Cellobiose dehydrogenase (CDH) is secreted by ascomycete and basidiomycetes fungi and catalyzes the oxidation of sugars. Its native substrate is cellobiose, a -1,4-linked disaccharide forming cellulose, thus, making CDH and important player in the biodegradation of wood. In this process, the oxidation of the natural substrate cellobiose is followed by the reduction of quinones and re-oxidation of the enzyme. CDH is the only known extracellular flavocytochrome to date, consisting of an N-terminal cytochrome domain which is connected via a linker to a larger C-terminal flavodehydrogenase domain. Heterologous expression of Myriococcum thermophilum CDH from in Pichia pastoris and its purification were core elements of this study. The highly purified enzyme with a ratio A420/A280 of 0.64 was then subjected to pre steady-state analysis. This technique allows the direct determination of intramolecular electron transfer (IET), measured by the rate constant for heme b reduction. The kinetic constants Kd and klim for cellobiose, lactose, glucose and maltose were determined. The catalytic efficiency of the heme b reduction process for glucose is outstanding, showing 3.2-fold higher value as with the natural substrate cellobiose. Highest observed rate constant of the heme b was 0.93 s-1 at pH 4.0, whereas for FAD the highest observed rate constant was 38.0 s-1 at pH 7.0. The variants D547K/E550K and D547K/E550K/E603K were found to be the most promising surface-charge variants, as they exhibit satisfying reduction performance of FAD with outstanding heme b rate constants at pH 7.5. As the surface-charge variants of CDH from Myriococcum thermophilum showed little glucose discrimination and outstanding reduction rates for heme b, they are facilitating direct electron transfer (DET) between the redox center of an enzyme and a polarized electrode and, thus, have the potential to be used in third generation biosensors.