Reaction of Reduced Flavins and Flavoproteins with Diphenyliodonium Chloride

Abstract

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 ϳ20% 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.

Peroxisome Proliferator–Activated Receptor α Induces NADPH Oxidase Activity in Macrophages, Leading to the Generation of LDL with PPAR-α Activation Properties

“…However, the increase in ROS appeared to be attributable to the induction of the membrane NADPH oxidase, because it was inhibited by a specific inhibitor of flavin, an important component of NADPH oxidase. 30 The increase in ROS production was associated with an increase in the expression of the NADPH oxidase protein subtypes p47 phox , p67 phox , and gp91 phox but not p22 phox , as well as membrane p47 phox protein levels. Moreover, PPAR-␣ agonists failed to activate ROS generation in p47 phoxϪ/Ϫ or gp91 phoxϪ/Ϫ bone marrow-derived macrophages, which indicates that the generation of ROS by PPAR-␣ depends on p47 phox and gp91 phox expression.…”

Dual Role of NOX2 in Respiratory Syncytial Virus- and Sendai Virus-Induced Activation of NF-κB in Airway Epithelial Cells

“…Although the effect of DPI is widely considered as an indicator of a potential role of a NADPH oxidase, other ROS-generating flavoproteins, including the mitochondrial respiratory chain, could explain the effects observed with DPI (56). Therefore, to definitively conclude on the involvement of NADPH-oxidase activity, we used selective RNAi against NOX1, NOX2, and NOX5, and provide the first direct proof that a NOX2-containing enzyme specifically participates in the redox-sensitive regulation of RSV-and SeV-induced NF-B signaling in A549 and NHBE.…”

Initial Reaction(s) in Biotransformation of CL-20 Is Catalyzed by Salicylate 1-Monooxygenase from Pseudomonas sp. Strain ATCC 29352

“…Inhibition with DPI, an inhibitor of flavoenzymes that acts by forming a flavin-phenyl adduct (6), was assessed by incubating salicylate 1-monooxygenase (1 mg) with DPI (0 to 0.5 mM) at room temperature for 15 to 20 min. After incubation, CL-20 biotransformation activities of the enzyme were determined by monitoring the residual CL-20 as described above.…”