Nitrite Reductase Activity of Nonsymbiotic Hemoglobins from Arabidopsis thaliana

Plant nonsymbiotic hemoglobins possess hexacoordinate heme geometry similar to that of the heme protein neuroglobin. We recently discovered that deoxygenated neuroglobin converts nitrite to nitric oxide (NO), an important signaling molecule involved in many processes in plants. We sought to determine whether Arabidopsis thaliana nonsymbiotic hemoglobins classes 1 and 2 (AHb1 and AHb2, respectively) might function as nitrite reductases. We found that the reaction of nitrite with deoxygenated AHb1 and AHb2 generates NO gas and iron-nitrosyl-hemoglobin species. The bimolecular rate constants for reduction of nitrite to NO are 19.8 ± 3.2 and 4.9 ± 0.2 M(-1) s(-1), respectively, at pH 7.4 and 25 °C. We determined the pH dependence of these bimolecular rate constants and found a linear correlation with the concentration of protons, indicating the requirement for one proton in the reaction. The release of free NO gas during the reaction under anoxic and hypoxic (2% oxygen) conditions was confirmed by chemiluminescence detection. These results demonstrate that deoxygenated AHb1 and AHb2 reduce nitrite to form NO via a mechanism analogous to that observed for hemoglobin, myoglobin, and neuroglobin. Our findings suggest that during severe hypoxia and in the anaerobic plant roots, especially in species submerged in water, nonsymbiotic hemoglobins provide a viable pathway for NO generation via nitrite reduction.     

Characterization of the Mechanism and Magnitude of Cytoglobin-mediated Nitrite Reduction and Nitric Oxide Generation under Anaerobic Conditions

Cytoglobin (Cygb) is a recently discovered cytoplasmic heme-binding globin. Although multiple hemeproteins have been reported to function as nitrite reductases in mammalian cells, it is unknown whether Cygb can also reduce nitrite to nitric oxide (NO). The mechanism, magnitude, and quantitative importance of Cygb-mediated nitrite reduction in tissues have not been reported. To investigate this pathway and its quantitative importance, EPR spectroscopy, spectrophotometric measurements, and chemiluminescence NO analyzer studies were performed. Under anaerobic conditions, mixing nitrite with ferrous-Cygb triggered NO formation that was trapped and detected using EPR spin trapping. Spectrophotometric studies revealed that nitrite binding to ferrous-Cygb is followed by formation of ferric-Cygb and NO. The kinetics and magnitude of Cygb-mediated NO formation were characterized. It was observed that Cygb-mediated NO generation increased linearly with the increase of nitrite concentration under anaerobic conditions. This Cygb-mediated NO production greatly increased with acidosis and near-anoxia as occur in ischemic conditions. With the addition of nitrite, soluble guanylyl cyclase activation was significantly higher in normal smooth muscle cells compared with Cygb knocked down cells with Cygb accounting for ∼40% of the activation in control cells and ∼60% in cells subjected to hypoxia for 48 h. Overall, these studies show that Cygb-mediated nitrite reduction can play an important role in NO generation and soluble guanylyl cyclase activation under hypoxic conditions, with this process regulated by pH, oxygen tension, nitrite concentration, and the redox state of the cells.     

The emerging roles of nitric oxide (NO) in plant mitochondria

In recent years nitric oxide (NO) has been recognized as an important signal molecule in plants. Both, reductive and oxidative pathways and different subcellular compartments appear involved in NO production. The reductive pathway uses nitrite as substrate, which is exclusively generated by cytosolic nitrate reductase (NR) and can be converted to NO by the same enzyme. The mitochondrial electron transport chain is another site for nitrite to NO reduction, operating specifically when the normal electron acceptor, O₂, is low or absent. Under these conditions, the mitochondrial NO production contributes to hypoxic survival by maintaining a minimal ATP formation. In contrast, excessive NO production and concomitant nitrosative stress may be prevented by the operation of NO-scavenging mechanisms in mitochondria and cytosol. During pathogen attacks, mitochondrial NO serves as a nitrosylating agent promoting cell death; whereas in symbiotic interactions as in root nodules, the turnover of mitochondrial NO helps in improving the energy status similarly as under hypoxia/anoxia. The contribution of NO turnover during pathogen defense, symbiosis and hypoxic stress is discussed in detail.     

