flavin-adenine-dinucleotide and diphenyliodonium

Xanthine oxidase (XO) was shown to catalyze the reduction of nitrite to nitric oxide (NO), under anaerobic conditions, in the presence of either NADH or xanthine as reducing substrate. NO production was directly demonstrated by ozone chemiluminescence and showed stoichiometry of approximately 2:1 versus NADH depletion. With xanthine as reducing substrate, the kinetics of NO production were complicated by enzyme inactivation, resulting from NO-induced conversion of XO to its relatively inactive desulfo-form. Steady-state kinetic parameters were determined spectrophotometrically for urate production and NADH oxidation catalyzed by XO and xanthine dehydrogenase in the presence of nitrite under anaerobic conditions. pH optima for anaerobic NO production catalyzed by XO in the presence of nitrite were 7.0 for NADH and

Topics: Anaerobiosis; Animals; Biphenyl Compounds; Catalytic Domain; Cattle; Flavin-Adenine Dinucleotide; Flavoproteins; Kinetics; Metalloproteins; Milk; Molybdenum; NAD; Nitric Oxide; Nitrites; Onium Compounds; Oxidation-Reduction; Xanthine; Xanthine Dehydrogenase; Xanthine Oxidase

The redox core of the neutrophil NADPH oxidase complex is a membrane-bound flavocytochrome b in which FAD and heme b are the two prosthetic redox groups. Both FAD and heme b are able to react with diphenylene iodonium (DPI) and iodonium biphenyl (IBP), two inhibitors of NADPH oxidase activity. In this study, we show that the iodonium modification of heme b contributes predominantly to the inhibition of NADPH oxidase. This conclusion is based on the finding that both iodonium compounds decreased the absorbance of the Soret peak of flavocytochrome b in neutrophil membranes incubated with NADPH, and that this decrease was strictly correlated with the loss of oxidase activity. Furthermore, the heme component of purified flavocytochrome b reduced to no more than 95% by a limited amount of sodium dithionite could be oxidized by DPI or IBP. Butylisocyanide which binds to heme iron precludes heme b oxidation. In activated neutrophil membranes, competitive inhibition of O2 uptake by DPI or IBP occurred transiently and was followed by a noncompetitive inhibition. These results, together with those of EPR spectroscopy experiments, lead us to postulate that DPI or IBP first captures an electron from the reduced heme iron of flavocytochrome b to generate a free radical. Then, the binding of this radical to the proximate environment of the heme iron, most probably on the porphyrin ring, results in inhibition of oxidase activity. In the presence of an excess of sodium dithionite, DPI and IBP produced a biphasic decrease of the Soret band of flavocytochrome b, with a break in the dose effect curve occurring at 50% of the absorbance loss. This was consistent with the presence of two hemes in flavocytochrome b that differ by their sensitivity to DPI or IBP.

Topics: Animals; Biphenyl Compounds; Cattle; Cell Membrane; Cytochrome b Group; Dithionite; Electron Spin Resonance Spectroscopy; Enzyme Inhibitors; Flavin-Adenine Dinucleotide; Heme; NADPH Oxidases; Neutrophils; Onium Compounds; Oxidation-Reduction; Oxygen Consumption; Spectrometry, Fluorescence; Spectrophotometry

SiR-FP43, the NADPH- and FAD-binding domain of the Escherichia coli sulphite reductase flavoprotein component (SiR-FP), has been overexpressed and characterized. It folds independently, retaining FAD as a cofactor and the catalytic properties associated with the presence of this cofactor. Iodonium diphenyl chloride (IDP) was shown to be a very efficient inhibitor of SiR-FP43 and SiR-FP60, the monomeric form of SiR-FP, containing both FMN and FAD as cofactors (K(i) = 18.5 +/- 5 microM, maximal inactivation rate = 0.053 +/- 0.005 s(-1)). In both cases, inactivation was shown to result from covalent binding of a phenyl group to FAD exclusively, in marked contrast with previous results obtained with cytochrome P450 reductase (CPR), where FMN and a tryptophan were phenylated, but not FAD. However, our kinetic analyses are in agreement with the inhibition mechanism demonstrated with CPR [Tew (1993) Biochemistry 32, 10209-10215]. Nine different FAD phenylated adducts were isolated and, for the first time, two FAD phenylated adducts were identified directly after extraction from a protein. Taken together, our results have shown that flavoprotein inactivation by IDP is not a reliable indicator for a flavin radical intermediate in catalysis.

