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.