A flavin-mononucleotide-binding site in Hansenula anomala nicked flavocytochrome b2, requiring the association of two domains.
Previous experiments in our laboratory with Saccharomyces cervisiae flavocytochrom b2 indicated that both fragments alpha and beta of the enzyme after cleavage by yeast proteases are required to form the flavin site. More detailed experiments have not 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 beta, and alpha with its heme still bound. The characteristics of the various species: isolated alpha and beta and mixed alpha + beta 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 alpha and beta have a folded globular structure after renaturation. The flavin binding to the alpha + beta mixture was important (50-100%) with recovery of the flavodehydrogenase activity. In contrast, binding was not detectable (< 0.5%, Kf > 10 mM) for isolated alpha and beta. As far as mononucleotide binding is concerned, such a cooperative requirement for two folding domains has never 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. anomala flavocytochrome b2 protomers is triglobular 'n-x-beta' (n and x combined within alpha). The tetramer assembly, which remains intact as a nicked enzyme (alpha beta)4 after the first trypsin cleavage, is broken down following a second cleavage of the chain into four cytochrome cores (n) and a functional T-flavodehydrogenase entity, a tetramer of the type (x beta)4.
D001203:Ascomycota / Q000378:metabolism*; D001665:Binding Sites; D005486:Flavin Mononucleotide / Q000032:analysis*; D006418:Heme; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D042844:L-Lactate Dehydrogenase (Cytochrome); D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D010843:Pichia / Q000378:metabolism*; D011485:Protein Binding; D014357:Trypsin
D010446:Peptide Fragments; D006418:Heme; D005486:Flavin Mononucleotide; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome); D014357:Trypsin