Amino Acid Residues Controlling Domain Interaction and Interdomain Electron Transfer in Cellobiose Dehydrogenase

The function of cellobiose dehydrogenase (CDH) in biosensors, biofuel cells, and as a physiological redox partner of lytic polysaccharide monooxygenase (LPMO) is based on its role as an electron donor. Before donating electrons to LPMO or electrodes, an interdomain electron transfer from the catalytic FAD-containing dehydrogenase domain to the electron shuttling cytochrome domain of CDH is required. This study investigates the role of two crucial amino acids located at the dehydrogenase domain on domain interaction and interdomain electron transfer by structure-based engineering. The electron transfer kinetics of wild-type Myriococcum thermophilum CDH and its variants M309A, R698S, and M309A/R698S were analyzed by stopped-flow spectrophotometry and structural effects were studied by small-angle X-ray scattering. The data show that R698 is essential to pull the cytochrome domain close to the dehydrogenase domain and orient the heme propionate group towards the FAD, while M309 is an integral part of the electron transfer pathway - its mutation reducing the interdomain electron transfer 10-fold. Structural models and molecular dynamics simulations pinpoint the action of these two residues on the domain interaction and interdomain electron transfer. Cellobiose dehydrogenase serves as an auxiliary enzyme donating electrons to lytic polysaccharide monooxygenase in biomass depolymerization, as well as a biorecognition element in biosensors due to its electron transfer capability. The involvement of two amino acids in the interdomain electron transfer process is investigated in depth with stopped-flow spectrophotometry, small-angle X-ray scattering, multistate modeling, and molecular dynamics simulations.image

Automated summary

This study investigates the role of two crucial amino acids in cellobiose dehydrogenase (CDH) in the interaction between the dehydrogenase and cytochrome domains, as well as in the interdomain electron transfer. The results suggest that these two residues play an essential role in shaping the domain interaction and facilitating electron transfer.