The role of nitrite in nitric oxide homeostasis: A comparative perspective

Nitrite is endogenously produced as an oxidative metabolite of nitric oxide, but it also functions as a NO donor that can be activated by a number of cellular proteins under hypoxic conditions. This article discusses the physiological role of nitrite and nitrite-derived NO in blood flow regulation and cytoprotection from a comparative viewpoint, with focus on mammals and fish. Constitutive nitric oxide synthase activity results in similar plasma nitrite levels in mammals and fish, but nitrite can also be taken up across the gills in freshwater fish, which has implications for nitrite/NO levels and nitrite utilization in hypoxia. The nitrite reductase activity of deoxyhemoglobin is a major mechanism of NO generation from nitrite and may be involved in hypoxic vasodilation. Nitrite is readily transported across the erythrocyte membrane, and the transport is enhanced at low O₂ saturation in some species. Also, nitrite preferentially reacts with deoxyhemoglobin rather than oxyhemoglobin at intermediate O₂ saturations. The hemoglobin nitrite reductase activity depends on heme O₂ affinity and redox potential and shows species differences within mammals and fish. The NO forming capacity is elevated in hypoxia-tolerant species. Nitrite-induced vasodilation is well documented, and many studies support a role of erythrocyte/hemoglobin-derived NO. Vasodilation can, however, also originate from nitrite reduction within the vessel wall, and at present there is no consensus regarding the relative importance of competing mechanisms. Nitrite reduction to NO provides cytoprotection in tissues during ischemia-reperfusion events by inhibiting mitochondrial respiration and limiting reactive oxygen species. It is argued that the study of hypoxia-tolerant lower vertebrates and diving mammals may help evaluate mechanisms and a full understanding of the physiological role of nitrite.     

The haemoglobin/nitric oxide cycle: involvement in flooding stress and effects on hormone signalling

BACKGROUND: Class 1 haemoglobins (Hbs) are induced in plant cells under hypoxic conditions. They have a high affinity for oxygen, which is two orders of magnitude lower than that of cytochrome oxidase, permitting the utilization of oxygen by the molecule at extremely low oxygen concentrations. Their presence reduces the levels of nitric oxide (NO) that is produced from nitrate ion during hypoxia and improves the redox and energy status of the hypoxic cell. SCOPE: The mechanism by which Hb interacts with NO under hypoxic conditions in plants is examined, and the effects of Hb expression on metabolism and signal transduction are discussed. CONCLUSIONS: The accumulated evidence suggests that a metabolic pathway involving NO and Hb provides an alternative type of respiration to mitochondrial electron transport under limited oxygen. Hb in hypoxic plants acts as part of a soluble, terminal, NO dioxygenase system, yielding nitrate ion from the reaction of oxyHb with NO. NO is mainly formed due to anaerobic accumulation of nitrite. The overall reaction sequence, referred to as the Hb/NO cycle, consumes NADH and maintains ATP levels via an as yet unknown mechanism. Hb gene expression appears to influence signal transduction pathways, possibly through its effect on NO, as evidenced by phenotypic changes in normoxic Hb-varying transgenic plants. Ethylene levels are elevated when Hb gene expression is suppressed, which could be a factor leading to root aerenchyma formation during hypoxic stress.     

Nitrite-dependent vasodilation is facilitated by hypoxia and is independent of known NO-generating nitrite reductase activities

The reduction of circulating nitrite to nitric oxide (NO) has emerged as an important physiological reaction aimed to increase vasodilation during tissue hypoxia. Although hemoglobin, xanthine oxidase, endothelial NO synthase, and the bc(1) complex of the mitochondria are known to reduce nitrite anaerobically in vitro, their relative contribution to the hypoxic vasodilatory response has remained unsolved. Using a wire myograph, we have investigated how the nitrite-dependent vasodilation in rat aortic rings is controlled by oxygen tension, norepinephrine concentration, soluble guanylate cyclase (the target for vasoactive NO), and known nitrite reductase activities under hypoxia. Vasodilation followed overall first-order dependency on nitrite concentration and, at low oxygenation and norepinephrine levels, was induced by low-nitrite concentrations, comparable to those found in vivo. The vasoactive effect of nitrite during hypoxia was abolished on inhibition of soluble guanylate cyclase and was unaffected by removal of the endothelium or by inhibition of xanthine oxidase and of the mitochondrial bc(1) complex. In the presence of hemoglobin and inositol hexaphosphate (which increases the fraction of deoxygenated heme), the effect of nitrite was not different from that observed with inositol hexaphosphate alone, indicating that under the conditions investigated here deoxygenated hemoglobin did not enhance nitrite vasoactivity. Together, our results indicate that the mechanism for nitrite vasorelaxation is largely intrinsic to the vessel and that under hypoxia physiological nitrite concentrations are sufficient to induce NO-mediated vasodilation independently of the nitrite reductase activities investigated here. Possible reaction mechanisms for nitrite vasoactivity, including formation of S-nitrosothiols within the arterial smooth muscle, are discussed.     

