We have studied the reaction kinetics of ten manganese porphyrins, differing in their meso substituents, with peroxynitrite (ONOO ؊ ) and carbonate radical anion (CO 3 . ) using stopped-flow and pulse radiolysis, re- . by manganese porphyrins could occur in vivo because of the catalytic reduction at the expense of a number of cellular reductants. Additionally, we determined the pK a of the axial water molecules of the Mn(III) complexes at pH 7.5-13.2 by spectrophotometric titration. Results were consistent with two acid-base equilibria for most of the complexes studied. The pK a values also correlated with the resonance and inductive effects of the substituents. The correlations of E1 ⁄2 with the rate constants with ONOO ؊ and with the pK a values display a deviation from linearity when N-alkylpyridinium substituents included N-alkyl moieties longer than ethyl, which is interpreted in terms of a decrease in the local dielectric constant.Metalloporphyrins catalyze numerous redox reactions (1); in particular, manganese porphyrins have been used as redox catalysts in several model systems relevant to biochemistry, for instance, as superoxide dismutase (2, 3) and catalase (4) mimics. Some of the N-alkylpyridinium substituted complexes afforded protection of superoxide dismutase-deficient Escherichia coli from O 2 toxicity (5) and in several rodent models of transient brain ischemia (6, 7), diabetes (8), sickle cell disease (9), and radiation injury (10). Moreover, Mn III TCPP 1 has been effective in a number of model studies of oxidative stressmediated injury (for a review see Ref. 11) despite having low superoxide dismutase and catalase activities.Our group (12-14) and others (15) Given the ubiquity of CO 2 , its high concentration (1 to 2 mM in human tissues), and the reactivity of CO 3. (25), a useful peroxynitrite scavenger needs to out-compete the target molecules and CO 2 and/or be able to efficiently scavenge CO 3 . .We have proposed that complexes such as Mn III TM-2-PyP can efficiently inhibit peroxynitrite-mediated oxidations even in the presence of CO 2 (13). Moreover, the reaction of Mn III TM-2-PyP with ONOO Ϫ in the presence of CO 2 produced more oxidation of the metal complex than expected based on simple competition kinetics (13), suggesting a probable reaction of the complex with CO 3. . The possible reaction of Mn(III) porphyrins with CO 3 . has also been proposed recently (26) in experiments related to the effect of bicarbonate on the peroxidase activity of Cu,Zn superoxide dismutase. In aerated aqueous solution, the stable oxidation state of

Background: Nitroalkene fatty acids are electrophilic cell metabolites that mediate anti-inflammatory signaling actions. Results: Conjugated linoleic acid is the preferential unsaturated fatty acid substrate for nitration reactions during oxidative inflammatory conditions and digestion. Conclusion: Nitro-fatty acid formation in vivo occurs during metabolic and inflammatory reactions and modulates cell signaling. Significance: Nitro-conjugated linoleic acid transduces signaling actions of nitric oxide, nitrite, and conjugated linoleic acid.

Nitric oxide (NO • ) competitively inhibits oxygen consumption by mitochondria at cytochrome c oxidase and S-nitrosates thiol proteins. We developed mitochondria-targeted S-nitrosothiols (MitoSNOs) that selectively modulate and protect mitochondrial function. The exemplar MitoSNO1, produced by covalently linking an Snitrosothiol to the lipophilic triphenylphosphonium cation, was rapidly and extensively accumulated within mitochondria, driven by the membrane potential, where it generated NO • and Snitrosated thiol proteins. MitoSNO1-induced NO • production reversibly inhibited respiration at cytochrome c oxidase and increased extracellular oxygen concentration under hypoxic conditions. MitoSNO1 also caused vasorelaxation due to its NO • generation. Infusion of MitoSNO1 during reperfusion was protective against heart ischemia-reperfusion injury, consistent with a functional modification of mitochondrial proteins, such as complex I, following S-nitrosation. These results support the idea that selectively targeting NO • donors to mitochondria is an effective strategy to reversibly modulate respiration and to protect mitochondria against ischemia-reperfusion injury.nitric oxide ͉ S-nitrosation

