| Affiliation: | 1. University of Natural Resources and Life Sciences, Vienna, Department of Food Science and Technology, Institute of Food Technology, Muthgasse 18, 1190 Vienna, Austria;2. University of Natural Resources and Life Sciences, Vienna, Department of Food Science and Technology, Institute of Food Technology, Muthgasse 18, 1190 Vienna, Austria
Contribution: Data curation (supporting), Methodology (supporting);3. Max Planck Institut für Multidisciplinary Sciences, Department of Theoretical and Computational Biophysics, Am Fassberg 11, 37077 Göttingen, Germany;4. University of Natural Resources and Life Sciences, Vienna, Department of Biotechnology, Institute of Bioprocess Science and Engineering, Muthgasse 18, 1190 Vienna, Austria;5. University of Natural Resources and Life Sciences, Vienna, Department of Material Sciences and Process Engineering, Institute of Molecular Modeling and Simulation, Muthgasse 18, 1190 Vienna, Austria;6. Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Cyclotron road 1, 94720 Berkeley, California, USA |
| Abstract: | 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. |
