Nitric oxide binding to oxygenated hemoglobin under physiological conditions.

We have added nitric oxide (NO) to hemoglobin in 0.1 M and 0.01 M phosphate buffers as well as to whole blood, all as a function of hemoglobin oxygen saturation. We found that in all these conditions, the amount of nitrosyl hemoglobin (HbNO) formed follows a model where the rates of HbNO formation and methemoglobin (metHb) formation (via hemoglobin oxidation) are independent of oxygen saturation. These results contradict those of an earlier report where, at least in 0.01 M phosphate, an elevated amount of HbNO was formed at high oxygen saturations. A radical rethink of the reaction of oxyhemoglobin with NO under physiological conditions was called for based on this previous proposition that the primary product is HbNO rather than metHb and nitrate. Our results indicate that no such radical rethink is called for.     

Nitric oxide binding to oxygenated hemoglobin under physiological conditions

We have added nitric oxide (NO) to hemoglobin in 0.1 M and 0.01 M phosphate buffers as well as to whole blood, all as a function of hemoglobin oxygen saturation. We found that in all these conditions, the amount of nitrosyl hemoglobin (HbNO) formed follows a model where the rates of HbNO formation and methemoglobin (metHb) formation (via hemoglobin oxidation) are independent of oxygen saturation. These results contradict those of an earlier report where, at least in 0.01 M phosphate, an elevated amount of HbNO was formed at high oxygen saturations. A radical rethink of the reaction of oxyhemoglobin with NO under physiological conditions was called for based on this previous proposition that the primary product is HbNO rather than metHb and nitrate. Our results indicate that no such radical rethink is called for.     

Nitrosylated Hemoglobin Levels in Human Venous Erythrocytes Correlate with Vascular Endothelial Function Measured by Digital Reactive Hyperemia

Impaired nitric oxide (NO)–dependent endothelial function is associated with the development of cardiovascular diseases. We hypothesized that erythrocyte levels of nitrosylated hemoglobin (HbNO-heme) may reflect vascular endothelial function in vivo. We developed a modified subtraction method using Electron Paramagnetic Resonance (EPR) spectroscopy to identify the 5-coordinate α-HbNO (HbNO) concentration in human erythrocytes and examined its correlation with endothelial function assessed by peripheral arterial tonometry (PAT). Changes in digital pulse amplitude were measured by PAT during reactive hyperemia following brachial arterial occlusion in a group of healthy volunteers (50 subjects). Erythrocyte HbNO levels were measured at baseline and at the peak of hyperemia. We digitally subtracted an individual model EPR signal of erythrocyte free radicals from the whole EPR spectrum to unmask and quantitate the HbNO EPR signals.

Results

Mean erythrocyte HbNO concentration at baseline was 219+/−12 nmol/L (n = 50). HbNO levels and reactive hyperemia (RH) indexes were higher in female (free of contraceptive pills) than male subjects. We observed a dynamic increase of HbNO levels in erythrocytes isolated at 1–2 min of post-occlusion hyperemia (120+/−8% of basal levels); post-occlusion HbNO levels were correlated with basal levels. Both basal and post-occlusion HbNO levels were significantly correlated with reactive hyperemia (RH) indexes (r = 0.58; P<0.0001 for basal HbNO).

Conclusion

The study demonstrates quantitative measurements of 5-coordinate α-HbNO in human venous erythrocytes, its dynamic physiologic regulation and correlation with endothelial function measured by tonometry during hyperemia. This opens the way to further understanding of in vivo determinants of NO bioavailability in human circulation.     

EPR spectroscopy studies on the structural transition of nitrosyl hemoglobin in the arterial-venous cycle of DEANO-treated rats as it relates to the proposed nitrosyl hemoglobin/nitrosothiol hemoglobin exchange

In vivo studies show a dynamic cycle in which alpha-nitrosylated hemoglobin is mainly in the relaxed state in arterial blood of rats treated with 2-(N,N-diethylamino)-diazenolate-2-oxide, but converts mainly to the tense state during the arterial-venous transit. A detailed analysis shows that different electron paramagnetic resonance spectra recorded for alpha-nitrosyl hemoglobin in arterial and venous blood at 77 K originate only from a different ratio between 5- and 6-coordinate heme without any change in the concentration of nitrosyl hemoglobin. In venous blood, the five- and six-coordination equilibrium of the alpha-nitrosyl heme is shifted in favor of the 5-coordinate state (58% venous vs. 20% arterial). These results are not consistent with the recently proposed exchange of nitrosyl heme with the beta-93 nitrosothiol group of hemoglobin during the arterial-venous cycle.     