Platelet inhibition by nitrite is dependent on erythrocytes and deoxygenation

BACKGROUND: Nitrite is a nitric oxide (NO) metabolite in tissues and blood, which can be converted to NO under hypoxia to facilitate tissue perfusion. Although nitrite is known to cause vasodilation following its reduction to NO, the effect of nitrite on platelet activity remains unclear. In this study, the effect of nitrite and nitrite+erythrocytes, with and without deoxygenation, on platelet activity was investigated. METHODOLOGY/FINDING: Platelet aggregation was studied in platelet-rich plasma (PRP) and PRP+erythrocytes by turbidimetric and impedance aggregometry, respectively. In PRP, DEANONOate inhibited platelet aggregation induced by ADP while nitrite had no effect on platelets. In PRP+erythrocytes, the inhibitory effect of DEANONOate on platelets decreased whereas nitrite at physiologic concentration (0.1 μM) inhibited platelet aggregation and ATP release. The effect of nitrite+erythrocytes on platelets was abrogated by C-PTIO (a membrane-impermeable NO scavenger), suggesting an NO-mediated action. Furthermore, deoxygenation enhanced the effect of nitrite as observed from a decrease of P-selectin expression and increase of the cGMP levels in platelets. The ADP-induced platelet aggregation in whole blood showed inverse correlations with the nitrite levels in whole blood and erythrocytes. CONCLUSION: Nitrite alone at physiological levels has no effect on platelets in plasma. Nitrite in the presence of erythrocytes inhibits platelets through its reduction to NO, which is promoted by deoxygenation. Nitrite may have role in modulating platelet activity in the circulation, especially during hypoxia.     

The Capacity of Red Blood Cells to Reduce Nitrite Determines Nitric Oxide Generation under Hypoxic Conditions

Nitric oxide (NO) is a key regulator of vascular tone. Endothelial nitric oxide synthase (eNOS) is responsible for NO generation under normoxic conditions. Under hypoxia however, eNOS is inactive and red blood cells (RBC) provide an alternative NO generation pathway from nitrite to regulate hypoxic vasodilation. While nitrite reductase activity of hemoglobin is well acknowledged, little is known about generation of NO by intact RBC with physiological hemoglobin concentrations. We aimed to develop and apply a new approach to provide insights in the ability of RBC to convert nitrite into NO under hypoxic conditions. We established a novel experimental setup to evaluate nitrite uptake and the release of NO from RBC into the gas-phase under different conditions. NO measurements were similar to well-established clinical measurements of exhaled NO. Nitrite uptake was rapid, and after an initial lag phase NO release from RBC was constant in time under hypoxic conditions. The presence of oxygen greatly reduced NO release, whereas inhibition of eNOS and xanthine oxidoreductase (XOR) did not affect NO release. A decreased pH increased NO release under hypoxic conditions. Hypothermia lowered NO release, while hyperthermia increased NO release. Whereas fetal hemoglobin did not alter NO release compared to adult hemoglobin, sickle RBC showed an increased ability to release NO. Under all conditions nitrite uptake by RBC was similar. This study shows that nitrite uptake into RBC is rapid and release of NO into the gas-phase continues for prolonged periods of time under hypoxic conditions. Changes in the RBC environment such as pH, temperature or hemoglobin type, affect NO release.     

Involvement of nitrite in the nitrate-mediated modulation of fermentative metabolism and nitric oxide production of soybean roots during hypoxia

