Synthesis of a Phosphorothioate Analogue of Flavin Mononucleotide: Reconstitution of the FMN-Free Form of NADPH-Cytochrome P-450 Reductase

Judith Porter Calhoun, James D. Otvos, Henry M. Miziorko, David P. Krum, Steven Ugent, Bettie Sue Siler Masters

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Abstract

The chemical synthesis of riboflavin 5’-phosphorothioate (5‘-FMNS) is described. 5’-FMNS is obtained from the alkaline hydrolysis of riboflavin 4/,5’-cyclic phosphorothioate, which is produced upon reaction of riboflavin (RB) with thiophosphoryl chloride in trimethyl phosphate. 5’-FMNS has been tested for enzymatic reconstitution of NADPH-cytochrome P-450 reductase (EC 1.6.2.4) depleted of its FMN prosthetic group, but containing its full complement (1 equiv) of FAD. The synthesis, purification, and characterization of 5’-FMNS are reported, and documentation of its efficacy in reconstituting the reductase by fluorometric and absorbance spectrophotometric measurements, as well as enzymatic activity, is presented. Data indicate that 5’-FMNS is totally competent in reconstituting NADPH-cytochrome c reductase activity, which requires the presence of both FAD and a flavin mononucleotide, and its fluorescence is completely quenched upon addition to FMN-free NADPH-cytochrome P-450 reductase.

Original languageEnglish (US)
Pages (from-to)5344-5350
Number of pages7
JournalBiochemistry
Volume26
Issue number17
DOIs
Publication statusPublished - Jan 1 1987

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ASJC Scopus subject areas

  • Biochemistry

Cite this

Calhoun, J. P., Otvos, J. D., Miziorko, H. M., Krum, D. P., Ugent, S., & Siler Masters, B. S. (1987). Synthesis of a Phosphorothioate Analogue of Flavin Mononucleotide: Reconstitution of the FMN-Free Form of NADPH-Cytochrome P-450 Reductase. Biochemistry, 26(17), 5344-5350. https://doi.org/10.1021/bi00391a019