Electron paramagnetic resonance analysis of nitrosylhemoglobin in humans during NO inhalation

The reactions of nitric oxide with hemoglobin play an important role in explaining the vascular biology of this free radical. It is perhaps surprising that the level of nitrosylhemoglobin (HbNO) in which NO is bound to the ferrous hemoglobin heme in whole human blood under basal and stimulated conditions is a matter of some controversy, with measurements ranging from <1 nm to close to 10 mum. In order to examine HbNO levels in human blood by using EPR spectroscopy, we have developed a regression-based spectral analysis technique that has a detection level of about 200 nm HbNO. We have utilized this methodology to detect the level of HbNO under basal conditions and during NO inhalation. The major findings of this study are as follows. (i) HbNO can be accurately detected and quantified in whole blood with a detection limit of approximately 200 nm. (ii) By using regression analysis, levels of HbNO as low as 0.5-1 mum can be deconvoluted into component species. (iii) HbNO is present at less than 200 nm at basal conditions in both arterial and venous blood and is formed at a level of 0.5-2.5 mum upon inhalation of 80 ppm NO. (iv) The levels of HbNO detected by EPR are remarkably close (within a factor of 2) to those detected by tri-iodide-based chemiluminescence and much smaller than those detected by photolysis chemiluminescence. (v) The half-time of HbNO in vivo is approximately 40 min.     

Mathematical model for the estimation of hemodynamic and oxygenation variables by tissue spectroscopy

This article presents a quasistatic, compartmental model of tissue-level hemodynamics and oxygenation that leads to a set of formulas, which is suitable to calculate important physiological variables from the mean tissue concentration and saturation of hemoglobin, measured by tissue spectroscopy. Dimensioned quantities are represented relative to their baseline value in the equations (relative value = perturbed/baseline). All model parameters are non-dimensional. The model is based and extends on a number of previous works: previous models of similar aim and scope are consolidated, and every critical assumptions and approximations are treated explicitly; extensions include for example the incorporation of the Fahraeus-effect and the separate estimation of the volume changes of the arterial and the venous compartments. The information content of spectroscopic data alone is shown to be valuable, but limited: the relative venous volume, the oxygen extraction fraction and the relative cellulovascular coupling (defined as the ratio of blood flow and oxygen consumption) can be calculated from these data, if the alterations in arterial blood volume are negligible. The number of variables estimated by the derived formulas can be increased if local blood flow is measured simultaneously: in this case, the relative arterial and venous volume and resistance, the oxygen extraction fraction, and the relative oxygen consumption can be determined. Given that this model considers arterial blood pressure, saturation and hematocrit as its inputs, when measured, the model becomes applicable in such conditions as hyper- or hypotension, hypoxic hypoxia, hemodilution and hemorrhage, where these variables do change. The estimation of the changes in arterial resistance can be applied to estimate the extent of an autoregulatory response.     

Attenuated hepatosplanchnic uptake of lactate during intense exercise in humans.

We evaluated whether the increase in blood lactate with intense exercise is influenced by a low hepatosplanchnic blood flow as assessed by indocyanine green dye elimination and blood sampling from an artery and the hepatic vein in eight men. The hepatosplanchnic blood flow decreased from a resting value of 1.6 +/- 0.1 to 0.7 +/- 0.1 (SE) l/min during exercise. Yet the hepatosplanchnic O2 uptake increased from 67 +/- 3 to 93 +/- 13 ml/min, and the output of glucose increased from 1.1 +/- 0.1 to 2.1 +/- 0.3 mmol/min (P < 0.05). Even at the lowest hepatosplanchnic venous hemoglobin O2 saturation during exercise of 6%, the average concentration of glucose in arterial blood was maintained close to the resting level (5.2 +/- 0.2 vs. 5.5 +/- 0.2 mmol/l), whereas the difference between arterial and hepatic venous blood glucose increased to a maximum of 22 mmol/l. In arterial blood, the concentration of lactate increased from 1.1 +/- 0.2 to 6.0 +/- 1.0 mmol/l, and the hepatosplanchnic uptake of lactate was elevated from 0.4 +/- 0.06 to 1.0 +/- 0.05 mmol/min during exercise (P < 0.05). However, when the hepatosplanchnic venous hemoglobin O2 saturation became low, the arterial and hepatosplanchnic venous blood lactate difference approached zero. Even with a marked reduction in its blood flow, exercise did not challenge the ability of the liver to maintain blood glucose homeostasis. However, it appeared that the contribution of the Cori cycle decreased, and the accumulation of lactate in blood became influenced by the reduced hepatosplanchnic blood flow.     

