Protein Conformational Change Is Essential for Reductive Activation of Lytic Polysaccharide Monooxygenase by Cellobiose Dehydrogenase

Large-scale
protein domain dynamics and electron transfer are often
associated. However, as protein motions span a broad range of time
and length scales, it is often challenging to identify and thus link
functionally relevant dynamic changes to electron transfer in proteins.
It is hypothesized that large-scale domain motions direct electrons
through a FAD and a heme b cofactor of the fungal
cellobiose dehydrogenase (CDH) enzymes to the type-II copper center
(T2Cu) of the polysaccharide-degrading lytic polysaccharide monooxygenases
(LPMOs). However, as of yet, domain motions in CDH have not been linked
formally to enzyme-catalyzed electron transfer reactions. The detailed
structural features of CDH, which govern the functional conformational
landscapes of the enzyme, have only been partially resolved. Here,
we use a combination of pressure, viscosity, ionic strength, and temperature
perturbation stopped-flow studies to probe the conformational landscape
associated with the electron transfer reactions of CDH. Through the
use of molecular dynamics simulations, potentiometry, and stopped-flow
spectroscopy, we investigated how a conserved Tyr99 residue plays
a key role in shaping the conformational landscapes for both the interdomain
electron transfer reactions of CDH (from FAD to heme) and the delivery
of electrons from the reduced heme cofactor to the LPMO T2Cu. Our
studies show how motions gate the electron transfer within CDH and
from CDH to LPMO and illustrate the conformational landscape for interdomain
and interprotein electron transfer in this extracellular fungal electron
transfer chain.