Sheila Frizzell

Nitric Oxide Scavenging by Red Blood Cell Microparticles and Cell-Free Hemoglobin as a Mechanism for the Red Cell Storage Lesion

Background— Intravascular red cell hemolysis impairs nitric oxide (NO)–redox homeostasis, producing endothelial dysfunction, platelet activation, and vasculopathy. Red blood cell storage under standard conditions results in reduced integrity of the erythrocyte membrane, with formation of exocytic microvesicles or microparticles and hemolysis, which we hypothesized could impair vascular function and contribute to the putative storage lesion of banked blood. Methods and Results— We now find that storage of human red blood cells under standard blood banking conditions results in the accumulation of cell-free and microparticle-encapsulated hemoglobin, which, despite 39 days of storage, remains in the reduced ferrous oxyhemoglobin redox state and stoichiometrically reacts with and scavenges the vasodilator NO. Using stopped-flow spectroscopy and laser-triggered NO release from a caged NO compound, we found that both free hemoglobin and microparticles react with NO about 1000 times faster than with intact erythrocytes. In complementary in vivo studies, we show that hemoglobin, even at concentrations below 10 &mgr;mol/L (in heme), produces potent vasoconstriction when infused into the rat circulation, whereas controlled infusions of methemoglobin and cyanomethemoglobin, which do not consume NO, have substantially reduced vasoconstrictor effects. Infusion of the plasma from stored human red blood cell units into the rat circulation produces significant vasoconstriction related to the magnitude of storage-related hemolysis. Conclusions— The results of these studies suggest new mechanisms for endothelial injury and impaired vascular function associated with the most fundamental of storage lesions, hemolysis.

Human Neuroglobin Functions as a Redox-regulated Nitrite Reductase

Neuroglobin is a highly conserved hemoprotein of uncertain physiological function that evolved from a common ancestor to hemoglobin and myoglobin. It possesses a six-coordinate heme geometry with proximal and distal histidines directly bound to the heme iron, although coordination of the sixth ligand is reversible. We show that deoxygenated human neuroglobin reacts with nitrite to form nitric oxide (NO). This reaction is regulated by redox-sensitive surface thiols, cysteine 55 and 46, which regulate the fraction of the five-coordinated heme, nitrite binding, and NO formation. Replacement of the distal histidine by leucine or glutamine leads to a stable five-coordinated geometry; these neuroglobin mutants reduce nitrite to NO ∼2000 times faster than the wild type, whereas mutation of either Cys-55 or Cys-46 to alanine stabilizes the six-coordinate structure and slows the reaction. Using lentivirus expression systems, we show that the nitrite reductase activity of neuroglobin inhibits cellular respiration via NO binding to cytochrome c oxidase and confirm that the six-to-five-coordinate status of neuroglobin regulates intracellular hypoxic NO-signaling pathways. These studies suggest that neuroglobin may function as a physiological oxidative stress sensor and a post-translationally redox-regulated nitrite reductase that generates NO under six-to-five-coordinate heme pocket control. We hypothesize that the six-coordinate heme globin superfamily may subserve a function as primordial hypoxic and redox-regulated NO-signaling proteins.

Reactive oxygen and nitrogen species in pulmonary hypertension

Pulmonary vascular disease can be defined as either a disease affecting the pulmonary capillaries and pulmonary arterioles, termed pulmonary arterial hypertension, or a disease affecting the left ventricle, called pulmonary venous hypertension. Pulmonary arterial hypertension (PAH) is a disorder of the pulmonary circulation characterized by endothelial dysfunction, as well as intimal and smooth muscle proliferation. Progressive increases in pulmonary vascular resistance and pressure impair the performance of the right ventricle, resulting in declining cardiac output, reduced exercise capacity, right-heart failure, and ultimately death. While the primary and heritable forms of the disease are thought to affect over 5000 patients in the United States, the disease can occur secondary to congenital heart disease, most advanced lung diseases, and many systemic diseases. Multiple studies implicate oxidative stress in the development of PAH. Further, this oxidative stress has been shown to be associated with alterations in reactive oxygen species (ROS), reactive nitrogen species (RNS), and nitric oxide (NO) signaling pathways, whereby bioavailable NO is decreased and ROS and RNS production are increased. Many canonical ROS and NO signaling pathways are simultaneously disrupted in PAH, with increased expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and xanthine oxidoreductase, uncoupling of endothelial NO synthase (eNOS), and reduction in mitochondrial number, as well as impaired mitochondrial function. Upstream dysregulation of ROS/NO redox homeostasis impairs vascular tone and contributes to the pathological activation of antiapoptotic and mitogenic pathways, leading to cell proliferation and obliteration of the vasculature. This paper will review the available data regarding the role of oxidative and nitrosative stress and endothelial dysfunction in the pathophysiology of pulmonary hypertension, and provide a description of targeted therapies for this disease.

