Dithionite treatment of flavins: spectral evidence for covalent adduct formation and effect on in vitro bacterial bioluminescence.
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)
Mager
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Department of Biochemical and Biophysical Sciences, University of Houston, Texas 77204-5500.
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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*
D005415:Flavins; D013447:Sulfites; D004227:Dithionite; D005486:Flavin Mononucleotide; D006861:Hydrogen Peroxide; D008156:Luciferases