Nitric oxide ( ⅐ NO)-derived reactive species nitrate unsaturated fatty acids, yielding nitroalkene derivatives, including the clinically abundant nitrated oleic and linoleic acids. The olefinic nitro group renders these derivatives electrophilic at the carbon ␤ to the nitro group, thus competent for Michael addition reactions with cysteine and histidine. By using chromatographic and mass spectrometric approaches, we characterized this reactivity by using in vitro reaction systems, and we demonstrated that nitroalkene-protein and GSH adducts are present in vivo under basal conditions in healthy human red cells. Nitro-linoleic acid (9-, 10-, 12-, and 13-nitro-9,12-octadecadienoic acids) (m/z 324.2) and nitro-oleic acid (9-and 10-nitro-9-octadecaenoic acids) (m/z 326.2) reacted with GSH (m/z 306.1), yielding adducts with m/z of 631.3 and 633.3, respectively. At physiological concentrations, nitroalkenes inhibited glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which contains a critical catalytic Cys (Cys-149). GAPDH inhibition displayed an IC 50 of ϳ3 M M for both nitroalkenes, an IC 50 equivalent to the potent thiol oxidant peroxynitrite (ONOO ؊ ) and an IC 50 30-fold less than H 2 O 2 , indicating that nitroalkenes are potent thiol-reactive species. Liquid chromatography-mass spectrometry analysis revealed covalent adducts between fatty acid nitroalkene derivatives and GAPDH, including at the catalytic Cys-149. Liquid chromatography-mass spectrometry-based proteomic analysis of human red cells confirmed that nitroalkenes readily undergo covalent, thiol-reversible post-translational modification of nucleophilic amino acids in GSH and GAPDH in vivo. The adduction of GAPDH and GSH by nitroalkenes significantly increased the hydrophobicity of these molecules, both inducing translocation to membranes and suggesting why these abundant derivatives had not been detected previously via traditional high pressure liquid chromatography analysis. The occurrence of these electrophilic nitroalkylation reactions in vivo indicates that this reversible post-translational protein modification represents a new pathway for redox regulation of enzyme function, cell signaling, and protein trafficking. Nitric oxide ( ⅐ NO)5 exerts a broad influence on cell and inflammatory signaling via both cGMP-dependent and -independent oxidative, nitrosative, and nitrative reactions (1, 2). The nitration of polyunsaturated fatty acids present in both membranes and lipoproteins is now emerging as a novel mechanism for transducing ⅐ NO-dependent redox signaling (3, 4). Recent evidence indicates that all major unsaturated fatty acids present in human blood contain some proportion of alkenyl nitro derivatives (R 1 HCϭC(NO 2 )R 2 ), also termed nitroalkenes. Because of the prevalence of fatty acid nitroalkenes in healthy humans, these species are now appreciated as an abundant pool of bioactive oxides of nitrogen in the vasculature (5). The two most clinically abundant nitroalkene fatty acid derivatives, nitro-oleic acid (9-and 10-nitro-9-cis-octadeca...

