Chimeric Cellobiose Dehydrogenases Reveal the Function of Cytochrome Domain Mobility for the Electron Transfer to Lytic Polysaccharide Monooxygenase

The natural function of cellobiose dehydrogenase (CDH) to donate electrons from its catalytic flavodehydrogenase (DH) domain via its cytochrome (CYT) domain to lytic polysaccharide monooxygenase (LPMO) is an example of a highly efficient extracellular electron transfer chain. To investigate the function of the CYT domain movement in the two occurring electron transfer steps, two CDHs from the ascomycete ( CDHIIA and CDHIIB) and five chimeric CDH enzymes created by domain swapping were studied in combination with the fungus' own LPMOs ( LPMO9C and LPMO9F). Kinetic and electrochemical methods and hydrogen/deuterium exchange mass spectrometry were used to study the domain movement, interaction, and electron transfer kinetics. Molecular docking provided insights into the protein-protein interface, the orientation of domains, and binding energies. We find that the first, interdomain electron transfer step from the catalytic site in the DH domain to the CYT domain depends on steric and electrostatic interface complementarity and the length of the protein linker between both domains but not on the redox potential difference between the FAD and heme cofactors. After CYT reduction, a conformational change of CDH from its closed state to an open state allows the second, interprotein electron transfer (IPET) step from CYT to LPMO to occur by direct interaction of the -type heme and the type-2 copper center. Chimeric CDH enzymes favor the open state and achieve higher IPET rates by exposing the heme cofactor to LPMO. The IPET, which is influenced by interface complementarity and the heme redox potential, is very efficient with bimolecular rates between 2.9 × 10 and 1.1 × 10 M s .