flavin-adenine-dinucleotide and 5-5-dimethyl-1-pyrroline-1-oxide

The nicotinamide adenine dinucleotide (NADH)/nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and the xanthine oxidase (XOD) systems generate reactive oxygen species (ROS). In the present study, to characterize the difference between the two systems, the kinetics of ROS generated by both the NADH oxidase and XOD systems were analysed by an electron spin resonance (ESR) spin trapping method using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), 5-(diethoxyphosphoryl)-5-methyl-pyrroline N-oxide (DEPMPO) and 5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO). As a result, two major differences in ROS kinetics were found between the two systems: (i) the kinetics of (•)OH and (ii) the kinetics of hydrogen peroxide. In the NADH oxidase system, the interaction of hydrogen peroxide with each component of the enzyme system (NADPH, NADH oxidase and FAD) was found to generate (•)OH. In contrast, (•)OH generation was found to be independent of hydrogen peroxide in the XOD system. In addition, the hydrogen peroxide level in the NADPH-NADH oxidase system was much lower than measured in the XOD system. This lower level of free hydrogen peroxide is most likely due to the interaction between hydrogen peroxide and NADPH, because the hydrogen peroxide level was reduced by ~90% in the presence of NADPH.

Topics: Cyclic N-Oxides; Flavin-Adenine Dinucleotide; Hydrogen Peroxide; Kinetics; NADP; NADPH Oxidases; Pyrroles; Reactive Oxygen Species; Spin Trapping; Superoxides; Xanthine Oxidase

Increased O(2)(*-) and NO production is a key mechanism of mitochondrial dysfunction in myocardial ischemia/reperfusion injury. In complex II, oxidative impairment and enhanced tyrosine nitration of the 70 kDa FAD-binding protein occur in the post-ischemic myocardium and are thought to be mediated by peroxynitrite (OONO(-)) in vivo [Chen, Y.-R., et al. (2008) J. Biol. Chem. 283, 27991-28003]. To gain deeper insights into the redox protein thiols involved in OONO(-)-mediated oxidative post-translational modifications relevant in myocardial infarction, we subjected isolated myocardial complex II to in vitro protein nitration with OONO(-). This resulted in site-specific nitration at the 70 kDa polypeptide and impairment of complex II-derived electron transfer activity. Under reducing conditions, the gel band of the 70 kDa polypeptide was subjected to in-gel trypsin/chymotrypsin digestion and then LC-MS/MS analysis. Nitration of Y(56) and Y(142) was previously reported. Further analysis revealed that C(267), C(476), and C(537) are involved in OONO(-)-mediated S-sulfonation. To identify the disulfide formation mediated by OONO(-), nitrated complex II was alkylated with iodoacetamide. In-gel proteolytic digestion and LC-MS/MS analysis were conducted under nonreducing conditions. The MS/MS data were examined with MassMatrix, indicating that three cysteine pairs, C(306)-C(312), C(439)-C(444), and C(288)-C(575), were involved in OONO(-)-mediated disulfide formation. Immuno-spin trapping with an anti-DMPO antibody and subsequent MS was used to define oxidative modification with protein radical formation. An OONO(-)-dependent DMPO adduct was detected, and further LC-MS/MS analysis indicated C(288) and C(655) were involved in DMPO binding. These results offered a complete profile of OONO(-)-mediated oxidative modifications that may be relevant in the disease model of myocardial infarction.

Topics: Amino Acid Sequence; Animals; Cell Hypoxia; Cyclic N-Oxides; Cysteine; Disulfides; Electron Transport Complex II; Flavin-Adenine Dinucleotide; Humans; Molecular Sequence Data; Molecular Weight; Muscle Cells; Myocardial Infarction; Oxidation-Reduction; Peroxynitrous Acid; Protein Subunits; Rats; Rats, Sprague-Dawley; Tyrosine

Self-regulation of the 2-oxo acid dehydrogenase complexes during catalysis was studied. Radical species as side products of catalysis were detected by spin trapping, lucigenin fluorescence and ferricytochrome c reduction. Studies of the complexes after converting the bound lipoate or FAD cofactors to nonfunctional derivatives indicated that radicals are generated via FAD. In the presence of oxygen, the 2-oxo acid, CoA-dependent production of the superoxide anion radical was detected. In the absence of oxygen, a protein-bound radical concluded to be the thiyl radical of the complex-bound dihydrolipoate was trapped by alpha-phenyl-N-tert-butylnitrone. Another, carbon-centered, radical was trapped in anaerobic reaction of the complex with 2-oxoglutarate and CoA by 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO). Generation of radical species was accompanied by the enzyme inactivation. A superoxide scavenger, superoxide dismutase, did not protect the enzyme. However, a thiyl radical scavenger, thioredoxin, prevented the inactivation. It was concluded that the thiyl radical of the complex-bound dihydrolipoate induces the inactivation by 1e- oxidation of the 2-oxo acid dehydrogenase catalytic intermediate. A product of this oxidation, the DMPO-trapped radical fragment of the 2-oxo acid substrate, inactivates the first component of the complex. The inactivation prevents transformation of the 2-oxo acids in the absence of terminal substrate, NAD+. The self-regulation is modulated by thioredoxin which alleviates the adverse effect of the dihydrolipoate intermediate, thus stimulating production of reactive oxygen species by the complexes. The data point to a dual pro-oxidant action of the complex-bound dihydrolipoate, propagated through the first and third component enzymes and controlled by thioredoxin and the (NAD+ + NADH) pool.

Topics: 3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); Catalysis; Coenzyme A; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Enzyme Activation; Flavin-Adenine Dinucleotide; Free Radicals; Ketone Oxidoreductases; Multienzyme Complexes; NAD; Nitrogen Oxides; Spin Labels; Superoxides; Thioctic Acid; Thioredoxins