14-3-3 Binding and Phosphorylation of Neuroglobin during Hypoxia Modulate Six-to-Five Heme Pocket Coordination and Rate of Nitrite Reduction to Nitric Oxide

Background: Neuroglobin protects neurons from hypoxia; however, the underlying mechanisms for this effect remain poorly understood. Results: Hypoxia increases neuroglobin phosphorylation, binding to 14-3-3, and nitrite reduction to form nitric oxide. Conclusion: Hypoxia-dependent post-translational modifications to neuroglobin regulate the six-to-five heme pocket equilibrium and heme access to ligands. Significance: Hypoxia-regulated neuroglobin may contribute to the cellular adaptation to hypoxia. Neuroglobin protects neurons from hypoxia in vitro and in vivo; however, the underlying mechanisms for this effect remain poorly understood. Most of the neuroglobin is present in a hexacoordinate state with proximal and distal histidines in the heme pocket directly bound to the heme iron. At equilibrium, the concentration of the five-coordinate neuroglobin remains very low (0.1–5%). Recent studies have shown that post-translational redox regulation of neuroglobin surface thiol disulfide formation increases the open probability of the heme pocket and allows nitrite binding and reaction to form NO. We hypothesized that the equilibrium between the six- and five-coordinate states and secondary reactions with nitrite to form NO could be regulated by other hypoxia-dependent post-translational modification(s). Protein sequence models identified candidate sites for both 14-3-3 binding and phosphorylation. In both in vitro experiments and human SH-SY5Y neuronal cells exposed to hypoxia and glucose deprivation, we observed that 1) neuroglobin phosphorylation and protein-protein interactions with 14-3-3 increase during hypoxic and metabolic stress; 2) neuroglobin binding to 14-3-3 stabilizes and increases the half-life of phosphorylation; and 3) phosphorylation increases the open probability of the heme pocket, which increases ligand binding (CO and nitrite) and accelerates the rate of anaerobic nitrite reduction to form NO. These data reveal a series of hypoxia-dependent post-translational modifications to neuroglobin that regulate the six-to-five heme pocket equilibrium and heme access to ligands. Hypoxia-regulated reactions of nitrite and neuroglobin may contribute to the cellular adaptation to hypoxia.

Exploring the Mechanisms of the Reductase Activity of Neuroglobin by Site-Directed Mutagenesis of the Heme Distal Pocket

Neuroglobin (Ngb) is a six-coordinate globin that can catalyze the reduction of nitrite to nitric oxide. Although this reaction is common to heme proteins, the molecular interactions in the heme pocket that regulate this reaction are largely unknown. We have shown that the H64L Ngb mutation increases the rate of nitrite reduction by 2000-fold compared to that of wild-type Ngb [Tiso, M., et al. (2011) J. Biol. Chem. 286, 18277–18289]. Here we explore the effect of distal heme pocket mutations on nitrite reduction. For this purpose, we have generated mutations of Ngb residues Phe28(B10), His64(E7), and Val68(E11). Our results indicate a dichotomy in the reactivity of deoxy five- and six-coordinate globins toward nitrite. In hemoglobin and myoglobin, there is a correlation between faster rates and more negative potentials. However, in Ngb, reaction rates are apparently related to the distal pocket volume, and redox potential shows a poor relationship with the rate constants. This suggests a relationship between the nitrite reduction rate and heme accessibility in Ngb, particularly marked for His64(E7) mutants. In five-coordinate globins, His(E7) facilitates nitrite reduction, likely through proton donation. Conversely, in Ngb, the reduction mechanism does not rely on the delivery of a proton from the histidine side chain, as His64 mutants show the fastest reduction rates. In fact, the rate observed for H64A Ngb (1120 M–1 s–1) is to the best of our knowledge the fastest reported for a heme nitrite reductase. These differences may be related to a differential stabilization of the iron–nitrite complexes in five- and six-coordinate globins.

Normoxic cyclic GMP-independent oxidative signaling by nitrite enhances airway epithelial cell proliferation and wound healing.

