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 ofNeurospora crassaCDH. 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.