Abstract
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.
Highlights
To probe the functional relevance of protein domain dynamics in the electron transfer reactions of cellobiose dehydrogenase (CDH) from C. hotsonii (ChCDH), we used a number of solvent perturbation methods (Figures 2 and S1)
Unless an increase in hydrostatic pressure leads to the population of conformational states that are catalytically incompetent, these closed conformations should support CDH-catalyzed intraprotein electron transfer reactions
Our observations suggest that long-range dynamical motions from the bulk solvent through the protein entrance channel to the DH active site might influence the rate of FAD reduction in CDH
Summary
Protein conformational changes are often associated with enzyme catalysis[1,2] and, in many cases, they control and coordinate biological electron transfer reactions.[3−6] The fungal flavocytochrome cellobiose dehydrogenase [CDH; EC: 1.1.99.18; Carbohydrate Active enZYmes (CAZy; www.cazy.org) family: AA3_1] is an example of a dynamic redox enzyme that is thought to undergo large-scale structural changes in the transfer of electrons originating from a cellobiose substrate to a range of small molecules and proteinogenic redox partners.[7,8]At a structural level, CDH contains a mobile heme b-binding cytochrome (CYT) domain, which is tethered to a catalytic FAD-containing dehydrogenase (DH) domain via a flexible linker of varying length (15−35 amino acids).[9]. Protein conformational changes are often associated with enzyme catalysis[1,2] and, in many cases, they control and coordinate biological electron transfer reactions.[3−6] The fungal flavocytochrome cellobiose dehydrogenase [CDH; EC: 1.1.99.18; Carbohydrate Active enZYmes Org) family: AA3_1] is an example of a dynamic redox enzyme that is thought to undergo large-scale structural changes in the transfer of electrons originating from a cellobiose substrate to a range of small molecules and proteinogenic redox partners.[7,8]. CDH is known to deliver electrons to fungal, cellulose-degrading and copper-containing lytic polysaccharide monooxygenases (LPMOs; EC 1.14.99.54, 1.14.99.56; CAZy family AA9).[8,10−13] Like many interprotein interactions, electron transfer between CDH and LPMO is thought to occur through the formation of a transient encounter
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