed. This study elucidates the regulatory mechanism of the interdomain linker on electron transfer by determining the minimum linker length, observing the effects of elongated linkers, and testing the covalent stabilization of a linker part to the flavodehydrogenase domain. The evolutionary guided, rational design of the interdomain linker provides a strategy to optimize electron transfer rates in multidomain enzymes and maximize their bioelectrocatalytic performance.
© 2023 The Authors. Published by American Chemical Society.