Reaction of reduced flavins and flavoproteins with diphenyliodonium chloride.
The reaction of diphenyliodonium chloride with free reduced flavins has been studied by stopped flow spectrophotometry under anaerobic conditions, and second order rate constants were determined as a function of pH. The reactive flavin species was identified as the reduced anion, based on an observed reaction pK of 6.7. The product mixture was independent of the initial concentration of reactant and contained approximately 20% oxidized flavin. The results can be modeled quantitatively on a modification of the mechanism proposed by Tew (Tew, D. G. (1993) Biochemistry 32, 10209-10215). The composition of the complex reaction mixture has been analyzed, and four flavin-phenyl adducts with distinctive absorbance and fluorescence characteristics have been identified, involving substitution at the flavin C4a, N5, and C8 positions. Inactivation of flavoprotein enzymes by diphenyliodonium has also been studied, and several examples were found where inactivation occurs readily, despite noninvolvement of radical intermediates in their reaction mechanisms. It can be concluded that inactivation by phenyliodonium species is not a valid indicator of catalytic mechanism involving radical intermediates. One of the several factors determining inactivation is maintenance of the enzyme flavin in the reduced form in the steady state of catalysis, the other factors being redox potential and accessibility of the inhibitor to the flavin active site.
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606, USA. sumita@umich.edu
Sumita
S
Chakraborty
Vincent
V
Massey
D015109:4-Hydroxybenzoate-3-Monooxygenase / Q000737:chemistry; D001665:Binding Sites; D001713:Biphenyl Compounds / Q000737:chemistry*; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000737:chemistry; D005415:Flavins / Q000737:chemistry*; D005418:Flavodoxin / Q000737:chemistry; D005420:Flavoproteins / Q000737:chemistry*; D005949:Glucose Oxidase / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D009252:NADPH Dehydrogenase / Q000737:chemistry; D009861:Onium Compounds / Q000737:chemistry*; D010084:Oxidation-Reduction; D013053:Spectrophotometry / Q000379:methods; D014969:Xanthine Oxidase / Q000737:chemistry
D001713:Biphenyl Compounds; D004791:Enzyme Inhibitors; D005415:Flavins; D005418:Flavodoxin; D005420:Flavoproteins; D009861:Onium Compounds; C031291:diphenyliodonium; D005949:Glucose Oxidase; D015109:4-Hydroxybenzoate-3-Monooxygenase; D014969:Xanthine Oxidase; D009252:NADPH Dehydrogenase