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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 Neurospora crassa (NcCDHIIA and NcCDHIIB) and five chimeric CDH enzymes created by domain swapping were studied in combination with the fungus' own LPMOs (NcLPMO9C and NcLPMO9F). 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 b 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 b-type heme and the type-2 copper center. Chimeric CDH enzymes favor the open state and achieve higher IPET rates by exposing the heme b cofactor to LPMO. The IPET, which is influenced by interface complementarity and the heme b redox potential, is very efficient with bimolecular rates between 2.9 × 105 and 1.1 × 106 M-1 s-1.

The binding mechanisms of two reactive sizing agents, alkenyl succinic anhydride (ASA) and maleated sunflower oil high-oleic (MSOHO), with cellulose were studied. While ASA is produced from olefins out of fossil resources, MSOHO is a green sizing agent based on renewable plant materials. In contrast to common assumptions, that ASA is mostly covalently bound to cellulose, this study showed the largest part of ASA to be bound only by physisorption and only a rather small fraction, typically about 0.5 %, to be covalently attached by ester bonds. In the case of MSOHO, the covalent binding was only slightly higher, with about 3 % of the total amount being covalently linked. In both cases, covalently bound sizing agents were found to be almost uniformly distributed over the whole DP range of the cellulose as seen by carboxyl-selective fluorescence labeling in combination with size exclusion chromatography (“FDAM method”).