Spectrofluorometric and Molecular Modeling Studies on Binding of Nitrite Ion with Bovine Hemoglobin: Effect of Nitrite Ion on Amino Acid Residues (original) (raw)
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Chemical Research in Toxicology, 2010
The green nitrihemoglobin (R 2 2 tetramer, NHb) was prepared by the aerobic reaction of excess nitrite with human hemoglobin A 0 under mildly acidic conditions. A rate equation was determined and found to depend on nitrite, hydrogen ion, and oxygen concentrations:-d[HbNO 2 ]/dt) [k 1 + k 2 (K a [HNO 2 ])-[O 2 ] 1/2 ][HbNO 2 ], where k 1) (2.4 (0.9) × 10-4 s-1 , k 2) (1 (0.2) × 10 5 M-5/2 s-1 , and K a is the acid dissociation constant for nitrous acid (4.5 × 10-4 M). Also, the chemical properties of NHb are compared to those of the normal hemoglobin (including the addition products of common oxidation states with exogenous ligands, the alkaline transitions of the ferric forms, and the oxygen binding characteristics of the ferrous forms) and were found to be nearly indistinguishable. Therefore, the replacement of a single vinyl hydrogen with a nitro group on the periphery of each macrocycle in hemoglobin does not significantly perturb the interaction between the hemes and the heme pockets. Because nonphotochemical reaction chemistry must necessarily be most dependent on electronic ground states, it follows that the clearly visible difference in color between hemoglobin A 0 and NHb must be associated primarily with the respective electronic excited states. The possibility of NHb formation in vivo and its likely consequences are considered.
Globins, such as hemoglobin (Hb) and myoglobin (Mb), have gained attention for their ability to reduce nitrite (NO 2 −) to nitric oxide (NO). The molecular interactions that regulate this chemistry are not fully elucidated, therefore we address this issue by investigating one part of the active site that may control this reaction. Here, the effects of the 2,4-heme substituents on the nitrite reductase (NiR) reaction, and on the structures and energies of the ferrous nitrite intermediates, are investigated using Mb as a model system. This is accomplished by studying Mbs with hemes that have different 2,4-R groups, namely diacetyldeuteroMb (-acetyl), protoMb (wild-type (wt) Mb,-vinyl), deuteroMb (-H), and mesoMb (-ethyl). While trends on the natural charge on Fe and O-atom of bound nitrite are observed among the series of Mbs, the Fe II –N Pyr (Pyr = pyrrole) and Fe II – N His93 (His = histidine) bond lengths do not significantly change. Kinetic analysis shows increasing NiR activity as follows: diacetyldeuteroMb b wt Mb b deuteroMb b mesoMb. Nitrite binding energy calculations of the different Mb II –nitrite conformations demonstrate the N-bound complexes to be more stable than the O-bound complexes for all the different types of heme structures, with diacetyldeuteroMb having the greatest nitrite binding affinity. Spectral deconvolution on the final product generated from the reaction between Mb II and NO 2 − for the reconstituted Mbs indicates the formation of 1:1 Mb III and Mb II –NO. The electronic changes induced by the –R groups on the 2,4-positions do not alter the stoichiometric ratio of the products, resembling wt Mb.
Electron Paramagnetic Resonance Investigation of Nitrite Binding in Myoglobin
bioRxiv, 2018
It has been proposed that myoglobin (Mb) may act as a nitrite reductase under hypoxic conditions. Any mechanism describing such activity should take into account the binding geometry of the ligand to the heme. Crystal structures of horse-heart Mb and human hemoglobin-nitrite complexes suggest that the anion adopts an uncommon O-nitrito binding mode. Electron Paramagnetic Resonance (EPR) spectroscopy was employed to investigate the nature of nitrite binding to Mb at pH values ranging from 6.5 to 10.8. Results suggest that for ferric Mb at low pH, nitrite binds in the O-bound nitrito mode resulting in a low-spin (LS) iron center. Further a high-spin (HS) iron center is observed at high pH in Mb-Nitrite with spectral values different to that of purely HS-Mb that is proposed to be due to an N-bound nitrite. The yields of these two species were found to be influenced by pH. Background Myoglobin has been theorized to have a role as a nitrite reductase. Results O-bound nitrite produces a l...
Nitrite binding to globins: linkage isomerism, EPR silence and reductive chemistry
Nitric Oxide, 2014
The nitrite adducts of globins can potentially bind via O-or N-linkage to the heme iron. We have used EPR (electron paramagnetic resonance) and DFT (density functional theory) to explore these binding modes to myoglobin and hemoglobin. We demonstrate that the nitrite adducts of both globins have detectable EPR signals; we provide an explanation for the difficulty in detecting these EPR features, based on uniaxial state considerations. The EPR and DFT data show that both nitrite linkage isomers can be present at the same time and that the two isomers are readily interconvertible in solution. The millisecondscale process of nitrite reduction by Hb is investigated in search of the elusive Fe(II)-nitrite adduct.
Conformation of bovine nitrosylhemoglobins: an ESR study
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1989
The structural properties of nitrosylhemoglobins from two bovine species, namely cow and buffalo, have been investigated using electron spin resonance spectroscopy. Bovine hemoglobins show sensitivity to the presence of chloride ions and organic phosphates. ESR spectral features ind;cate a stable deoxy quaternary conformation of the molecule when compared to normal adult human hemoglobin A. Amiao acid substitutions at the amino terminal end of the ,O chain and at other sites of the a and ~ chains seems to shift the allosteric equilibrium towards the T state in bovine hemoglobins. The results also confirm the intrinsically low oxygen affinity of bovine hemoglobins under physiological conditions.