Electron microscopic studies of the intracellular polymerization of sickle hemoglobin

Transmission electron microscopy has been used to study intracellular sickle hemoglobin polymer in unfractionated cells from the arterial and venous blood of patients and after external deoxygenation. We detect polymerized hemoglobin in up to 10% of the cells in the venous circulation, especially in cells that are "cigar-shaped" and appear to be irreversibly sickled. We could not see well-defined polymer in mixed arterial samples; nevertheless, we found electron opaque spots, which could be ferritin granules, hemosiderin, or small aggregates of hemoglobin S. However, upon sequential chemical deoxygenation using 1.0% sodium metabisulphite, polymer formation was seen at oxygen saturation values of 75%-85%. Cells that were physically deoxygenated using gas mixtures containing nitrogen-carbon dioxide-oxygen mixtures were found to contain distinct polymers of deoxyhemoglobin S at oxyhemoglobin saturation values of 50%-75%. As deoxygenation increases, we detect short, randomly arranged polymer in a loose network, with occasional long polymers. Upon further deoxygenation, the length and number of polymer forms increased. Between 0% and 50% saturation, most erythrocytes were full of long, parallel, closely packed polymers that tend to align and run parallel to the cell membrane. In both chemical and physically deoxygenated blood samples, cells were seen at 50%-75% oxyhemoglobin saturation that retained their normal biconcave disc shape, although they contained significant amounts of polymer. The structural changes in sickle erythrocytes seen in vitro due to physical or chemical deoxygenation of cells, may reflect in vivo intracellular changes in the sickle cell patient.     

Respiratory responses to short term hypoxia in the snapping turtle, Chelydra serpentina

Among vertebrates, turtles are able to tolerate exceptionally low oxygen tensions. We have investigated the compensatory mechanisms that regulate respiration and blood oxygen transport in snapping turtles during short exposure to hypoxia. Snapping turtles started to hyperventilate when oxygen levels dropped below 10% O(2). Total ventilation increased 1.75-fold, essentially related to an increase in respiration frequency. During normoxia, respiration occurred in bouts of four to five breaths, whereas at 5% O(2), the ventilation pattern was more regular with breathing bouts consisting of a single breath. The increase in the heart rate between breaths during hypoxia suggests that a high pulmonary blood flow may be maintained during non-ventilatory periods to improve arterial blood oxygenation. After 4 days of hypoxia at 5% O(2), hematocrit, hemoglobin concentration and multiplicity and intraerythrocytic organic phosphate concentration remained unaltered. Accordingly, oxygen binding curves at constant P(CO(2)) showed no changes in oxygen affinity and cooperativity. However, blood pH increased significantly from 7.50+/-0.05 under normoxia to 7.72+/-0.03 under hypoxia. The respiratory alkalosis will produce a pronounced in vivo left-shift of the blood oxygen dissociation curve due to the large Bohr effect and this is shown to be critical for arterial oxygen saturation.     

Blood-gas properties of plateau zokor (Myospalax baileyi)

Plateau zokor (Myospalax baileyi) is one of the blind subterranean mole rats that spend their life solely underground in sealed burrows. It is one of the special species of the Qinghai-Tibet plateau. In their burrows, oxygen is low and carbon dioxide is high and their contents fluctuate with the change of seasons, soil types, rain and depth of burrows. However, plateau zokors show successful adaptation to that extreme environment. In this study, their adapting mechanisms to the hypoxic hypercapnic environment were analyzed through the comparison of their blood-gas properties with that of pikas (Ochotona curzniae) and Sprague-Dawley rats. The results indicated that plateau zokors had higher red blood corpuscle counts (8.11+/-0.59 (10(12)/L)) and hemoglobin concentrations (147+/-9.85 g/L), but hematocrit (45.9+/-3.29%) and mean corpuscular volume (56.67+/-2.57 fL) were lower than the other rodents. Their arterial blood and venous blood pH were 7.46+/-0.07 and 7.27+/-0.07. Oxygen pressure in arterial blood of plateau zokors was about 1.5 times higher than that of pikas and rats, and it was 0.36 and 0.26 times in their venous blood. Partial pressure for carbon dioxide in arterial and venous blood of plateau zokors was 1.5-fold and 2.0-fold higher, respectively, than in rats and pikas. Oxygen saturation of plateau zokors was 5.7 and 9.3 times lower in venous blood than that of pikas and rats, respectively. As result, the difference of oxygen saturation in arterial blood to venous blood was 2- and 4.5-fold higher in plateau zokors as that of pikas and rats, respectively. In conclusion, plateau zokors had a high tolerance to pH changes in tissues, together with strong capabilities to obtain oxygen from their hypoxic-hypercapnic environment.     

