Resolving domain positions of cellobiose dehydrogenase by small angle X-ray scattering.

The interdomain electron transfer between the catalytic flavodehydrogenase domain and the electron transferring cytochrome domain of cellobiose dehydrogenase plays an essential role in biocatalysis, biosensors, and biofuel cells as well as for its natural function as an auxiliary enzyme of lytic polysaccharide monooxygenase. We investigated the mobility of the cytochrome and dehydrogenase domains of cellobiose dehydrogenase (CDH), which is hypothesised to limit interdomain electron transfer in solution by small angle X-ray scattering (SAXS). CDH from Myriococcum thermophilum (syn. Crassicarpon hotsonii, syn. Thermothelomyces myriococcoides) was probed by SAXS to study the CDH mobility at different pH and in the presence of divalent cations. By comparison of the experimental SAXS data, using pair-distance distribution functions and Kratky plots, we show an increase of CDH mobility at higher pH, indicating alterations of domain mobility. To further visualise CDH movement in solution, we performed SAXS-based multi-state modelling. Glycan structures present on CDH partially masked the resulting SAXS shapes, we diminished these effects by deglycosylation and studied the effect of glycoforms by modelling. The modelling shows that with increasing pH, the cytochrome domain adopts a more flexible state with significant separation from the dehydrogenase domain. On the contrary, the presence of calcium ions decreases the mobility of the cytochrome domain. Experimental SAXS data, multi-state modelling, and previously reported kinetic data show how pH and divalent ions impact the closed state necessary for the interdomain electron transfer governed by the movement of the CDH cytochrome domain.