The airway epithelium provides important barrier and host defense functions. Recent studies reveal that nitrite is an endocrine reservoir of nitric oxide (NO) bioactivity that is converted to NO by enzymatic reductases along the physiological oxygen gradient. Nitrite signaling has been described as NO dependent activation mediated by reactions with deoxygenated redox active hemoproteins, such as hemoglobin, myoglobin, neuroglobin, xanthine oxidoreductase (XO) and NO synthase at low pH and oxygen tension. However, nitrite can also be readily oxidized to nitrogen dioxide (NO(2)·) via heme peroxidase reactions, suggesting the existence of alternative oxidative signaling pathways for nitrite under normoxic conditions. In the present study, we examined normoxic signaling effects of sodium nitrite on airway epithelial cell wound healing. In an in vitro scratch injury model under normoxia, we exposed cultured monolayers of human airway epithelial cells to various concentrations of sodium nitrite and compared responses to NO donor. We found sodium nitrite potently enhanced airway epithelium wound healing at physiological concentrations (from 1 μM). The effect of nitrite was blocked by the NO and NO(2)· scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO). Interestingly, nitrite treatment did not increase cyclic guanosine monophosphate (cGMP) levels under these normoxic conditions, even in the presence of a phosphodiesterase 5 inhibitor, suggesting cGMP independent signaling. Consistent with an oxidative signaling pathway requiring hydrogen peroxide (H(2)O(2))/heme-peroxidase/NO(2)· signaling, the effects of nitrite were potentiated by superoxide dismutase (SOD) and low concentration H(2)O(2), whereas inhibited completely by catalase, followed by downstream extracellular-signal-regulated kinase (ERK) 1/2 activation. Our data represent the first description of normoxic nitrite signaling on lung epithelial cell proliferation and wound healing and suggest novel oxidative signaling pathways involving nitrite-H(2)O(2) reactions, possibly via the intermediary, NO(2)·.

Nitrite and Heme Globins: Reaction Mechanisms and Physiological Targets

Publisher Summary This chapter focuses on the mechanism and implications of hemoglobin-dependent nitrite reduction. It outlines the chemistry of this reaction, its subcellular targets, and the physiological implications of nitrite reduction. Nitrite is a dynamic endocrine storage form of NO and hemoglobin plays an integral role in reducing it to bioavailable NO. While the chemistry of hemoglobin- and myoglobin-dependent nitrite reduction is the same, the structural and redox properties of these two proteins determine their differing reaction kinetics. In addition, the physiological targets of these reactions differ due to their localization in different biological compartments. While hemoglobin-mediated nitrite reduction is responsible for hypoxic vasodilation, mitochondria are a major target for myoglobin-dependent nitrite reduction. Nitrite is an important mediator of many physiological responses. The family of hemoglobin proteins – including hemoglobin, neuroglobin, and myoglobin – play a central role in the bioactivation of nitrite by reacting with the molecule to reduce it to bioavailable NO under physiological and pathological hypoxic conditions. Hemoglobin- and myoglobin-dependent nitrite reduction to NO has been hypothesized to contribute to fundamental physiological responses, including hypoxic vasodilation and the regulation of hypoxic mitochondrial function. Future studies will determine whether neuroglobin and cytoglobin act as functional nitrite reductases in vivo and whether the nitrite reductase activity of all these globins can be harnessed for therapeutic applications.Publisher Summary This chapter focuses on the mechanism and implications of hemoglobin-dependent nitrite reduction. It outlines the chemistry of this reaction, its subcellular targets, and the physiological implications of nitrite reduction. Nitrite is a dynamic endocrine storage form of NO and hemoglobin plays an integral role in reducing it to bioavailable NO. While the chemistry of hemoglobin- and myoglobin-dependent nitrite reduction is the same, the structural and redox properties of these two proteins determine their differing reaction kinetics. In addition, the physiological targets of these reactions differ due to their localization in different biological compartments. While hemoglobin-mediated nitrite reduction is responsible for hypoxic vasodilation, mitochondria are a major target for myoglobin-dependent nitrite reduction. Nitrite is an important mediator of many physiological responses. The family of hemoglobin proteins – including hemoglobin, neuroglobin, and myoglobin – play a central role in the bioactivation of nitrite by reacting with the molecule to reduce it to bioavailable NO under physiological and pathological hypoxic conditions. Hemoglobin- and myoglobin-dependent nitrite reduction to NO has been hypothesized to contribute to fundamental physiological responses, including hypoxic vasodilation and the regulation of hypoxic mitochondrial function. Future studies will determine whether neuroglobin and cytoglobin act as functional nitrite reductases in vivo and whether the nitrite reductase activity of all these globins can be harnessed for therapeutic applications.