Mass spectrometric analysis of human plasma and urine revealed abundant nitrated derivatives of all principal unsaturated fatty acids. Nitrated palmitoleic, oleic, linoleic, linolenic, arachidonic and eicosapentaenoic acids were detected in concert with their nitrohydroxy derivatives. Two nitroalkene derivatives of the most prevalent fatty acid, oleic acid, were synthesized (9-and 10-nitro-9-cis-octadecenoic acid; OA-NO 2 ), structurally characterized and determined to be identical to OA-NO 2 found in plasma, red cells, and urine of healthy humans. These regioisomers of OA-NO 2 were quantified in clinical samples using 13 C isotope dilution. Plasma free and esterified OA-NO 2 concentrations were 619 ؎ 52 and 302 ؎ 369 nM, respectively, and packed red blood cell free and esterified OA-NO 2 was 59 ؎ 11 and 155 ؎ 65 nM. The OA-NO 2 concentration of blood is ϳ50% greater than that of nitrated linoleic acid, with the combined free and esterified blood levels of these two fatty acid derivatives exceeding 1 M. OA-NO 2 is a potent ligand for peroxisome proliferator activated receptors at physiological concentrations. CV-1 cells co-transfected with the luciferase gene under peroxisome proliferator-activated receptor (PPAR) response element regulation, in concert with PPAR␥, PPAR␣, or PPAR␦ expression plasmids, showed dose-dependent activation of all PPARs by OA-NO 2 . PPAR␥ showed the greatest response, with significant activation at 100 nM, while PPAR␣ and PPAR␦ were activated at ϳ300 nM OA-NO 2 . OA-NO 2 also induced PPAR␥-dependent adipogenesis and deoxyglucose uptake in 3T3-L1 preadipocytes at a potency exceeding nitrolinoleic acid and rivaling synthetic thiazolidinediones. These data reveal that nitrated fatty acids comprise a class of nitric oxide-derived, receptor-dependent, cell signaling mediators that act within physiological concentration ranges.The oxidation of unsaturated fatty acids converts lipids, otherwise serving as cellular metabolic precursors and structural components, into potent signaling molecules including prostaglandins, leukotrienes, isoprostanes, and hydroxy-and hydroperoxyeicosatetraenoates. These enzymatic and auto-catalytic oxidation reactions yield products that orchestrate immune responses, neurotransmission, and the regulation of cell growth. For example, prostaglandins are cyclooxygenase-derived lipid mediators that induce receptor-dependent regulation of inflammatory responses, vascular function, initiation of parturition, cell survival, and angiogenesis (1). In contrast, the various isoprostane products of arachidonic acid auto-oxidation exert vasoconstrictive and pro-inflammatory signaling actions via receptor-dependent and -independent mechanisms (2). A common element of these diverse lipid signaling reactions is that nitric oxide ( ⅐ NO) 6 and other oxides of nitrogen significantly impact lipid mediator formation and bioactivities.The ability of ⅐ NO and ⅐ NO-derived species to oxidize, nitrosate, and nitrate biomolecules serves as the molecular basis for how ⅐ NO influences the sy...

Helicobacter pylori, an oxygen-sensitive microaerophile, contains an alkyl hydroperoxide reductase homologue (AhpC, HP1563) that is more closely related to 2-Cys peroxiredoxins of higher organisms than to most other eubacterial AhpC proteins. Allelic replacement mutagenesis revealed ahpC to be essential, suggesting a critical role for AhpC in defending H. pylori against oxygen toxicity. Characterization of the ahpC promoter region divulged two putative regulatory elements and identified the transcription initiation site, which was mapped to 96 and 94 bp upstream of the initiation codon. No homologue of ahpF, which encodes the dedicated AhpC reductase in most eubacteria, was found in the H. pylori genome. Instead, homologues of Escherichia coli thioredoxin (Trx) reductase (TrxR, HP0825) and Trx (Trx1, HP0824) formed a reductase system for H. pylori AhpC. A second Trx homologue (Trx2, HP1458) was identified but was incapable of AhpC reduction, although Trx2 exhibited disulfide reductase activity with other substrates [insulin and 5,5-dithiobis(2-nitrobenzoic acid)]. AhpC interactions with each substrate, Trx1 and hydroperoxide, were bimolecular and nonsaturable (infinite V max and K m values) but rapid enough (at 1 ؋ 10 5 to 2 ؋ 10 5 M ؊1 s ؊1 ) to suggest an important role for AhpC in cellular peroxide metabolism. AhpC also exhibited a wide specificity for hydroperoxide substrates, which, taken together with the above results, suggests a minimal binding site for hydroperoxides composed of little more than the cysteinyl (Cys49) active site. H. pylori AhpC was not reduced by Salmonella typhimurium AhpF and was slightly more active with E. coli TrxR and Trx1 than was S. typhimurium AhpC, demonstrating the specialized catalytic properties of this peroxiredoxin.

