Iron nitrosyl hemoglobin formation from the reaction of hydroxylamine and hemoglobin under physiological conditions

Sickle cell disease patients receiving hydroxyurea (HU) therapy have shown increases in the production of nitric oxide (NO) metabolites, which include iron nitrosyl hemoglobin (HbNO), nitrite, and nitrate. However, the exact mechanism by which HU forms HbNO in vivo is not understood. Previous studies indicate that the reaction of oxyhemoglobin (oxyHb) or deoxyhemoglobin (deoxyHb) with HU are too slow to account for in vivo HbNO production. In this study, we show that the reaction of methemoglobin (metHb) with HU to form HbNO could potentially be fast enough to account for in vivo HbNO formation but competing reactions of either excess oxyHb or deoxyHb during the reaction reduces the likelihood that HbNO will be produced from the metHb-HU reaction. Using electron paramagnetic resonance (EPR) spectroscopy we have detected measurable amounts of HbNO and metHb during the reactions of oxyHb, deoxyHb, and metHb with excess hydroxylamine (HA). We also demonstrate HbNO and metHb formation from the reactions of excess oxyHb, deoxyHb, or metHb and HA, conditions that are more likely to mimic those in vivo. These results indicate that the reaction of hydroxylamine with hemoglobin produces HbNO and lend chemical support for a potential role for hydroxylamine in the in vivo metabolism of hydroxyurea.     

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.     

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.     

Resonance Raman and EPR of nitrosyl human hemoglobin and chains, carp hemoglobin, and model compounds. Implications for the nitrosyl heme coordination state.

We report the joint resonance Raman (RR) and electron paramagnetic resonance (epr) study of five- and six-coordinate nitrosyl heme model compounds and of the titled nitrosyl hemoproteins. Both epr and RR spectra fall into two types which, in the models, correspond to five- and six-coordinate nitrosyl hemes. However, neither RR nor epr spectroscopy is highly sensitive to the nature of the bond between a nitrosyl heme and a coordinated nitrogenous base, nor do the results of one technique uniformly correlate with those of the other. It is not possible to use epr spectroscopy as a test for the coordination state of a nitrosyl heme. The position of the highest frequency (depolarized) RR band possibly provides such a test. Any breaking of the very weak bond between nitrosyl heme and proximal histidine in T state human HbNO is more a consequence of tertiary structural features unique to the human alphaNO chains than it is of properties of the T quaternary conformation.     

Iron absorption from concentrated hemoglobin hydrolysate by rat

Although heme iron is highly bioavailable, the low iron content of hemoglobin prevents its use for dietary fortification; on the other hand, purified heme has low solubility and absorption rate. The present study was designed to assess the interactions between concentrated heme iron and peptides released during globin hydrolysis and cysteine and their relation with iron absorption. Hemoglobin was hydrolyzed by pepsin or subtilisin, and then, heme iron was concentrated by ultrafiltration. Iron absorption was studied in a Ussing chamber; gluconate was used as control. Iron uptake from nonconcentrated pepsin hydrolysate and gluconate was lower than from other groups. Cysteine significantly enhanced iron uptake except from the concentrated subtilisin hydrolysate. There was no significant difference between cysteine-supplemented groups. According to the different hydrolysis pathways of enzymes, it is assumed that the presence of hydrophobic peptides and the strength of heme-peptide interactions are both determining factors of heme iron absorption. These interactions occur mainly before iron uptake, as emphasized by the effect of cysteine.     

The reaction of deoxy-sickle cell hemoglobin with hydroxyurea.

In addition to its capacity to increase fetal hemoglobin levels, other mechanisms are implicated in hydroxyurea's ability to provide beneficial effects to patients with sickle cell disease. We hypothesize that the reaction of hemoglobin with hydroxyurea may play a role. It is shown that hydroxyurea reacts with deoxy-sickle cell hemoglobin (Hb) to form methemoglobin (metHb) and nitrosyl hemoglobin (HbNO). The products of the reaction as well as the kinetics are followed by absorption spectroscopy and electron paramagnetic resonance (EPR) spectroscopy. Analysis of the kinetics shows that the reaction can be approximated by a pseudo-first order rate constant of 3.7x10(-4) (1/(s.M)) for the disappearance of deoxy-sickle cell hemoglobin. Further analysis shows that HbNO is formed at an observed average rate of 5.25x10(-5) (1/s), three to four times slower than the rate of formation of metHb. EPR spectroscopy is used to show that the formation of HbNO involves the specific transfer of NO from the NHOH group of hydroxyurea. The potential importance of this reaction is discussed in the context of metHb and HbNO being able to increase the delay time for sickle cell hemoglobin polymerization and HbNO's vasodilating capabilities through conversion to S-nitrosohemoglobin.     