It is widely accepted that nitrate but not ammonium improves tolerance of plants to hypoxic stress, although the mechanisms related to this beneficial effect are not well understood. Recently, nitrite derived from nitrate reduction has emerged as the major substrate for the synthesis of nitric oxide (NO), an important signaling molecule in plants. Here, we analyzed the effect of different nitrogen sources (nitrate, nitrite and ammonium) on the metabolic response and NO production of soybean roots under hypoxia. Organic acid analysis showed that root segments isolated from nitrate-cultivated plants presented a lower accumulation of lactate and succinate in response to oxygen deficiency in relation to those from ammonium-cultivated plants. The more pronounced lactate accumulation by root segments of ammonium-grown plants was followed by a higher ethanol release in the medium, evidencing a more intense fermentation under oxygen deficiency than those from nitrate-grown plants. As expected, root segments from nitrate-cultivated plants produced higher amounts of nitrite and NO during hypoxia compared to ammonium cultivation. Exogenous nitrite supplied during hypoxia reduced both ethanol and lactate production and stimulated cyanide-sensitive NO emission by root segments from ammonium-cultivated plants, independent of nitrate. On the other hand, treatments with a NO donor or a NO scavenger did not affect the intensity of fermentation of soybean roots. Overall, these results indicate that nitrite participates in the nitrate-mediated modulation of the fermentative metabolism of soybean roots during oxygen deficiency. The involvement of mitochondrial reduction of nitrite to NO in this mechanism is discussed.     

Characterization of the magnitude and kinetics of xanthine oxidase-catalyzed nitrite reduction. Evaluation of its role in nitric oxide generation in anoxic tissues

Xanthine oxidase (XO)-catalyzed nitrite reduction with nitric oxide (NO) production has been reported to occur under anaerobic conditions, but questions remain regarding the magnitude, kinetics, and biological importance of this process. To characterize this mechanism and its quantitative importance in biological systems, electron paramagnetic resonance spectroscopy, chemiluminescence NO analyzer, and NO electrode studies were performed. The XO reducing substrates xanthine, NADH, and 2,3-dihydroxybenz-aldehyde triggered nitrite reduction to NO, and the molybdenum-binding XO inhibitor oxypurinol inhibited this NO formation, indicating that nitrite reduction occurs at the molybdenum site. However, at higher xanthine concentrations, partial inhibition was seen, suggesting the formation of a substrate-bound reduced enzyme complex with xanthine blocking the molybdenum site. Studies of the pH dependence of NO formation indicated that XO-mediated nitrite reduction occurred via an acid-catalyzed mechanism. Nitrite and reducing substrate concentrations were important regulators of XO-catalyzed NO generation. The substrate dependence of anaerobic XO-catalyzed nitrite reduction followed Michaelis-Menten kinetics, enabling prediction of the magnitude of NO formation and delineation of the quantitative importance of this process in biological systems. It was determined that under conditions occurring during no-flow ischemia, myocardial XO and nitrite levels are sufficient to generate NO levels comparable to those produced from nitric oxide synthase. Thus, XO-catalyzed nitrite reduction can be an important source of NO generation under ischemic conditions.     

Alternative pathways of nitric oxide formation in Lactobacilli: Evidence for nitric oxide synthase activity by EPR

The study of the ability of Lactobacillus plantarum 8P-A3 to synthesize nitric oxide (NO) showed that this strain lacks nitrite reductase. However, analysis by the EPR method revealed the presence of nitric oxide synthase activity in this strain. Like mammalian nitric oxide synthase, lactobacillar NO synthase is involved in the formation of nitric oxide from L-arginine. L. plantarum 8P-A3 does not produce NO in the denitrification process. The regulatory role of NO in symbiotic bacteria is emphasixed.     

New insights into the mitochondrial nitric oxide production pathways

Considerable evidence has appeared over the past few years that nitric oxide (NO) is an important anoxic metabolite and a potent signal molecule in plants. Several pathways operative in different cell compartments, lead to NO production. Mitochondria, being a major NO producing compartment, can generate it by either nitrite reduction occurring at nearly anoxic conditions or by the oxidative route via nitric oxide synthase (NOS). Recently we compared both pathways by ozone collision chemiluminescence and by DAF fluorescence. We found that nitrite reduction to NO is associated with the mitochondrial membrane fraction but not with the matrix. In case of the nitric oxide synthase pathway, an L-arginine dependent fluorescence was detected but its response to NOS inhibitors and substrates was untypical. Therefore the existence of NOS or NOS-like activity in barley root mitochondria is very doubtful. We also found that mitochondria scavenge NO. In addition, we found indirect evidence that mitochondria are able to convert NO to gaseous intermediates like NO₂, N₂O and N₂O₃.Key words: nitrate reductase, nitric oxide synthase, nitric oxide, mitochondria, DAF fluorescenceMitochondria are known as powerhouses of the cell. These organelles harbour the citric acid cycle and electron transport chain. Almost all the eukaryotic mitochondria share these basic functions. In addition to the energy generation, mitochondria are one of the major producers of reactive oxygen species¹ and involved in retrograde signalling.² Recent evidence suggests that mitochondria are one of the major producers of nitric oxide (NO) in plants.^3,4 Since nitric oxide has gained high importance, this novel property of mitochondria stimulated interest in NO signalling research.Eukaryotic mitochondria may produce NO by two distinct pathways. One is an oxidative pathway which uses L-arginine as a substrate and produces NO and citrulline⁷ and the other is a reductive pathway which uses nitrite as a substrate and produces NO at low oxygen conditions.^5,6     

