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 ϳ2000 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-tofive-coordinate heme pocket control. We hypothesize that the sixcoordinate heme globin superfamily may subserve a function as primordial hypoxic and redox-regulated NO-signaling proteins.

Nitrite reacts with deoxyhemoglobin to form nitric oxide (NO) and methemoglobin. Though this reaction is experimentally associated with NO generation and vasodilation, kinetic analysis suggests that NO should not be able to escape inactivation in the erythrocyte. We have discovered that products of the nitrite-hemoglobin reaction generate dinitrogen trioxide (N2O3) via a novel reaction of NO and nitrite-bound methemoglobin. The oxygen-bound form of nitrite-methemoglobin shows a degree of ferrous nitrogen dioxide (Fe(II)-NO2*) character, so it may rapidly react with NO to form N2O3. N2O3 partitions in lipid, homolyzes to NO and readily nitrosates thiols, all of which are common pathways for NO escape from the erythrocyte. These results reveal a fundamental heme globin- and nitrite-catalyzed chemical reaction pathway to N2O3, NO and S-nitrosothiol that could form the basis of in vivo nitrite-dependent signaling. Because the reaction redox-cycles (that is, regenerates ferrous heme) and the nitrite-methemoglobin intermediate is not observable by electron paramagnetic resonance spectroscopy, this reaction has been 'invisible' to experimentalists over the last 100 years.

The reaction rate between nitric oxide and intraerythrocytic hemoglobin plays a major role in nitric oxide bioavailability and modulates homeostatic vascular function. It has previously been demonstrated that the encapsulation of hemoglobin in red blood cells restricts its ability to scavenge nitric oxide. This effect has been attributed to either factors intrinsic to the red blood cell such as a physical membrane barrier or factors external to the red blood cell such as the formation of an unstirred layer around the cell. We have performed measurements of the uptake rate of nitric oxide by red blood cells under oxygenated and deoxygenated conditions at different hematocrit percentages. Our studies include stopped-flow measurements where both the unstirred layer and physical barrier potentially participate, as well as competition experiments where the potential contribution of the unstirred layer is limited. We find that deoxygenated erythrocytes scavenge nitric oxide faster than oxygenated cells and that the rate of nitric oxide scavenging for oxygenated red blood cells increases as the hematocrit is raised from 15% to 50%. Our results 1) confirm the critical biological phenomenon that hemoglobin compartmentalization within the erythrocyte reduces reaction rates with nitric oxide, 2) show that extraerythocytic diffusional barriers mediate most of this effect, and 3) provide novel evidence that an oxygen-dependent intrinsic property of the red blood cell contributes to this barrier activity, albeit to a lesser extent. These observations may have important physiological implications within the microvasculature and for pathophysiological disruption of nitric oxide homeostasis in diseases. Nitric oxide (NO)3 is an endothelium-derived relaxation factor that is synthesized in endothelial cells (1-4). To elicit its vasodilatory activity, NO must diffuse to the smooth muscle cells and activate soluble guanylate cyclase. In 1994, Lancaster suggested that the proximity of the endothelium to the millimolar concentrations of hemoglobin (Hb), an avid NO scavenger, would severely compromise the efficiency of the NO/soluble guanylate cyclase pathway (5). However, later studies have indicated that the physical compartmentalization of hemoglobin within the red blood cell (RBC) effectively reduces the apparent rate at which NO is consumed by Hb (6 -15). One contributory element to this effect is a RBC-free zone at the blood/endothelium interface that is present during laminar flow (7, 9, 10). In addition, the rate of NO consumption has been reported to occur up to 1000 times more slowly by red blood cells than by an equivalent concentration of cell-free hemoglobin. Two potential mechanisms for this effect involve either the presence of an unstirred layer surrounding the red blood cell that is formed as a result of NO diffusion (6, 13) or a physical barrier to NO diffusion that is integral to the protein-rich RBC submembrane (11). The faster effective reaction of NO with cell-free Hb compared with RBC-encapsulated hemoglobin may ...

Recent studies reveal a novel role for hemoglobin as an allosterically regulated nitrite reductase that may mediate nitric oxide (NO)-dependent signaling along the physiological oxygen gradient. Nitrite reacts with deoxyhemoglobin in an allosteric reaction that generates NO and oxidizes deoxyhemoglobin to methemoglobin. NO then reacts at a nearly diffusion-limited rate with deoxyhemoglobin to form iron-nitrosyl-hemoglobin, which to date has been considered a highly stable adduct and, thus, not a source of bioavailable NO. However, under physiological conditions of partial oxygen saturation, nitrite will also react with oxyhemoglobin, and although this complex autocatalytic reaction has been studied for a century, the interaction of the oxy-and deoxy-reactions and the effects on NO disposition have never been explored. We have now characterized the kinetics of hemoglobin oxidation and NO generation at a range of oxygen partial pressures and found that the deoxy-reaction runs in parallel with and partially inhibits the oxy-reaction. In fact, intermediates in the oxy-reaction oxidize the heme iron of ironnitrosyl-hemoglobin, a product of the deoxy-reaction, which releases NO from the iron-nitrosyl. This oxidative denitrosylation is particularly striking during cycles of hemoglobin deoxygenation and oxygenation in the presence of nitrite. These chemistries may contribute to the oxygen-dependent disposition of nitrite in red cells by limiting oxidative inactivation of nitrite by oxyhemoglobin, promoting nitrite reduction to NO by deoxyhemoglobin, and releasing free NO from iron-nitrosyl-hemoglobin.