Effects of hyperchloremia on blood oxygen binding in healthy calves

Three different levels of hyperchloremia wereinduced in healthy Friesian calves to study the effects of chloride onblood oxygen transport. By infusion, the calves received either 5 ml/kg of 0.9% NaCl (low-level hyperchloremia; groupA), 5 ml/kg of 7.5% NaCl (moderate hyperchloremia;group B), or 7.5 ml/kg of 7.5% NaCl(high-level hyperchloremia; groupC). Blood was sampled from the jugular vein and thebrachial artery. Chloride concentration, hemoglobin content, arterialand venous pH, PCO₂, and PO₂ were determined. At each timepoint (0, 15, 30, 60, and 120 min), the whole blood oxygen equilibriumcurve (OEC) was measured under standard conditions. Ingroups B andC, hyperchloremia was accompanied by asustained rightward shift of the OEC, as indicated by the significantincrease in the standard PO₂ at 50%hemoglobin saturation. Infusion of hypertonic saline also inducedrelative acidosis. The arterial and venous OEC were calculated, withbody temperature, pH, and PCO₂ valuesin arterial and venous blood taken into account. The degree of blooddesaturation between the arterial and the venous compartments[O₂ exchange fraction(OEF%)] and the amount of oxygen released at tissue level by 100 ml of bovine blood (OEF vol%) were calculated from the arterial andvenous OEC combined with the PO₂ andhemoglobin concentration. The chloride-induced rightward shift of theOEC was reinforced by the relative acidosis, but the alteredPO₂ values combined with the lowerhemoglobin concentration explained the absence of any significantdifference in OEF (% and vol%). We conclude that infusion ofhypertonic saline induces hyperchloremia and acidemia, which canexplain the OEC rightward shift observed in arterial and peripheralvenous blood.

     

Lactate uptake by the fetal sheep liver

Lactate is produced by the sheep placenta and is an important metabolic substrate for fetal sheep. However, lactate uptake and release by the fetal liver have not been assessed directly. We measured lactate flux across the liver in 16 fetal sheep at 129 (120-138) days gestation that had catheters chronically maintained in the fetal descending aorta, inferior vena cava, right or left hepatic vein, and umbilical vein. Lactate and hemoglobin concentrations and oxygen saturation were measured in blood drawn from all vessels. Umbilical venous, portal venous, and hepatic blood flow were measured by injecting radionuclide-labeled microspheres into the umbilical vein while obtaining a reference sample from the descending aorta. We found net hepatic uptake of lactate (5.0 +/- 4.4 mg/min per 100 g liver). A large quantity of lactate was delivered to the liver (94.2 +/- 78.1 mg/min per 100 g), so that the hepatic extraction of lactate was only 7.7 +/- 6.5%. Hepatic oxygen consumption was 3.18 +/- 3.3 ml/min per 100 g, and the hepatic lactate/oxygen quotient was 2.07 +/- 1.54. There was no significant correlation between hepatic lactate uptake and hepatic lactate or glucose delivery, hepatic oxygen consumption, hepatic blood flow, hepatic glucose flux, total body oxygen consumption, arterial pH, oxygen content, or oxygen saturation. There was, however, a significant correlation between hepatic lactate uptake and umbilical lactate uptake (r = 0.74, P less than 0.005) such that net hepatic lactate uptake was nearly equivalent to that produced across the umbilical-placental circulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of chemical kinetics on oxygen delivery to tissue.

