title: Dithionite treatment of flavins: spectral evidence for covalent adduct formation and effect on in vitro bacterial bioluminescence.
abstract: Intrigued by the apparent requirement of dithionite for FMN reduction (as opposed to photoreduction or catalytic hydrogenation) in the H2O2-initiated bacterial bioluminescence reaction, we chose 5-ethyl-3-methyllumiflavinium cation I as a model to investigate possible flavin adduct formation by treatment with dithionite or (bi)sulfite. In the range of pH 5-8, the reaction of dithionite with 5-ethyl-3-methyllumiflavinium cation, which is in equilibrium with the 5-ethyl-4a-hydroxy-3-methyl-4a, 5-dihydrolumiflavin pseudobase II (X = OH), is not limited to the formation of flavosemiquinone and dihydroflavin following two one-electron steps. Several parallel and sequential reactions may take place involving the intermediacy of covalent flavin adducts. Addition of (bi)sulfite gave a 4a-sulfiteflavin adduct II (X = SO3-). Consistent with the S2O4(2-) in equilibrium with 2 SO2-. equilibrium, the reaction of dithionite and II (X = OH; SO3-) gave rise to two flavin adducts in competitive nucleophilic displacements: a 4a-sulfoxylate-flavin radical (II, X = SO2.) and a 4a-dithioniteflavin adduct (II, X = S2O4-), respectively. On increasing the (S2O4(2-), SO2.-)/flavin ratio under N2, the formation of the 4a-sulfoxylate-flavin radical became predominant. The II (X = SO2.) so formed was in equilibrium with the flavosemiquinone and bisulfate and can be trapped by reacting with hydroxylamine. In the initial presence of oxygen, II (X = SO2.) was highly reactive toward O2, giving a fast oxidation to II (X = SO3-) and effectively suppressing the formation of the flavosemiquinone.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemical and Biophysical Sciences, University of Houston, Texas 77204-5500.
authors.forename: H I
authors.initials: HI
authors.lastname: Mager
authors.forename: S C
authors.initials: SC
authors.lastname: Tu
mesh_terms: D004227:Dithionite / Q000494:pharmacology*; D005486:Flavin Mononucleotide / Q000378:metabolism / Q000494:pharmacology; D005415:Flavins / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism*; D049449:Luminescence; D013053:Spectrophotometry; D013447:Sulfites / Q000494:pharmacology*; D014733:Vibrio / Q000201:enzymology*
chemical_list: D005415:Flavins; D013447:Sulfites; D004227:Dithionite; D005486:Flavin Mononucleotide; D006861:Hydrogen Peroxide; D008156:Luciferases