Spectral transitions of nitrosyl hemes during ligand binding to hemoglobin.

Human deoxyhemoglobin has been titrated with nitric oxide at several pH values ranging from 6.0 to 9.0, in the presence and absence of the allosteric effector inositol hexaphosphate at 25 degrees C. Samples were frozen for EPR measurements or analyzed optically within 30 s after mixing to ensure a kinetic population of intermediates. Fractions of pentacoordinate alpha-NO heme groups were determined by fitting EPR and absorbance difference spectra in terms of linear combinations of standard signals. Equivalent results were obtained by these techniques. The fraction of alpha-NO heme exhibiting pentacoordinate character in Hb4NO increases from 0.07 to 0.73 in going from pH 9 to 6. The fraction of alpha hemes which are pentacoordinate in fully saturated nitrosyl hemoglobin, Hb4(NO), increases from 0.0 to 0.41 over the same pH range. Only in the presence of bound inositol-P6 are all 4 the alpha-NO hemes pentacoordinate. Thus, the expression of modified NO heme character is not simply a reflection of the formation of low affinity quaternary conformations. Rather, within this conformation the alpha chain iron atoms exhibit an equilibrium between hexa- and pentacoordinate structures which is perturbed markedly by both proton and phosphate binding. No intermediate coordination structure of the type suggested by Chevion et al. (Chevion, M., Stern, A., Peisach, J., Blumberg, W.E., and Simon, S. (1978) Biochemistry 17, 1745-1750) appears to occur since the observed alpha-NO heme spectra can always by represented quantitatively as a linear combination of the normal hexacoordinate and pentacoordinate signals. The formation of pentacoordinate alpha-NO causes this subunit to exhibit a higher affinity for nitric oxide. Thus on standing at low levels of saturation, there is a slow (t1/2 approximately equal to 8 min at pH 7, 25 degrees C) re-equilibration of ligand from beta to alpha subunits. The final ratio of alpha-NO to beta-NO is 2 to 1 in the absence of phosphates and greater than 10 to 1 in the presence of inositol hexaphosphate.     

EPR spectral changes of nitrosyl hemes and their relation to the hemoglobin T-R transition

EPR spectra of nitrosyl hemes were used to study the quaternary structure of hemoglobin. Human adult hemoglobin has been titrated with nitric oxide at pH 7.0 and 25 degrees C. After the equilibration of NO among the alpha and beta subunits the samples were frozen for EPR measurements. The spectra were fitted by linear combinations of three standard signals: the first arising from NO-beta-hemes and the other two arising for NO-alpha-hemes of molecules in the high- and low-affinity conformations. The fractional amounts of alpha subunits exhibiting the high-affinity spectrum fitted the two-state model (Edelstein, S.J. (1974) Biochemistry 13, 4998-5002) with the allosteric constant L = 7.10(6) and relative affinities cNO alpha and cNO beta approx. 0.01. Hemoglobin has been marked with nitric oxide one chain using low-saturation amounts of nitric oxide. The EPR spectra was studied as a function of oxygen saturation. Linear combinations of the three standard signals above fitted these spectra. The fractions of molecules exhibiting the high-affinity spectrum fitted the two-state model with L = 7 . 10(6), c)2 = 0.0033 and cNO alpha = 0.08, instead of cNO alpha = 0.01. Thus, the two-state model is not adequate to describe the conformational transition of these hybrids. The results present evidence of the non-equivalence between oxygen and nitric oxide as ligands.     

Electrochemical assay for determining nitrosyl derivatives of human hemoglobin: nitrosylhemoglobin and S-nitrosylhemoglobin

Nitric oxide (NO) is an important biological regulator. It can bind to heme iron and form NO+, involved in the synthesis of S-nitrosothiols (-SNOs). NO reacts with human hemoglobin (Hb) to produce the derivatives: S-nitrosylhemoglobin (-SNOHb) and nitrosylhemoglobin (HbNO). At neutral pH values, free NO does not react directly with the -SH groups of Hb. The reductive nitrosylation of Fe(III) heme upon reaction with NO has long been studied, but it is not yet completely known. To quantify the reaction of NO with Hb, we developed a new, sensitive (nanomolar concentration range) electrochemical assay to selectively measure HbNO and -SNOHb. The assay also allows the monitoring of free NO during the reaction with human Fe(III)Hb and Fe(II)HbO(2).     