Introductory ParagraphThe coupling of hemoglobin sensing of physiological oxygen gradients to stimulation of nitric oxide (NO) bioactivity is an established principle of hypoxic blood flow. One mechanism proposed to explain this O 2 sensing/NO bioactivity linkage postulates an essential role for the conserved hemoglobin β93Cys residue and, specifically, for S-nitrosation of β93Cys to form S-nitrosohemoglobin (SNO-Hb) 1 . The SNO-Hb hypothesis, which conceptually linked hemoglobin and NO biology, has been debated intensely in recent years 2,3 . This debate has precluded a consensus on physiological mechanisms and on assessment of the potential role of SNO-Hb in pathology. Here we describe novel mouse models that express exclusively either human wild type hemoglobin or human hemoglobin in which the β93cys residue is replaced with alanine to assess the role of SNO-Hb in red cell mediated hypoxic vasodilation. Substitution of this residue, precluding hemoglobin S-nitrosation, did not change total red cell S-nitrosothiol levels but shifted S-nitrosothiol distribution to lower MWt species, consistent with the loss of SNO-Hb. Loss of β93cys resulted in no deficits in systemic nor pulmonary hemodynamics under basal conditions and, importantly, did not affect isolated red cell dependent hypoxic vasodilation. These results demonstrate that SNO-Hb is not essential for the physiologic coupling of erythrocyte deoxygenation with increased NO-bioactivity in vivo. *Co corresponding Authors: Rakesh P Patel, PhD, Department of Pathology, University of Alabama at Birmingham, 901 19 th street south, BMR 2, room 302, Birmingham, AL 35294, E mail: E-mail: rakeshp@uab.edu. Tim M Townes, PhD, Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Kaul Human Genetics Bldg, room 502, 720 20 th street south, Birmingham, AL 35294, E mail: E-mail: ttownes@uab.edu. # these authors contributed equally to this work Author Contributions TSI, CWS, LCW, XT, DAV and KMP were responsible for performing experiments. TSI, CWS, DAV, RPP and TMT were responsible for planning all experiments, analyzing data and writing manuscript. MBR contributed to mass spectrometry assays, LS was responsible for exercise related studies, CGK and BGB for capillary density measurements, NP and JW contributed to blood pressure measurements and NA for assessment of pulmonary hemodynamics. JR did the ES cell injections to generate the chimeras. In addition to hemoglobin oxygen affinity, blood flow is a key component of the processes that match oxygen delivery to demand. Increased blood flow in response to hypoxia is a critical physiological response which does not correlate with dissolved oxygen tensions but does correlate with hemoglobin oxygen fractional saturation 4 . These observations have led to the concept that the red blood cell (RBC) itself is a regulator of flow and to the general paradigm that RBC/hemoglobin deoxygenation is coupled to the stimulation of vasodilation 1,5,6 . Three mechanisms for this coupling have been proposed (...

This article is available online at http://www.jlr.org biomolecule nitration ( 1 ). Nitroalkene substituents are electrophilic and promote Michael addition of fatty acids with biological nucleophiles such as cysteine and histidine. The extent, rate, and reversibility of these reactions will be dictated both by the concentration and reactivity of individual nucleophiles. In this regard, protein structure and compartmentalization affect the reactivity of individual nucleophilic centers and will defi ne the molecular targets of electrophilic fatty acids.While enzymatically-oxygenated unsaturated fatty acids typically transduce anti-infl ammatory actions via specifi c g protein-coupled receptor ligand activity ( 2, 3 ), transcriptional responses to electrophilic fatty acids reveal that a broader array of signaling events are instigated ( 4, 5 ). The basis for this pleiotropy resides in the facile Michael addition of electrophilic fatty acid derivatives with nucleophilic centers of proteins that regulate structure and function ( 6 ). Functionally-signifi cant protein targets of electrophilic fatty acids include the transcriptional regulatory protein complex nuclear factor kappa B (NFkB), the