Objective To test whether a relatively low single dose or week-long dosing of dietary inorganic nitrate can improve exercise tolerance in patients with Heart Failure with preserved ejection fraction (HFPEF). Background Exercise intolerance is the primary manifestation of HFPEF and is largely due to non-cardiac factors that reduce oxygen delivery to active skeletal muscles. A recent study showed improved exercise capacity in patients with HFPEF after a single, acute dose of beetroot juice (BRJ, 12.9 mmol inorganic nitrate) while another recent study showed neutral and negative effects of an organic nitrate. Methods Twenty HFPEF patients (age: 69 ± 7 years) were enrolled in an initial cross-over design comparing a single, acute dose of BRJ (6.1 mmol nitrate) to a nitrate-depleted, placebo BRJ. A second, one week of daily dosing, phase employed an all-treated design in which patients consumed BRJ for an average of 7 days. The primary outcome of the study was submaximal aerobic endurance, measured as cycling time to exhaustion at 75% of measured maximal power output. Results No adverse events were associated with the intervention. Submaximal aerobic endurance improved 24% after one week of daily BRJ dosing (p =0.02), but was not affected by the single, acute dose of the BRJ compared to placebo. Consumption of BRJ significantly reduced resting systolic blood pressure and increased plasma nitrate and nitrite in both dosing schemes. Conclusions One week of daily dosing with BRJ (6.1 mmol inorganic nitrate) significantly improves submaximal aerobic endurance and blood pressure in elderly HFPEF patients.

Dietary nitrate (NO3−) supplementation via beetroot juice has been shown to increase the exercise capacity of younger and older adults. The purpose of this study was to investigate the effects of acute NO3− ingestion on the submaximal constant work rate exercise capacity of COPD patients. Fifteen patients were assigned in a randomized, single-blind, crossover design to receive one of two treatments (beetroot juice then placebo or placebo then beetroot juice). Submaximal constant work rate exercise time at 75% of the patient’s maximal work capacity was the primary outcome. Secondary outcomes included plasma NO3− and nitrite (NO2−) levels, blood pressure, heart rate, oxygen consumption (VO2), dynamic hyperinflation, dyspnea and leg discomfort. Relative to placebo, beetroot ingestion increased plasma NO3− by 938% and NO2− by 379%. Median (+ interquartile range) exercise time was significantly longer (p = 0.031) following the ingestion of beetroot versus placebo (375.0 + 257.0 vs. 346.2 + 148.0 sec., respectively). Compared to placebo, beetroot ingestion significantly reduced iso-time (p = 0.001) and end exercise (p = 0.008) diastolic blood pressures by 6.4 and 5.6 mmHg, respectively. Resting systolic blood pressure was significantly reduced (p = 0.019) by 8.2 mmHg for the beetroot versus the placebo trial. No other variables were significantly different between the beetroot and placebo trials. These results indicate that acute dietary NO3− supplementation can elevate plasma NO3− and NO2− concentrations, improve exercise performance, and reduce blood pressure in COPD patients.

Small increases in physiological nitrite concentrations have now been shown to mediate a number of biological responses, including hypoxic vasodilation, cytoprotection after ischemia/ reperfusion, and regulation of gene and protein expression. Thus, while nitrite was until recently believed to be biologically inert, it is now recognized as a potentially important hypoxic signaling molecule and therapeutic agent. Nitrite mediates signaling through its reduction to nitric oxide, via reactions with several heme-containing proteins. In this report, we show for the first time that the mitochondrial electron carrier cytochrome c can also effectively reduce nitrite to NO. This nitrite reductase activity is highly regulated as it is dependent on pentacoordination of the heme iron in the protein and occurs under anoxic and acidic conditions. Further, we demonstrate that in the presence of nitrite, pentacoordinate cytochrome c generates bioavailable NO that is able to inhibit mitochondrial respiration. These data suggest an additional role for cytochrome c as a nitrite reductase that may play an important role in regulating mitochondrial function and contributing to hypoxic, redox, and apoptotic signaling within the cell.

Race, age group, year of discharge, associated complications, and cancer type were significantly associated with risk of longer los and mortality. These factors may potentially help in identifying high-risk patients who might benefit from targeted antibiotic therapy or prophylactic hematopoietic growth factor support.