Mössbauer and EPR study of nitrosyl hemoglobin
Hyperfine Interactions, 1994
Nitrosyl hemoglobin was prepared by bubbling fresh 57Fe-enriched rat hemoglobin with NO. S-and X-band EPR spectra at 77 K are typical for an S = 112 system with an anisotropic g-tensor and exhibit hyperfine interactions of 14N with the electronic spin. M6ssbauer spectra at 4.2 and 100 K consist of a superposition of spectra from high-and low-spin Fe(III), deoxygenated hemoglobin and a component corresponding to S = 1/2, g = 2, hyperfine constants A~lgnfl n =Ayylgnfl n = -19.6 T, A=lgnfl n = 6.8 T, quadrupole splitting AEQ = 1.5 mm s -1, isomer shift I s = 0.42 mm s -t and linewidth 0.4 mm s -l. The spin-lattice relaxation rate at 100 K is <2 x 10 6 s -1.
Crystallographic Analysis of the Interaction of Nitric Oxide with Quaternary-T Human Hemoglobin
Biochemistry, 2004
In addition to interacting with hemoglobin as a heme ligand to form nitrosylhemoglobin, NO can react with cysteine sulfhydryl groups to form S-nitrosocysteine or cysteine oxides such as cysteinesulfenic acid. Both modes of interaction are very sensitive to the quaternary structure of hemoglobin. To directly view the interaction of NO with quaternary-T deoxyhemoglobin, crystallographic studies were carried out on crystals of deoxyhemoglobin that were exposed to gaseous NO under a variety of conditions. Consistent with previous spectroscopic studies in solution, these crystallographic studies show that the binding of NO to the heme groups of crystalline wild-type deoxyhemoglobin ruptures the Fe-proximal histidine bonds of the R-subunits but not the-subunits. This finding supports Perutz's theory that ligand binding induces tension in the R Fe-proximal histidine bond. To test Perutz's theory, deoxy crystals of the mutant hemoglobin W37E were exposed to NO. This experiment was carried out because previous studies have shown that this mutation greatly reduces the quaternary constraints that oppose the ligandinduced movement of the R-heme Fe atom into the plane of the porphyrin ring. As hypothesized, the Fe-proximal histidine bonds in both the-and the R-subunits remain intact in crystalline W37E after exposure to NO. With regard to S-nitrosocysteine or cysteine oxide formation, no evidence for the reaction of NO with any cysteine residues was detected under anaerobic conditions. However, when deoxyhemoglobin crystals are first exposed to air and then to NO, the appearance of additional electron density indicates that Cys93(F9) has been modified, most likely to cysteinesulfenic acid. This modification of Cys93(F9) disrupts the intrasubunit salt bridge between His146(HC3) and Asp94(FG1) , a key feature of the quaternary-T hemoglobin structure. Also presented is a reanalysis of our previous crystallographic studies [Chan, N.-L., et al. (1998) Biochemistry 37, 16459-16464] of the interaction of NO with liganded hemoglobin in the quaternary-R2 structure. These studies showed additional electron density at Cys93-(F9) that was consistent with an NO adduct. However, for reasons discussed in this paper, we now believe that this adduct may be the Hb-S-N •-O-H radical intermediate and not Hb-S-NdO as previously suggested.
Aggregation of adult and fetal hemoglobin by ingested nitrate anions
Progress in Biological Sciences, 2015
The ingested nitrates sourced from tap water, food, chemicals and pharmaceuticals areconverted to nitrites in the body surfaces by bacteria and then, the nitrite ions can lead thestructural changing in hemoglobin. In the present work, aggregation of the purified hemoglobinin adult (HbA) and in fetus or newborn (HbF) in the presence of nitrite ions were studied.Hemoglobin aggregation was performed chemically in the presence of 10 mg/l nitrite ions andexamined by UV-Vis spectrophotometer at 360 nm wavelength. The extrinsic fluorimetricmeasurements indicated that repulsive electrostatic interaction between nitrite anions andnegative charged groups of both types of HbA and HbF molecules leads to expose thehydrophobic patch of the protein molecules. Moreover, the α-helix to β-strand transition in bothtypes of hemoglobins shown by circular dichroism support aggregation process among thisprotein. However, at natural pH, the protonated amino group of Gly in HbF tends to bind tonitrite anion...
Frontiers in Chemistry, 2017
Synthetic dyes are a very efficient class of dyes that are ingested or come into contact with the skin from numerous sources (cosmetics, textiles, leather, paper, and drugs). An important component of their safety profile is the interactions that they form after they enter the body. Hemoglobin is a functionally important protein that can form multiple interactions with soluble compounds present in the blood, and hence forms an important aspect of the toxicological or safety profile of the dyes. Here we study the interaction between bovine hemoglobin and organic dyes using UV-Vis absorbance and fluorescence spectroscopy. Molecular modeling was used to visualize the binding site and partners of the dye molecules, within the hemoglobin molecule. We find that all four dyes studied form sufficiently strong interactions with hemoglobin to allow for the formation of potentially toxic interactions. Molecular modeling showed that all four dyes bind within the central cavity of the hemoglobin molecule. However, binding partners could not be identified as multiple binding conformations with very similar energies were possible for each dye.