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A challenge in vascularized composite allotransplantation (VCA) is mitigating tissue damage within the composite secondary to brain death (BD). Loss of central nervous system function disrupts S-nitrosothiol (SNO) homeostasis to produce systemic hypoxia and ischemic injury during the donor support phase. We reasoned that addition of an S-nitrosylating agent to the preservation solution could correct this damage during ex vivo storage. Employing a swine BD preparation, we excised VC tissues (abdominal blocks and limbs) after a 16-h period of systemic support. The composites were perfused with/without the S-nitrosylating agent ethyl nitrite (ENO) present in the preservation solution. Flow rates and resistance were recorded during the storage period; tissue hypoxia was also quantified. BD decreased circulating SNO levels and reduced tissue oxygenation and muscle protein NO content. During storage, ENO increased flow and decreased resistance within the VCs. Muscle from ENO-treated VCs had lower protein levels of inducible nitric oxide synthase and the hypoxia marker Hif1α and higher levels of the anti-apoptotic protein Bcl2, all suggestive of enhancements of tissue oxygenation. As such, ex vivo S-nitrosylation therapy may be a means to correct BD-induced impairments in SNO-status to improve the quality of composite tissue grafts prior to transplantation.

Significance Coenzyme A (CoA) is a small-molecular-weight thiol that plays a central role in cellular metabolism. We have discovered a novel, phylogenetically conserved class of enzymes that reduce S-nitroso-CoA (SNO-CoA) and thereby regulate protein S-nitrosylation. These denitrosylases, identified as alcohol dehydrogenase 6 (Adh6) in yeast and aldo-keto reductase 1A1 in mammals, may be analogized to deacetylases, which regulate CoA-mediated protein acetylation. In yeast, Adh6 (previously without ascribed cellular function) regulates endogenous protein S-nitrosylation (heretofore unknown) including function-altering S-nitrosylation that impacts CoA-related metabolism. Thus, our findings establish a novel role for CoA in protein S-nitrosylation (operating through SNO-CoA), which is governed by specific enzymes. This mechanism may regulate the influence of nitric oxide on cellular metabolism in health and disease. Coenzyme A (CoA) mediates thiol-based acyl-group transfer (acetylation and palmitoylation). However, a role for CoA in the thiol-based transfer of NO groups (S-nitrosylation) has not been considered. Here we describe protein S-nitrosylation in yeast (heretofore unknown) that is mediated by S-nitroso-CoA (SNO-CoA). We identify a specific SNO-CoA reductase encoded by the alcohol dehydrogenase 6 ( ADH6 ) gene and show that deletion of ADH6 increases cellular S-nitrosylation and alters CoA metabolism. Further, we report that Adh6, acting as a selective SNO-CoA reductase, protects acetoacetyl–CoA thiolase from inhibitory S-nitrosylation and thereby affects sterol biosynthesis. Thus, Adh6-regulated, SNO-CoA–mediated protein S-nitrosylation provides a regulatory mechanism paralleling protein acetylation. We also find that SNO-CoA reductases are present from bacteria to mammals, and we identify aldo-keto reductase 1A1 as the mammalian functional analog of Adh6. Our studies reveal a novel functional class of enzymes that regulate protein S-nitrosylation from yeast to mammals and suggest that SNO-CoA–mediated S-nitrosylation may subserve metabolic regulation.

Current human donor care protocols following death by neurologic criteria (DNC) can stabilize macro-hemodynamic parameters but have minimal ability to preserve systemic blood flow and microvascular oxygen delivery. S-nitrosylated hemoglobin (SNO-Hb) within red blood cells (RBCs) is the main regulator of tissue oxygenation (StO 2 ). Based on various pre-clinical studies, we hypothesized that brain death (BD) would decrease post-mortem SNO-Hb levels to negatively-impact StO 2 and reduce organ yields. We tracked SNO-Hb and tissue oxygen in 61 DNC donors. After BD, SNO-Hb levels were determined to be significantly decreased compared to healthy humans ( p  = 0·003) and remained reduced for the duration of the monitoring period. There was a positive correlation between SNO-Hb and StO 2 ( p  < 0.001). Furthermore, SNO-Hb levels correlated with and were prognostic for the number of organs transplanted ( p  < 0.001). These clinical findings provide additional support for the concept that BD induces a systemic impairment of S-nitrosylation that negatively impacts StO 2 and reduces organ yield from DNC human donors. Exogenous S-nitrosylating agents are in various stages of clinical development. The results presented here suggest including one or more of these agents in donor support regimens could increase the number and quality of organs available for transplant.

