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

Abstract

Previous experiments in our laboratory with Sacchuromyces cerevisiae flavocytochrome hz indicated that both fragments x and /3 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 Hnnsenulu anonzulu enzyme obtained by tryptic cleavage. A method has been devised that gives a quantitative separation in 4 M urea of p, and a with its heme still bound. The characteristics of the various species: isolated a and B and mixed a + B 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 x and j have a folded globular structure after renaturation. The flavin binding to the CI + fl mixture was important (50-100%) with recovery of the flavodehydrogenase activity. In contrast, binding was not detectable (< 0.5 %, Kd