A Flavin-Mononucleotide-Binding Site in Hansenula anomala Nicked Flavocytochrome b2, Requiring the Association of Two Domains

Previous experiments in our laboratory with Saccharomyces cerevisiae flavocytochrome b2 indicated that both fragments α and β of the enzyme isolated after cleavage by yeast proteases are required to form the flavin site. More detailed experiments have now been carried out on the nicked Hansenula anomala enzyme obtained by tryptic cleavage. A method has been devised that gives a quantitative separation in 4 M urea of β, and α with its heme still bound. The characteristics of the various species: isolated α and β and mixed α+β were studied in 4 M urea and after elimination of this reagent by dialysis in the presence of FMN and 2-mercaptoethanol. Several methods, including heme spectroscopy, tryptophan fluorescence, sedimentation studies, and titration of bound flavin, were used. The results indicate that isolated α and β have a folded globular structure after renaturation. The flavin binding to the α+β mixture was important (50–100 %) with recovery of the flavodehydrogenase activity. In contrast, binding was not detectable ( 10 mM) for isolated α and β. As far as mononucleotide binding is concerned, such a cooperative requirement for two folding domains has never before been reported in other enzymes. The present results are discussed together with others obtained in our laboratory which demonstrate that, as deduced from their sensitivity to trypsin, the structure of S. cerevisiae and H. annomala flavocytochrome b2 protomers is triglobular ‘n-x-β’ (n and x combined within α). The tetramer assembly, which remains intact as a nicked enzyme (αβ)4 after the first trypsin cleavage, is broken down following a second cleavage of the chain into four cytochrotne cores (n) and a functional T-flavodelhydrogenase entity, a tetramer of the type (xβ)4.