Characterization of the heme iron in pyridoxylated hemoglobin cross-linked by glutaraldehyde using Mössbauer spectroscopy

The heme iron in human adult hemoglobin modified by both pyridoxal-5'-phosphate and glutaraldehyde was characterized by M?ssbauer spectroscopy and compared with non-modified hemoglobin. M?ssbauer spectra of the samples were measured at 87 and 295 K (1yophilized form) and at 87 K (frozen solution). The values of quadrupole splitting for the oxy-form of modified hemoglobin were found to be lower than those of the oxy-form of hemoglobin without modifications in lyophilized form and frozen solution, respectively. On the other hand, the values of quadrupole splitting for the deoxy-form of modified and non-modified hemoglobins in frozen solution were the same. The M?ssbauer spectra of the oxy-form of modified hemoglobin were also analyzed in terms of the heme iron non-equivalence in alpha- and beta-subunits of tetramer. The differences of the tendencies of temperature dependencies of quadrupole splitting for the oxy-form of modified and non-modified hemoglobins in lyophilized form were shown. These results indicated that the heme iron electronic structure and stereochemistry were changed in the oxy-form of pyridoxylated hemoglobin cross-linked by glutaraldehyde.     

Collective motion of iron and exogenous ligands in hemoglobin

M?ssbauer studies of deoxyhemoglobin (Hb), oxyhemoglobin (HbO2), and (carbonmonoxy)-hemoglobin (HbCO) have been performed providing information regarding the center shift (CS) up to 250 K and the recoil-free fraction (f) in the "solid state regime". The temperature dependence of the CS indicates that the effective mass of the iron in liganded hemoglobin includes the exogenous ligand, suggesting that vibrations of the iron are strongly coupled to those of the ligand. As a result of this coupled motion, the recoil-free fraction (f) provides a valuable probe for the shape of the potential energy surface in the ligand pocket. Thus HbO2 experiences a stronger restoring force than HbCO, which can be attributed to a steeper potential well for the oxygen into the ligand pocket.     

Dynamic structural disorder of the FeO<Subscript>2</Subscript> moiety in oxymyoglobin studied by nuclear resonant forward scattering of synchrotron radiation

Oxy- as well as deoxymyoglobin exhibit a pronounced temperature dependence of the quadrupole splitting of the heme iron as detected by conventional M?ssbauer spectroscopy. With nuclear resonant forward scattering (NFS) of synchrotron radiation, which can be viewed as M?ssbauer spectroscopy in the time domain, it is shown that this spectroscopic behavior, although it is phenomenologically similar in the two cases, is based on completely different physical mechanisms. It is demonstrated that stochastic fluctuations of the iron electric field gradient in MbO(2), which are due to the dynamic structural disorder of the FeO(2) moiety, are the reason for the temperature-dependent alterations of the coherent quantum beat pattern in the NFS spectra of MbO(2), in contrast to deoxyMb where transitions between orbital states of iron take place. This subtle spectroscopic difference cannot be inferred from conventional M?ssbauer spectroscopy.     

M?ssbauer study of beef heart cytochrome oxidase. Comparative study of the bovine enzyme and cytochrome c1aa3 from Thermus thermophilus

We have studied beef heart cytochrome c oxidase at 4.2 K with M?ssbauer spectroscopy using the 57Fe present in natural abundance. The spectra observed are very similar to those of the a- and a3-sites of cytochrome c1aa3 from Thermus thermophilus. Thus, many conclusions derived from studies of the bacterial oxidase (available with enriched 57Fe) also apply to the mammalian enzyme. In the resting (as isolated) state, cytochrome a3 of the mammalian enzyme exhibits a doublet with quadrupole splitting, delta EQ = 1.0 mm/s and isomer shift, delta = 0.48 mm/s. These parameters suggest a high spin ferric heme and rule out an Fe(IV) assignment. The absence of magnetic features in the 4.2 K spectrum is consistent with earlier proposals that cytochrome a3 is spin-coupled to a cupric ion. The absorption lines are rather broad, suggesting that the a3-site is heterogeneous in the resting enzyme. Reduced cytochrome a3 has delta EQ = 1.85 mm/s and delta = 0.93 mm/s, demonstrating that the heme iron is high spin ferrous. The observed value for delta EQ is smaller than those of hemoglobin (2.4 mm/s), myoglobin (2.2 mm/s), and cytochrome a3 from T. thermophilus (2.06 mm/s). The M?ssbauer spectra of oxidized cytochrome a3-CN show that the heme iron is low spin ferric and that the ground state has integer spin S greater than or equal to 1, which plausibly results from ferromagnetic coupling of the S = 1/2 heme to an S = 1/2 cupric ion. Reduced cytochrome a is low spin ferrous, with parameters similar to those of cytochrome b5 and cytochrome c.     