A mathematical model has been formulated to analyze the effect of nonequilibrium kinetics on oxygen delivery to tissue. The model takes into account molecular diffusion, facilitated diffusion in the capillary blood, convection, chemical kinetics of O2 with hemoglobin, and the rate of metabolic consumption. A line iterative technique is described to solve numerically the resulting coupled system of nonlinear partial differential equations with physiologically relevant boundary and entrance conditions. With nonequilibrium kinetics the end-capillary PO2 is found to be lower than that in the venous blood. The effect is more pronounced during hypoxia and anemia. It is found that the tissue PO2 at the lethal corner decreases with the decrease in blood velocity, arterial PO2, hemoglobin concentration, P50, and increase in COHb concentration or metabolic rate, while the difference between end-capillary PO2 and venous PO2 increases, which reflects the effect of nonequilibrium kinetics on the delivery of O2 to tissue. Thus, the consideration of venous PO2 as an indicator of tissue PO2 in clinical and experimental studies may be questionable.     

Oxygenation of Native or UV-Modified Human Hemoglobin in the Presence of Nitric Oxide

The effect of 0.1–10% nitrosohemoglobin (HbNO) on the functional properties of human oxyhemoglobin (HbO₂) was studied before and after UV irradiation at 151–453 J/m². Oxygen binding analysis showed that HbNO intensified the first stage of oxygenation and weakened the cooperative interactions in the tetramer, decreasing the affinity of hemoglobin for oxygen at physiologically important oxygen partial pressures (40–100 mm Hg). Mixtures of HbO₂ and HbNO were highly resistant to therapeutic doses of UV irradiation. Since the functional activity of hemoglobin depended nonlinearly on the concentration of HbNO in the mixture, it was assumed that sophisticated interactions of HbO₂ and HbNO yielded a new product differing in properties from the initial components.     

Urease enhances the formation of iron nitrosyl hemoglobin in the presence of hydroxyurea

Although it has been shown that hydroxyurea (HU) therapy produces measurable amounts of nitric oxide (NO) metabolites, including iron nitrosyl hemoglobin (HbNO) in patients with sickle cell disease, the in vivo mechanism for formation of these is not known. Much in vitro data and some in vivo data indicates that HU is the NO donor, but other studies suggest a role for nitric oxide synthase (NOS). In this study, we confirm that the NO-forming reactions of HU with hemoglobin (Hb) or other blood constituents is too slow to account for NO production measured in vivo. We hypothesize that, in vivo, HU is partially metabolized to hydroxylamine (HA), which quickly reacts with Hb to form methemoglobin (metHb) and HbNO. We show that addition of urease, which converts HU to HA, to a mixture of blood and HU, greatly enhances HbNO formation.     

Unloading oxygen in a capillary vessel under a pathological condition

In this work, we study theoretically the unloading of oxygen from a hemoglobin molecule to the wall of a typical capillary vessel, considering that the hemoglobin under pathological conditions, obeys the rheological Maxwell model. Based on recent experimental evidences in hypertension, we consider that the red blood cells (RBCs) are composed by a single continuous medium in contrast with the classical particulate or discrete RBC models, which are only valid under normal physiological conditions. The analysis considers the hemodynamic interactions between the plasma and the hemoglobin, both circulating in a long horizontal capillary. We apply numerical and analytical methods to obtain the main fluid-dynamic characteristics for both fluids in the limit of low Reynolds and Womersley numbers. A diffusion boundary layer formulation for the oxygen transport in the combined plasma-hemoglobin core region is presented. The main aspects derived are the time and spatial evolution of the membrane. The hemoglobin and plasma velocities and the pressure distributions are shown. For the oxygen unloading the results are the oxy-hemoglobin saturation, the oxygen flux and the oxygen concentration in the cell-free plasma layer. The volume fraction of red blood cells and the Strouhal number have a great influence on the hemodynamic interactions.     

Oxidation and nitrosylation of oxyhemoglobin by S-nitrosoglutathione via nitroxyl anion

The reaction between low molecular weight S-nitrosothiols and hemoglobin is often used to synthesize S-nitrosohemoglobin, a form of hemoglobin suggested to be involved in the regulation of vascular oxygen delivery. However, this reaction has not been studied in detail, and several groups have reported a variable co-formation of oxidized methemoglobin (metHb) during synthesis. This study examines the mechanism of metHb formation and shows that nitrosylhemoglobin (HbNO) can also be formed. Generation of metHb and HbNO is largely dependent on the presence of protein thiol groups. We present evidence for a mechanism for the formation of metHb and HbNO involving the intermediacy of nitroxyl anion. Specifically, the reaction of nitroxyl with S-nitrosothiols to liberate nitric oxide and reduced thiol is proposed to be central to the reaction mechanism.     

