Proteolytic Cleavage of Hansenula anomala Flavocytochrome b 2 into Its Two Functional Domains

In a previous work, we have described the tryptic cleavage of yeast flavocytochrome b 2 into its two functional domains: a cytochrome b 2 core and a flavodehydrogenase. The lactate dehydrogenase efficiency of the latter was, however, dramatically low, only about 1% that of intact flavocytochrome b 2 . Our present study concerns a new flavodehydrogenase derivative of Hansenula anomala flavocytochrome b 2 which spontaneously dissociates from the cytochrome domain when the polypeptide bridge connecting them is cleaved by Staphylococcus aureus V8 protease I. This flavodehydrogenase was purified and some of its functional and structural properties were studied. It presents an exceptionally high lactate dehydrogenase activity, about 80% that of flavocytochrome b 2 . This result clearly demonstrates that the cytochrome domain is not necessary for the lactate dehydrogenase function and suggests an autonomous folding for both domains. Our results are discussed in terms of ‘gene fusion’.