M?ssbauer investigations of high-spin ferrous heme proteins. I. Cytochrome P-450.

Anaerobically reduced samples of cytochrome P-450 from Pseudomonas putida were studied by M?ssbauer spectroscopy. In the presence of an applied magnetic field the high-spin ferrous heme iron showed an intricate pattern of electric and magnetic hyperfine interactions which could be parametrized successfully in terms of a spin Hamiltonian formalism. The results imply a very low (triclinic) symmetry of the heme iron. The effects of the ligand environment and of spin-orbit coupling result in a large zero-field splitting of the electronic ground state. The electronic ground state. The electric-field gradient tensor is characterized by a large asymmetry parameter, and its principal axes are rotated substantially from the frame that defines the zero-field splitting. This study shows that high-field M?ssbauer spectroscopy provides a unique tool for structural investigations of high-spin ferrous compounds and can substitute, under suitable conditions, for magnetic susceptibility measurements. The present paper focuses on the methodology and data analysis; in the subsequent paper the data obtained for P-450 are compared with new results obtained for hemoglobin, chloroperoxidase, and horseradish peroxidase.     

Nitrogenase. VIII. M?ssbauer and EPR spectroscopy. The MoFe protein component from Azotobacter vinelandii OP.

We have studied the molybdenum-iron protein (MoFe protein, also known as component I) from Azobacter vinelandi using M?ssbauer spectroscopy and electron paramagnetic resonance on samples enriched with 57Fe. These spectra can be interpreted in terms of two EPR active centers, each of which is reducible by one electron. A total of four different chemical environments of Fe can be discerned. One of them is a cluster of Fe atoms with a net electronic spin of 3/2, one of them is high-spin ferrous iron and the remaining two are iron in a reduced state (probably in clusters). The results are as follows: Chemical analysis yields 11.5 Fe atoms and 12.5 labile sulfur atoms per molybdenum atom; the molecule contains two Mo atoms per 300 000 daltons. The EPR spectrum of the MoFe protein exhibits g values at 4.32, 3.65 and 2.01, associated with the ground state doublet of a S = 3/2 spin system. The spin Hamiltonian H = D(S2/z minus 5/4 + lambda(S2/x minus S2/y)) + gbeta/o S-H fits the experimental data for go = 2.00 and lambda = 0.055. Quantitative analysis of the temperature dependence of the EPR spectrum yields D/k = 7.5 degrees K and 0.91 spins/molybdenum atom, which suggests that the MoFe protein has two EPR active centers. Quantitative evaluation of M?ssbauer spectra shows that approximately 8 iron atoms give rise to one quadrupole doublet; at lower temperatures magnetic spectra, associated with the groud electronic doublet, are observed; at least two magnetically inequivalent sites can be distinguished. Taken together the data suggest that each EPR center contains 4 iron atoms. The EPR and M?ssbauer data can only be reconciled if these iron atoms reside in a spin-coupled (S = 3/2) cluster. Under nitrogen fixing conditions the magnetic M?ssbauer spectra disappeared concurrently with the EPR signal and quadrupole doublets are obserced at all temperatures. The data suggest that each EPR active center is reduced by one electron. The M?ssbauer investigation reveals three other spectral components characteristic of iron nuclei in an environment of integer or zero electronic spin, i.e. they reside in complexes which are "EPR-silent". One of the components (3-4 iron atoms) has M?ssbauer parameters characteristic of the high-spin ferrous iron as in reduced ruberdoxin. However, measurements in strong fields indicate a diamagnetic environment. Another component, representing 9-11 iron atoms, seems to be diamagnetic also. It is suggested that these atoms are incorporated in spin-coupled clusters.     