Oxidation-reduction reactions of hemoglobin A, hemoglobin M Iwate, and hemoglobin M Hyde Park

The kinetics and equilibrium of the redox reactions of hemoglobin A, hemoglobin M Iwate, and hemoglobin M Hyde Park using the iron (II) and iron (III) complexes of trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetate (CDTA4-) as the reducing and oxidizing agents have been studied. With respect to the equilibrium it was found that hemoglobin M Iwate (where the beta chains were reduced) was more readily reduced than hemoglobin M Hyde Park (where the alpha chains are reduced). This difference was shown to be a result of a difference in the rate constant for reduction but not oxidation. The observed rate contants for the reduction of all three hemoglobins were shown to decrease with increasing pH. This was attributed to a decrease in the [T]/[R] ratio. The observed rate contants for the oxidation reaction were shown to increase with increasing pH. Accompanying this increase was a change in the kinetic profile for hemoglobin A from pseudo first order to one in which the rate increased as the extent of reaction increased. Inositol hexaphosphate had no effect on the rate of oxidation of deoxyhemoglobin A. This was a result of binding of FeCDTA2- or HCDTA3- to the protein. However, in the presence of inositol hexaphosphate, the reduction of methemoglobin A exhibited biphasic kinetics. This result was interpreted in terms of the production of a small amount of a conformation which was more readily reduced.     

The effect of inositol hexaphosphate on the absorption spectra of alpha and beta chains in nitrosyl hemoglobin.

The spectral changes of nitrosyl hemoglobin on addition of inositol hexaphosphate were studied in hybrid-heme hemoglobins. The results showed that the decrease in absorption in the Soret region was mainly due to a spectral change in alpha chains, and that the tension on heme in the quaternary T structure was much stronger in alpha than in beta chains.     

Copper-induced change in the ESR signal of hemoglobin nitrosyl complexes in wound by the action of copper nanoparticles

The results concerning changes in the ESR signal of hemoglobin nitrosyl complexes in wound tissues in the course of healing by the action of ointments with copper nanoparticles (patent N2460532, Russia) are presented. It is shown that the wound healing process modified by the influence of copper nanoparticles demonstrates the increase in the ESR signal amplitude for hemoglobin nitrosyl complexes as compared with controls (the ointment base without nanoparticles). Planimetric measurements of wound area through reparation course indicate an active process of wound healing for injuries treated with copper nanoparticles in the ointment, resulting in lessening half-reparation time up to 5.0 times as compared with controls (treatment with the ointment base). The paper discusses the role of copper nanoparticles, NO and their potential synergistic effect on the skin wound regeneration.     

Iron normal mode dynamics in (nitrosyl)iron(II)tetraphenylporphyrin from X-ray nuclear resonance data

The complete iron atom vibrational spectrum has been obtained by refinement of normal mode calculations to nuclear inelastic x-ray absorption data from (nitrosyl)iron(II)tetraphenylporphyrin, FeTPP(NO), a useful model for heme dynamics in myoglobin and other heme proteins. Nuclear resonance vibrational spectroscopy (NRVS) provides a direct measurement of the frequency and iron amplitude for all normal modes involving significant displacement of (57)Fe. The NRVS measurements on isotopically enriched single crystals permit determination of heme in-plane and out-of-plane modes. Excellent agreement between the calculated and experimental values of frequency and iron amplitude for each mode is achieved by a force-field refinement. Significantly, we find that the presence of the phenyl groups and the NO ligand leads to substantial mixing of the porphyrin core modes. This first picture of the entire iron vibrational density of states for a porphyrin compound provides an improved model for the role of iron atom dynamics in the biological functioning of heme proteins.     

An electron paramagnetic resonance investigation of the oxygen dependence of the arterial-venous gradient of nitrosyl hemoglobin in blood circulation

Whether there is a nitrosyl hemoglobin (HbNO) gradient between the venous and the arterial parts of the circulatory system is a very controversial issue in nitric oxide research. We have carefully evaluated the measurement of HbNO concentration in blood using EPR generated in vivo by the NO donor DEANO under various oxygen tensions. We found that the absolute concentrations of HbNO in venous and arterial blood were the same within experimental error, independent of hemoglobin saturation; only the ratios of 5-coordinate and 6-coordinate HbNO differed. The HbNO concentration increased when the oxygen concentration breathed by the rats decreased in a manner that was linear in hemoglobin saturation. These results do not support the existence of an arterial-venous gradient of HbNO under our experimental conditions.