Mitochondrial cytochrome oxidase produces nitric oxide under hypoxic conditions: implications for oxygen sensing and hypoxic signaling in eukaryotes

Eukaryotic cells respond to low-oxygen concentrations by upregulating hypoxic nuclear genes (hypoxic signaling). Although it has been shown previously that the mitochondrial respiratory chain is required for hypoxic signaling, its underlying role in this process has been unclear. Here, we find that yeast and rat liver mitochondria produce nitric oxide (NO) at dissolved oxygen concentrations below 20 microM. This NO production is nitrite (NO2-) dependent, requires an electron donor, and is carried out by cytochrome c oxidase in a pH-dependent fashion. Mitochondrial NO production in yeast is influenced by the YHb flavohemoglobin NO oxidoreductase, stimulates expression of the hypoxic nuclear gene CYC7, and is accompanied by an increase in protein tyrosine nitration. These findings demonstrate an alternative role for the mitochondrial respiratory chain under hypoxic or anoxic conditions and suggest that mitochondrially produced NO is involved in hypoxic signaling, possibly via a pathway that involves protein tyrosine nitration.     

Production and storage of nitric oxide in adaptation to hypoxia.

Adaptation to hypobaric hypoxia is known to exert multiple protective effects related with nitric oxide (NO). However the effect of adaptation to hypoxia on NO metabolism has remained unclear in many respects. In the present work we studied the interrelation between NO production and storage in the process of adaptation to hypoxia. The NO production was determined by the total nitrite/nitrate concentration in rats plasma. The volume of NO store was evaluated in vitro by the magnitude of isolated aorta relaxation to diethyldithiocarbamate. It was shown that both the nitrite/nitrate level and the NO store increased as adaptation to hypoxia developed. Furthermore, the NO store volume significantly correlated with plasma nitrite/nitrate. Therefore, adaptation to hypoxia stimulates NO production and storage and these effects can potentially underlie NO-dependent beneficial effects of adaptation.     

Dramatic increase of nitrite levels in hearts of anoxia-exposed crucian carp supporting a role in cardioprotection

Nitrite (NO(2)(-)) functions as an important nitric oxide (NO) donor under hypoxic conditions. Both nitrite and NO have been found to protect the mammalian heart and other tissues against ischemia (anoxia)-reoxygenation injury by interacting with mitochondrial electron transport complexes and limiting the generation of reactive oxygen species upon reoxygenation. The crucian carp naturally survives extended periods without oxygen in an active state, which has made it a model for studying how evolution has solved the problems of anoxic survival. We investigated the role of nitrite and NO in the anoxia tolerance of this fish by measuring NO metabolites in normoxic, anoxic, and reoxygenated crucian carp. We also cloned and sequenced crucian carp NO synthase variants and quantified their mRNA levels in several tissues in normoxia and anoxia. Despite falling levels of blood plasma nitrite, the crucian carp showed massive increases in nitrite, S-nitrosothiols (SNO), and iron-nitrosyl (FeNO) compounds in anoxic heart tissue. NO(2)(-) levels were maintained in anoxic brain, liver, and gill tissues, whereas SNO and FeNO increased in a tissue-specific manner. Reoxygenation reestablished normoxic values. We conclude that NO(2)(-) is shifted into the tissues where it acts as NO donor during anoxia, inducing cytoprotection under anoxia/reoxygenation. This can be especially important in the crucian carp heart, which maintains output in anoxia. NO(2)(-) is currently tested as a therapeutic drug against reperfusion damage of ischemic hearts, and the present study provides evolutionary precedent for such an approach.     