Nitrite and Heme Globins

Publisher Summary This chapter focuses on the mechanism and implications of hemoglobin-dependent nitrite reduction. It outlines the chemistry of this reaction, its subcellular targets, and the physiological implications of nitrite reduction. Nitrite is a dynamic endocrine storage form of NO and hemoglobin plays an integral role in reducing it to bioavailable NO. While the chemistry of hemoglobin- and myoglobin-dependent nitrite reduction is the same, the structural and redox properties of these two proteins determine their differing reaction kinetics. In addition, the physiological targets of these reactions differ due to their localization in different biological compartments. While hemoglobin-mediated nitrite reduction is responsible for hypoxic vasodilation, mitochondria are a major target for myoglobin-dependent nitrite reduction. Nitrite is an important mediator of many physiological responses. The family of hemoglobin proteins – including hemoglobin, neuroglobin, and myoglobin – play a central role in the bioactivation of nitrite by reacting with the molecule to reduce it to bioavailable NO under physiological and pathological hypoxic conditions. Hemoglobin- and myoglobin-dependent nitrite reduction to NO has been hypothesized to contribute to fundamental physiological responses, including hypoxic vasodilation and the regulation of hypoxic mitochondrial function. Future studies will determine whether neuroglobin and cytoglobin act as functional nitrite reductases in vivo and whether the nitrite reductase activity of all these globins can be harnessed for therapeutic applications.Publisher Summary This chapter focuses on the mechanism and implications of hemoglobin-dependent nitrite reduction. It outlines the chemistry of this reaction, its subcellular targets, and the physiological implications of nitrite reduction. Nitrite is a dynamic endocrine storage form of NO and hemoglobin plays an integral role in reducing it to bioavailable NO. While the chemistry of hemoglobin- and myoglobin-dependent nitrite reduction is the same, the structural and redox properties of these two proteins determine their differing reaction kinetics. In addition, the physiological targets of these reactions differ due to their localization in different biological compartments. While hemoglobin-mediated nitrite reduction is responsible for hypoxic vasodilation, mitochondria are a major target for myoglobin-dependent nitrite reduction. Nitrite is an important mediator of many physiological responses. The family of hemoglobin proteins – including hemoglobin, neuroglobin, and myoglobin – play a central role in the bioactivation of nitrite by reacting with the molecule to reduce it to bioavailable NO under physiological and pathological hypoxic conditions. Hemoglobin- and myoglobin-dependent nitrite reduction to NO has been hypothesized to contribute to fundamental physiological responses, including hypoxic vasodilation and the regulation of hypoxic mitochondrial function. Future studies will determine whether neuroglobin and cytoglobin act as functional nitrite reductases in vivo and whether the nitrite reductase activity of all these globins can be harnessed for therapeutic applications.

Chapter 19 – Nitrite and Heme Globins: Reaction Mechanisms and Physiological Targets

Publisher Summary This chapter focuses on the mechanism and implications of hemoglobin-dependent nitrite reduction. It outlines the chemistry of this reaction, its subcellular targets, and the physiological implications of nitrite reduction. Nitrite is a dynamic endocrine storage form of NO and hemoglobin plays an integral role in reducing it to bioavailable NO. While the chemistry of hemoglobin- and myoglobin-dependent nitrite reduction is the same, the structural and redox properties of these two proteins determine their differing reaction kinetics. In addition, the physiological targets of these reactions differ due to their localization in different biological compartments. While hemoglobin-mediated nitrite reduction is responsible for hypoxic vasodilation, mitochondria are a major target for myoglobin-dependent nitrite reduction. Nitrite is an important mediator of many physiological responses. The family of hemoglobin proteins – including hemoglobin, neuroglobin, and myoglobin – play a central role in the bioactivation of nitrite by reacting with the molecule to reduce it to bioavailable NO under physiological and pathological hypoxic conditions. Hemoglobin- and myoglobin-dependent nitrite reduction to NO has been hypothesized to contribute to fundamental physiological responses, including hypoxic vasodilation and the regulation of hypoxic mitochondrial function. Future studies will determine whether neuroglobin and cytoglobin act as functional nitrite reductases in vivo and whether the nitrite reductase activity of all these globins can be harnessed for therapeutic applications.Publisher Summary This chapter focuses on the mechanism and implications of hemoglobin-dependent nitrite reduction. It outlines the chemistry of this reaction, its subcellular targets, and the physiological implications of nitrite reduction. Nitrite is a dynamic endocrine storage form of NO and hemoglobin plays an integral role in reducing it to bioavailable NO. While the chemistry of hemoglobin- and myoglobin-dependent nitrite reduction is the same, the structural and redox properties of these two proteins determine their differing reaction kinetics. In addition, the physiological targets of these reactions differ due to their localization in different biological compartments. While hemoglobin-mediated nitrite reduction is responsible for hypoxic vasodilation, mitochondria are a major target for myoglobin-dependent nitrite reduction. Nitrite is an important mediator of many physiological responses. The family of hemoglobin proteins – including hemoglobin, neuroglobin, and myoglobin – play a central role in the bioactivation of nitrite by reacting with the molecule to reduce it to bioavailable NO under physiological and pathological hypoxic conditions. Hemoglobin- and myoglobin-dependent nitrite reduction to NO has been hypothesized to contribute to fundamental physiological responses, including hypoxic vasodilation and the regulation of hypoxic mitochondrial function. Future studies will determine whether neuroglobin and cytoglobin act as functional nitrite reductases in vivo and whether the nitrite reductase activity of all these globins can be harnessed for therapeutic applications.