Systemic hypoxia is characterized by peripheral vasodilation and pulmonary vasoconstriction. However, the system-wide mechanism for signaling hypoxia remains unknown. Accumulating evidence suggests that hemoglobin (Hb) in RBCs may serve as an O2 sensor and O2-responsive NO signal transducer to regulate systemic and pulmonary vascular tone, but this remains unexamined at the integrated system level. One residue invariant in mammalian Hbs, β-globin cysteine93 (βCys93), carries NO as vasorelaxant S-nitrosothiol (SNO) to autoregulate blood flow during O2 delivery. βCys93Ala mutant mice thus exhibit systemic hypoxia despite transporting O2 normally. Here, we show that βCys93Ala mutant mice had reduced S-nitrosohemoglobin (SNO-Hb) at baseline and upon targeted SNO repletion and that hypoxic vasodilation by RBCs was impaired in vitro and in vivo, recapitulating hypoxic pathophysiology. Notably, βCys93Ala mutant mice showed marked impairment of hypoxic peripheral vasodilation and developed signs of pulmonary hypertension with age. Mutant mice also died prematurely with cor pulmonale (pulmonary hypertension with right ventricular dysfunction) when living under low O2. Altogether, we identify a major role for RBC SNO in clinically relevant vasodilatory responses attributed previously to endothelial NO. We conclude that SNO-Hb transduces the integrated, system-wide response to hypoxia in the mammalian respiratory cycle, expanding a core physiological principle.

Inhaled nitric oxide (NO) exerts a variety of effects through metabolites and these play an important role in regulation of hemodynamics in the body. A detailed investigation into the generation of these metabolites has been overlooked. We investigated the kinetics of nitrite and S-nitrosothiol-hemoglobin (SNO-Hb) in plasma derived from inhaled NO subjects and how this modifies the cutaneous microvascular response. We enrolled 15 healthy volunteers. Plasma nitrite levels at baseline and during NO inhalation (15 minutes at 40 ppm) were 102 (86-118) and 114 (87-129) nM, respectively. The nitrite peak occurred at 5 minutes of discontinuing NO (131 (104-170) nM). Plasma nitrate levels were not significantly different during the study. SNO-Hb molar ratio levels at baseline and during NO inhalation were 4.7E-3 (2.5E-3-5.8E-3) and 7.8E-3 (4.1E-3-13.0E-3), respectively. Levels of SNO-Hb continued to climb up to the last study time point (30 min: 10.6E-3 (5.3E-3-15.5E-3)). The response to acetylcholine iontophoresis both before and during NO inhalation was inversely associated with the SNO-Hb level (r: -0.57, p = 0.03, and r: -0.54, p = 0.04, respectively). Both nitrite and SNO-Hb increase during NO inhalation. Nitrite increases first, followed by a more sustained increase in Hb-SNO. Nitrite and Hb-SNO could be a mobile reservoir of NO with potential implications on the systemic microvasculature.