Functional arterio-venous anastomoses between the testicular artery and the pampiniform plexus in the spermatic cord of rams

Blood flow to the testis, haemoglobin oxygen saturation and testosterone concentration in arterial and venous testicular blood vessels were studied in Texel rams in the breeding and non-breeding season. Blood flow in the proximal and distal testicular artery was measured electromagnetically. The mean flow in the proximal testicular artery was 18.5 ml/min and in the distal testicular artery 7.5 ml/min, and there was no detectable seasonal influence. Haemoglobin oxygen saturation and testosterone concentration were measured in the saphenous artery and vein, the distal testicular artery and vein, and in the proximal testicular vein. The haemoglobin oxygen saturation in the proximal testicular vein was significantly higher than in the distal testicular vein in both seasons. The mean testosterone concentration was significantly lower in the proximal testicular vein than in the distal testicular vein in both seasons. Based on haemoglobin oxygen saturation and testosterone data, it was calculated that between 28 and 46% of the testicular arterial blood was bypassing the testis and was directly flowing through arterio-venous anastomoses towards the pampiniform plexus in the spermatic cord of conscious rams. In anaesthetized rams 55 and 64% of the blood was flowing directly from the testicular artery to the pampiniform plexus based on blood flow data. Transfer of testosterone and oxygen by passive diffusion from the testicular artery to the pampiniform plexus and vice versa in the spermatic cord was not detected.     

Evaluation of systemic blood NO dynamics by EPR spectroscopy: HbNO as an endogenous index of NO

The measurement of hemoglobin-nitric oxide (NO) adduct (HbNO) in whole blood by the electron paramagnetic resonance (EPR) method seems relevant for the assessment of systemic NO levels. However, ceruloplasmin and unknown radical species overlap the same magnetic field as that of HbNO. To reveal the EPR spectrum of HbNO, we then introduced the EPR signal subtraction method, which is based on the computer-assisted subtraction of the digitized EPR spectrum of HbNO-depleted blood from that of sample blood using the software. Rats were treated with N(omega)-nitro-L-arginine methyl ester (L-NAME; 120 mg. kg-1. day-1) for 1 wk to obtain HbNO-depleted blood. When this method was applied to the analysis of untreated fresh whole blood, the five-coordinate state of HbNO was observed. HbNO concentration in pentobarbital-anesthetized rats was augmented (change in [HbNO] = 1.6-5.5 microM) by infusion of L-arginine (0.2-0.6 g/kg) but not D-arginine. Using this method, we attempted to evaluate the effects of temocapril on HbNO dynamics in an L-NAME-induced rat endothelial dysfunction model. The oral administration of L-NAME for 2 wk induced a serious hypertension, and the HbNO concentration was reduced (change in [HbNO] = 5.7 microM). Coadministration of temocapril dose dependently improved both changes in blood pressure and the systemic HbNO concentration. In this study, we succeeded in measuring the blood HbNO level as an index of NO by the EPR HbNO signal subtraction method. We also demonstrated that temocapril improves abnormalities of NO dynamics in L-NAME-induced endothelial dysfunction rats using the EPR HbNO signal subtraction method.     

Structure of nitric oxide hemoglobin

We have compared the structure of horse nitric oxide hemoglobin (HbNO) and methemoglobin in the oxy quaternary structure by difference Fourier analysis at 2.8 Å resolution. Both nitric oxide and oxygen assume bent co-ordination geometry and form low-spin complexes in binding to heme; on the basis of preferred ligand and heme stereochemistry, HbNO is the closest analog of HbO₂ (oxyhemoglobin) examined to date. To the resolution of the X-ray data, the stereochemistry of the heme-NO complex in hemoglobin and the corresponding free heme complex appears similar. In contrast, the ligand pockets in hemoglobin hinder binding of cyanide and carbon monoxide in their preferred linear axial co-ordination modes and force them to assume a strained off-axis binding stereochemistry. The structural similarity between HbNO and HbO₂ is reflected in their kinetic behavior, which is similar, and distinct from that of carboxyhemoglobin.