Topics: Base Sequence; Binding Sites; Biphenyl Compounds; Chromatography, High Pressure Liquid; DNA Primers; Enzyme Inhibitors; Escherichia coli; Flavin-Adenine Dinucleotide; Gene Expression; Mass Spectrometry; Onium Compounds; Oxidoreductases Acting on Sulfur Group Donors; Recombinant Proteins; Sulfite Reductase (NADPH)

In the human keratinocyte cell line HaCaT, reactive oxygen species (ROS) were generated in a dose- and time-dependent manner in response to epidermal growth factor (EGF), bradykinin, thapsigargin, and the Ca(2+)-ionophore A23187, agonists that interact with different primary cell targets. ROS formation was assessed by both chemiluminescence- and fluorescence-based methods. The ROS evoked by EGF and bradykinin decayed within 8 and 4 min, respectively, this transient effect resulting probably from down-regulation of the specific agonist receptors or dissipation of the secondary signals. In contrast, the response to thapsigargin and A23187 was sustained for at least 15 min. Extracellular Ca2+ and a rise in intracellular Ca2+ concentration ([Ca2+]i) proved essential for ROS production. Chelation by BAPTA suppressed ROS formation. Direct measurement of [Ca2+]i using fura fluorescence revealed that EGF and bradykinin evoked a modest, transient [Ca2+]i elevation of less than twofold, whereas with thapsigargin and A23187 there was a sustained two- to fourfold elevation. For each agonist, the kinetics of the rise and decay of [Ca2+]i were similar to those of ROS. The enzyme(s) involved in ROS formation were inhibited by diphenyleneiodonium, indicating dependence on FAD. Our results suggest a close link between ROS and changes in [Ca2+]i generated by growth factors and hormones. This is a particularly interesting connection because elevation of ROS and/ or [Ca2+]i has been linked to cell proliferation, differentiation, and apoptosis.

Topics: Biphenyl Compounds; Bradykinin; Calcimycin; Calcium; Cell Line; Epidermal Growth Factor; Flavin-Adenine Dinucleotide; Humans; Hydrogen Peroxide; Ionophores; Keratinocytes; Luminescent Measurements; Onium Compounds; Reactive Oxygen Species; Receptors, Cell Surface; Thapsigargin

The thioredoxin-catalyzed insulin reduction by dihydrolipoate was applied to study the 2-oxoacid: lipoate oxidoreductase activity of 2-oxoacid dehydrogenase complexes. The enzymatic and non-enzymatic mechanisms of the transfer of reducing equivalents from the complexes to free lipoic acid (alpha-lipoic acid, 6,8-thiooctic acid) were distinguished using the high stereoselectivity of the complex enzymes to the R-enantiomer of lipoate. Unlike these enzymes, thioredoxin from E. coli exhibited no stereoselectivity upon reduction with chemically obtained dihydrolipoate. However, coupled to the dihydrolipoate production by the dehydrogenase complexes, the process was essentially sensitive both to the enantiomer used and the dihydrolipoyl dehydrogenase activity of the complexes. These results indicated the involvement of the third complex component, dihydrolipoyl dehydrogenase, in the 2-oxoacid-dependent dihydrolipoate formation. The implication of the investigated reaction for a connection between thioredoxin and the 2-oxoacid dehydrogenase complexes in the mitochondrial metabolism are discussed.

Topics: 3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); Biphenyl Compounds; Chemical Precipitation; Escherichia coli; Flavin-Adenine Dinucleotide; Insulin; Ketoglutarate Dehydrogenase Complex; Ketoglutaric Acids; Ketone Oxidoreductases; Multienzyme Complexes; NAD; Onium Compounds; Pyruvate Dehydrogenase Complex; Stereoisomerism; Thioctic Acid; Thioredoxins

Diphenyliodonium has been shown to be an irreversible, time-dependent inhibitor of NADPH cytochrome P450 oxidoreductase (EC 1.6.2.4) with the Ki for diphenyliodonium chloride being 2.8 mM. Kinetic studies have indicated that diphenyliodonium interacts with the reduced enzyme and NADPH is essential for inactivation to take place. Cytochrome c acts as a competitive substrate. The use of radiolabeled diphenyliodonium has enabled two sites of covalent modification to be identified. Isolation of radiolabeled cofactor followed by mass spectrometry has shown that a phenyl group is added to FMN while the FMN is effectively trapped in the reduced state. Trypsin digestion of S-carboxymethylated P450 reductase after inhibition with radiolabeled inhibitor shows covalent modification of the protein. Purification of a single radiolabelled peptide followed by automated Edman degradation has enabled identification of the second site of covalent attachment as Trp 419.

Topics: Animals; Binding, Competitive; Biphenyl Compounds; Chromatography, High Pressure Liquid; Cytochrome c Group; Enzyme Induction; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Kinetics; Male; Mathematics; Mitochondria, Liver; NADP; NADPH-Ferrihemoprotein Reductase; Onium Compounds; Phenobarbital; Rats; Rats, Wistar; Spectrometry, Mass, Fast Atom Bombardment