Red blood cells prevent inhibition of hypoxic pulmonary vasoconstriction by nitrite in isolated, perfused rat lungs

Nitrite reduction to nitric oxide (NO) may be potentiated by a nitrite reductase activity of deoxyHb and contribute to systemic hypoxic vasodilation. The effect of nitrite on the pulmonary circulation has not been well characterized. We explored the effect of nitrite on hypoxic pulmonary vasoconstriction (HPV) and the role of the red blood cell (RBC) in nitrite reduction and nitrite-mediated vasodilation. As to method, isolated rat lungs were perfused with buffer, or buffer with RBCs, and subjected to repeated hypoxic challenges, with or without nitrite. As a result, in buffer-perfused lungs, HPV was reduced at nitrite concentrations of 7 muM and above. Nitrite inhibition of HPV was prevented by excess free Hb and RBCs, suggesting that vasodilation was mediated by free NO. Nitrite-inhibition of HPV was not potentiated by mild acidosis (pH = 7.2) or xanthine oxidase activity. RBCs at 15% but not 1% hematocrit prevented inhibition of HPV by nitrite (maximum nitrite concentration of approximately 35 muM) independent of perfusate Po(2). Degradation of nitrite was accelerated by hypoxia in the presence of RBCs but not during buffer perfusion. In conclusion, low micromolar concentrations of nitrite inhibit HPV in buffer-perfused lungs and when RBC concentration is subphysiological. This effect is lost when RBC concentration approaches physiological levels, despite enhanced nitrite degradation in the presence of RBCs. These data suggest that, although deoxyHb may generate NO from nitrite, insufficient NO escapes the RBC to cause vasodilation in the pulmonary circulation under the dynamic conditions of blood flow through the lungs and that RBCs are net scavengers of NO.     

Nitric oxide emission from tobacco leaves and cell suspensions: rate limiting factors and evidence for the involvement of mitochondrial electron transport

Quantitative data on nitric oxide (NO) production by plants, and knowledge of participating reactions and rate limiting factors are still rare. We quantified NO emission from tobacco (Nicotiana tabacum) wild-type leaves, from nitrate reductase (NR)- or nitrite reductase (NiR)-deficient leaves, from WT- or from NR-deficient cell suspensions and from mitochondria purified from leaves or cells, by following NO emission through chemiluminescence detection. In all systems, NO emission was exclusively due to the reduction of nitrite to NO, and the nitrite concentration was an important rate limiting factor. Using inhibitors and purified mitochondria, mitochondrial electron transport was identified as a major source for reduction of nitrite to NO, in addition to NR. NiR and xanthine dehydrogenase appeared to be not involved. At equal respiratory activity, mitochondria from suspension cells had a much higher capacity to produce NO than leaf mitochondria. NO emission in vivo by NiR-mutant leaves (which was not nitrite limited) was proportional to photosynthesis (high in light +CO(2), low in light -CO(2), or in the dark). With most systems including mitochondrial preparations, NO emission was low in air (and darkness for leaves), but high under anoxia (nitrogen). In contrast, NO emission by purified NR was not much different in air and nitrogen. The low aerobic NO emission of darkened leaves and cell suspensions was not due to low cytosolic NADH, and appeared only partly affected by oxygen-dependent NO scavenging. The relative contribution of NR and mitochondria to nitrite-dependent NO production is estimated.     

Assessment of the functional diversity of human myoglobin

Myoglobin is presumably the most studied protein in biology. Its functional properties as a dioxygen storage and facilitator of dioxygen transport are widely acknowledged. Experimental evidence also implicates an essential role for myoglobin in the heart in regulating nitric oxide homeostasis. Under normoxia, oxygenated myoglobin can scavenge excessive nitric oxide, while under hypoxia, deoxygenated myoglobin can reduce nitrite, an oxidative product of nitric oxide, to bioactive nitric oxide. Myoglobin-driven nitrite reduction can protect the heart from ischemia and reperfusion injury. While horse and mouse myoglobin have been previously described to reduce nitrite under these conditions, a comparable activity has not been detected in human myoglobin. We here show that human myoglobin is a fully functional nitrite reductase. To study the role of human myoglobin for nitric oxide homeostasis we used repeated photometric wavelength scans and chemiluminescence based experiments. The results revealed that oxygenated human myoglobin reacts with nitrite-derived nitric oxide to form ferric myoglobin and that deoxygenated human myoglobin acts as a nitrite reductase in vitro and in situ. Rates of both nitric oxide scavenging and nitrite reduction were significantly higher in human compared to horse myoglobin. These data extend the existing knowledge about the functional properties of human myoglobin and are the basis for further translational studies towards the importance of myoglobin for nitric oxide metabolism in humans.