Isolation of the flavodehydrogenase domain of Hansenula anomala flavocytochrome b2 after mild proteolysis by an H. anomala proteinase

Abstract

The protomeric chain of Hansenula anomala flavocytochrome b₂ was previously shown to be built as the covalent association of two functional domains: an l‐Lactate dehydrogenase domain and a cytochrome c reductase domain, joined together by a proteolytically sensitive zone. This paper concerns the specific cleavage of this latter zone with a H. anomala proteinase(s) preparation and the purification of the purification of the resulting l‐lactate: dehydrogenase moiety of the molecule with at least 25% recovery, (i.e. one order of magnitude more than for the previously published method). A preliminary characterization of this dehydrogenase domain indicates that it is a tetramer (M_r= 4 × 39000) containing FMN as expected and not heme. It has high l‐lactate:ferricyanide oxidoreductase activity (about 70% that of the whole flavocytochrome b₂, but it has no l‐lactate:cytochrome c oxidoreductase activity. Its flavin semiquinone is stabilized in the presence of pyruvate as in flavocytochrome b₂. The subcellular origin of the H. anomala proteinase in the preparation has not yet been elucidated.