Extra virgin olive oil (EVOO) and olives, key sources of unsaturated fatty acids in the Mediterranean diet, provide health benefits to humans. Nitric oxide (•NO) and nitrite (NO2 −)-dependent reactions of unsaturated fatty acids yield electrophilic nitroalkene derivatives (NO2-FA) that manifest salutary pleiotropic cell signaling responses in mammals. Herein, the endogenous presence of NO2-FA in both EVOO and fresh olives was demonstrated by mass spectrometry. The electrophilic nature of these species was affirmed by the detection of significant levels of protein cysteine adducts of nitro-oleic acid (NO2-OA-cysteine) in fresh olives, especially in the peel. Further nitration of EVOO by NO2 − under acidic gastric digestive conditions revealed that human consumption of olive lipids will produce additional nitro-conjugated linoleic acid (NO2-cLA) and nitro-oleic acid (NO2-OA). The presence of free and protein-adducted NO2-FA in both mammalian and plant lipids further affirm a role for these species as signaling mediators. Since NO2-FA instigate adaptive anti-inflammatory gene expression and metabolic responses, these redox-derived metabolites may contribute to the cardiovascular benefits associated with the Mediterranean diet.

A gap in our understanding of the beneficial systemic responses to dietary constituents nitrate (NO3−), nitrite (NO2−) and conjugated linoleic acid (cLA) is the identification of the downstream metabolites that mediate their actions. To examine these reactions in a clinical context, investigational drug preparations of 15N-labeled NO3− and NO2− were orally administered to healthy humans with and without cLA. Mass spectrometry analysis of plasma and urine indicated that the nitrating species nitrogen dioxide was formed and reacted with the olefinic carbons of unsaturated fatty acids to yield the electrophilic fatty acid, nitro-cLA (NO2-cLA). These species mediate the post-translational modification (PTM) of proteins via reversible Michael addition with nucleophilic amino acids. The PTM of critical target proteins by electrophilic lipids has been described as a sensing mechanism that regulates adaptive cellular responses, but little is known about the endogenous generation of fatty acid nitroalkenes and their metabolites. We report that healthy humans consuming 15N-labeled NO3− or NO2−, with and without cLA supplementation, produce 15NO2-cLA and corresponding metabolites that are detected in plasma and urine. These data support that the dietary constituents NO3−, NO2− and cLA promote the further generation of secondary electrophilic lipid products that are absorbed into the circulation at concentrations sufficient to exert systemic effects before being catabolized or excreted.

Acyloxy nitroso compounds hydrolyze to nitroxyl (HNO), a nitrogen monoxide with distinct chemistry and biology. Ultraviolet-visible spectroscopy and mass spectrometry show hydrolysis rate depends on pH and ester group structure with the observed rate being trifluoroacetate (3) > acetate (1) > pivalate (2). Under all conditions, 3 rapidly hydrolyzes to HNO. A combination of spectroscopic, kinetic and product studies show that addition of thiols increases the decomposition rate of 1 and 2 leading to hydrolysis and HNO. Under conditions that favor thiolates, the thiolate directly reacts with the nitroso group yielding oximes without HNO formation. Biologically, 3 behaves like Angeli's salt demonstrating thiol-sensitive nitric oxide-mediated soluble guanylate cyclase-dependent vasorelaxation, suggesting HNO-mediated vasorelaxation. The slow HNO-donor 1 demonstrates weak thiol-insensitive vasorelaxation indicating HNO release kinetics determine HNO bioavailability and activity. These results show that acyloxy nitroso compounds represent new HNO donors capable of vasorelaxation depending on HNO release kinetics.