title: 8-thiocyanatoflavins as active-site probes for flavoproteins.
abstract: 8-Thiocyanatoflavins at the riboflavin, FMN, and FAD level were prepared via the diazonium salt of the corresponding 8-aminoflavin and some of the physical and chemical properties studied. 8-Thiocyanatoriboflavin has a UV-visible spectrum similar to that of the native flavin with absorbance maxima at 446 nm (epsilon = 14,900 M-1 cm-1) and 360 nm. Reaction with thiols such as dithiothreitol and mercaptoethanol gives rise to an 8-mercapto- and an 8-SR-flavin, whereas reaction with sulfide yields only the 8-mercaptoflavin. The 8-SCN-flavin binds to riboflavin-binding protein as the riboflavin derivative, to apoflavodoxin, apo-Old Yellow Enzyme, and apo-lactate oxidase as the FMN derivative, and to apo-D-amino acid oxidase, apo-p-hydroxybenzoate hydroxylase, apo-glucose oxidase, apo-anthranilate hydroxylase, and apo-general acyl-CoA dehydrogenase as the FAD derivative. In two cases, namely, with anthranilate hydroxylase and D-amino acid oxidase, the 8-SCN-FAD was spontaneously and completely converted to the 8-mercapto-FAD derivative, suggesting the presence of a nucleophile (most likely the thiol of a cysteine residue) in the vicinity of the 8-position. It was also found that flavodoxin stabilizes the neutral radical and Old Yellow Enzyme the anionic radical of 8-SCN-FMN. Further studies with Old Yellow Enzyme, established that fully (two electron) reduced 8-SCN-FMN undergoes photoelimination of cyanide.
authors.affiliation: Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109.
authors.forename: P
authors.initials: P
authors.lastname: Macheroux
authors.forename: V
authors.initials: V
authors.lastname: Massey
mesh_terms: D042964:Acyl-CoA Dehydrogenase; D044944:Acyl-CoA Dehydrogenases / Q000737:chemistry; D000345:Affinity Labels; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003605:D-Amino-Acid Oxidase / Q000737:chemistry; D005415:Flavins / Q000737:chemistry*; D005418:Flavodoxin / Q000737:chemistry; D005420:Flavoproteins / Q000737:chemistry*; D026901:Membrane Transport Proteins; D006899:Mixed Function Oxygenases / Q000737:chemistry; D009252:NADPH Dehydrogenase / Q000737:chemistry; D010084:Oxidation-Reduction; D012256:Riboflavin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry; D013057:Spectrum Analysis; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013861:Thiocyanates / Q000737:chemistry
chemical_list: D000345:Affinity Labels; D002352:Carrier Proteins; D005415:Flavins; D005418:Flavodoxin; D005420:Flavoproteins; D026901:Membrane Transport Proteins; D013438:Sulfhydryl Compounds; D013861:Thiocyanates; C019079:riboflavin-binding protein; C066913:8-thiocyanatoriboflavin; D006899:Mixed Function Oxygenases; C023440:anthranilate 2,3-dioxygenase(deaminating); D044944:Acyl-CoA Dehydrogenases; D042964:Acyl-CoA Dehydrogenase; D003605:D-Amino-Acid Oxidase; D009252:NADPH Dehydrogenase; D012256:Riboflavin