Nitric oxide (NO) bioactivity is mainly conveyed through reactions with iron and thiols, furnishing iron nitrosyls and S-nitrosothiols with wide-ranging stabilities and reactivities. Triiodide chemiluminescence methodology has been popularized as uniquely capable of quantifying these species together with NO byproducts, such as nitrite and nitrosamines. Studies with triiodide, however, have challenged basic ideas of NO biochemistry. The assay, which involves addition of multiple reagents whose chemistry is not fully understood, thus requires extensive validation: Few protein standards have in fact been characterized; NO mass balance in biological mixtures has not been verified; and recovery of species that span the range of NO-group reactivities has not been assessed. Here we report on the performance of the triiodide assay vs. photolysis chemiluminescence in side-by-side assays of multiple nitrosylated standards of varied reactivities and in assays of endogenous Fe- and S-nitrosylated hemoglobin. Although the photolysis method consistently gives quantitative recoveries, the yields by triiodide are variable and generally low (approaching zero with some standards and endogenous samples). Moreover, in triiodide, added chemical reagents, changes in sample pH, and altered ionic composition result in decreased recoveries and misidentification of NO species. We further show that triiodide, rather than directly and exclusively producing NO, also produces the highly potent nitrosating agent, nitrosyliodide. Overall, we find that the triiodide assay is strongly influenced by sample composition and reactivity and does not reliably identify, quantify, or differentiate NO species in complex biological mixtures.

We have previously reported that bacterial flavohemoglobin (HMP) catalyzes both a rapid reaction of heme-bound O2with nitric oxide (NO) to form nitrate [HMP-Fe(II)O2+ NO → HMP-Fe(III) + NO3 -] and, under anaerobic conditions, a slower reduction of heme-bound NO to an NO-equivalent (followed by the formation of N2O), thereby protecting against nitrosative stress under both aerobic and anaerobic conditions, and rationalizing our finding that NO is rapidly consumed across a wide range of O2concentrations. It has been alternatively suggested that HMP activity is inhibited at low pO2because the enzyme is then in the relatively inactive nitrosyl form [koff/konfor NO (0.000008 µM) « koff/konfor O2(0.012 µM) and KMfor O2= 30-100 µM]. To resolve this discrepancy, we have directly measured heme-ligand turnover and NADH consumption under various O2/NO concentrations. We find that, at biologically relevant O2concentrations, HMP preferentially binds NO (not O2), which it then reacts with oxygen to form nitrate (in essence NO-+ O2→ NO3 -). During steady-state turnover, the enzyme can be found in the ferric (FeIII) state. The formation of a heme-bound nitroxyl equivalent and its subsequent oxidation is a novel enzymatic function, and one that dominates the oxygenase activity under biologically relevant conditions. These data unify the mechanism of HMP/NO interaction with those recently described for the nematode Ascaris and mammalian hemoglobins, and more generally suggest that the peroxidase (FeIII)-like properties of globins have evolved for handling of NO.

Only a few intracellular S-nitrosylated proteins have been identified, and it is unknown if protein S-nitrosylation/denitrosylation is a component of signal transduction cascades. Caspase-3 zymogens were found to be S-nitrosylated on their catalytic-site cysteine in unstimulated human cell lines and denitrosylated upon activation of the Fas apoptotic pathway. Decreased caspase-3 S-nitrosylation was associated with an increase in intracellular caspase activity. Fas therefore activates caspase-3 not only by inducing the cleavage of the caspase zymogen to its active subunits, but also by stimulating the denitrosylation of its active-site thiol. Protein S-nitrosylation/denitrosylation can thus serve as a regulatory process in signal transduction pathways.

Nitric oxide (NO) biology has focused on the tightly regulated enzymatic mechanism that transforms L-arginine into a family of molecules, which serve both signaling and defense functions. However, very little is known of the pathways that metabolize these molecules or turn off the signals. The paradigm is well exemplified in bacteria where S-nitrosothiols (SNO)--compounds identified with antimicrobial activities of NO synthase--elicit responses that mediate bacterial resistance by unknown mechanisms. Here we show that Escherichia coli possess both constitutive and inducible elements for SNO metabolism. Constitutive enzyme(s) cleave SNO to NO whereas bacterial hemoglobin, a widely distributed flavohemoglobin of poorly understood function, is central to the inducible response. Remarkably, the protein has evolved a novel heme-detoxification mechanism for NO. Specifically, the heme serves a dioxygenase function that produces mainly nitrate. These studies thus provide new insights into SNO and NO metabolism and identify enzymes with reactions that were thought to occur only by chemical means. Our results also emphasize that the reactions of SNO and NO with hemoglobins are evolutionary conserved, but have been adapted for cell-specific function.