Escherichia coli thiol peroxidase (Tpx, p20, scavengase) is part of an oxidative stress defense system that uses reducing equivalents from thioredoxin (Trx1) and thioredoxin reductase to reduce alkyl hydroperoxides. Tpx contains three Cys residues, Cys 95 , Cys 82 , and Cys 61 , and the latter residue aligns with the N-terminal active site Cys of other peroxidases in the peroxiredoxin family. To identify the catalytically important Cys, we have cloned and purified Tpx and four mutants (C61S, C82S, C95S, and C82S,C95S). In rapid reaction kinetic experiments measuring steady-state turnover, C61S is inactive, C95S retains partial activity, and the C82S mutation only slightly affects reaction rates. Oxidative stress defenses combat reactive oxygen species (1, 2) such as superoxide (O 2 . ), hydrogen peroxide (H 2 O 2 ) and the hydroxyl radical (OH ⅐ ) generated by the host immune response, environmental factors, and the incomplete reduction of oxygen to water during aerobic respiration, all of which are hazardous to proteins, DNA, and lipids (3, 4). Escherichia coli protects cellular components from oxidative damage by employing a variety of antioxidant defense enzymes, such as hydroperoxidases (catalases) I and II (gene products of katG and katE, respectively) that decompose H 2 O 2 (5), and superoxide dismutases (manganese superoxide dismutase, sodA; iron superoxide dismutase, sodB; copper-zinc superoxide dismutase, sodC) (6) that eliminate O 2 . . Additional defenses in E. coli against alkyl and lipid hydroperoxides are provided by multiple, non-heme peroxidases including 1) the peroxidatic component from the alkyl hydroperoxide reductase system (AhpC) 1 (7); 2) a weakly active, thioredoxin (Trx)-dependent bacterioferritin-comigratory protein (BCP) (8); and 3) the periplasmic thiol peroxidase (Tpx, p20, scavengase) (9). In addition, a glutathione peroxidase homologue, the gene product of btuE (10), has been identified in E. coli, and preliminary investigations have indicated Trx-dependent peroxidatic activity against organic hydroperoxides (ROOH) and H 2 O 2 .2

Fatty acid nitration by nitric oxide-derived species yields electrophilic products that adduct protein thiols, inducing changes in protein function and distribution. Nitro-fatty acid adducts of protein and reduced glutathione (GSH) are detected in healthy human blood. Kinetic and mass spectrometric analyses reveal that nitroalkene derivatives of oleic acid (OA-NO 2 ) and linoleic acid (LNO 2 ) rapidly react with GSH and Cys via Michael addition reaction. Rates of OA-NO 2 and LNO 2 reaction with GSH, determined via stopped flow spectrophotometry, displayed second-order rate constants of 183 M ؊1 s ؊1 and 355 M ؊1 s ؊1 , respectively, at pH 7.4 and 37°C. These reaction rates are significantly greater than those for GSH reaction with hydrogen peroxide and non-nitrated electrophilic fatty acids including 8-iso-prostaglandin A 2 and 15-deoxy-⌬ 12,14 -prostaglandin J 2 . Increasing reaction pH from 7.4 to 8.9 enhanced apparent second-order rate constants for the thiol reaction with OA-NO 2 and LNO 2 , showing dependence on the thiolate anion of GSH for reactivity. Rates of nitroalkene reaction with thiols decreased as the pK a of target thiols increased. Increasing concentrations of the detergent octyl-␤-D-glucopyranoside decreased rates of nitroalkene reaction with GSH, indicating that the organization of nitro-fatty acids into micellar or membrane structures can limit Michael reactivity with more polar nucleophilic targets. In aggregate, these results reveal that the reversible adduction of thiols by nitro-fatty acids is a mechanism for reversible post-translational regulation of protein function by nitro-fatty acids.