Study of normal human fetal and adult hemoglobins by M?ssbauer spectroscopy

Human normal fetal and adult hemoglobins were investigated by M?ssbauer spectroscopy. The differences discovered in quadrupole splitting are connected with distinctions in protein molecular structures. An approximation of M?ssbauer spectra of hemoglobins in supposition of nonequivalence of Fe(II) electronic structure in alpha-, beta- and gamma-subunits in tetramers was proposed. Qualitative analysis of the relationship of Fe(II) electronic structure, molecular structure of the active site and functionality of hemoglobin was carried out.     

Response to oxidative stress as a welfare parameter in swine

Summary

Pro-oxidant effects of hemoglobin-derived heme and iron contribute to the progressive damage observed in β thalassemic and sickle (HbS) red blood cells. Agents that prevent heme/iron release and inhibit their redox activity might diminish such injury. Consequently, the inhibitory effects of chloroquine (CQ), a heme-binding antimalarial drug, and a novel dichloroquine compound (CQ-D2) on iron release and lipid peroxidation were investigated. In contrast to normal hemoglobin, significant amounts of iron were released from both purified hemin and α-hemoglobin chains during incubations with exogenous reduced glutathione (GSH) and/or H₂O₂. Addition of either CQ or CQ-D2 effectively inhibited GSH- and GSH/H₂O₂-mediated iron release from hemin (P<0.001). During prolonged incubations (6 h), both CQ and CQ-D2 significantly decreased the release of heme-free iron from both purified hemoglobin and α-hemoglobin chains. Interestingly, CQ and CQ-D2 differentially affected the redox availability of the heme-bound iron. The CQ: heme complex significantly enhanced membrane lipid peroxidation whereas CQ-D2 dramatically (P<0.001) inhibited heme-dependent peroxidation to almost baseline levels. In summary, CQ-derivatives which render heme redox inert and prevent the release of free iron from heme might be beneficial in the treatment of certain hemoglobinopathies and, perhaps, other pathologies promoted by delocalized heme/iron.     

Refined structure of cytochrome c3 at 1.8 A resolution

The structure of cytochrome c3 from the sulfate-reducing bacterium Desulfovibrio vulgaris Miyazaki has been successfully refined at 1.8 A resolution. The crystallographic R factor is 0.176 for 9907 significant reflections. The isotropic temperature factors of individual atoms were refined and a total of 47 water molecules located on the difference map were incorporated in the refinement. The four heme groups are closely packed, with adjacent pairs of heme planes being nearly perpendicular to each other. The fifth and the sixth ligands of the heme iron atoms are histidine residues with N epsilon 2-Fe distances ranging from 1.88 A to 2.12 A. The histidine co-ordination to the heme iron is different for each heme group. The heme groups are all highly exposed to solvent, although the actual regions exposed differ among the hemes. The four heme groups are located in different environments, and the heme planes are deformed from planarity. The differences in the heme structures and their environments indicate that the four heme groups are non-equivalent. The chemical as well as the physical properties of cytochrome c3 should be interpreted in terms of the structural non-equivalence of the heme groups. The characteristic secondary structural non-equivalence of the heme groups. The characteristic secondary structures of the polypeptide chain of this molecule are three short alpha-helices, two short beta-strands and ten reverse turns.     

The importance of quantitative M?ssbauer spectroscopy of MoFe-protein from Azotobacter vinelandii

The M?ssbauer spectra of MoFe-protein of Azotobacter vinelandii, as isolated under dithionite and taken at temperatures from 125 K to 175 K, are the sums of four resolved quadrupole doublets. Our results indicate that the currently accepted interpretation of these doublets can be questioned. Our data reduction method converts the M?ssbauer transmission spectra to source lineshape deconvolved absorption spectra linear in iron. We used these absorption spectra to determine the stoichiometry of the Fe clusters in MoFe-protein and we obtained much better fits if we assumed that there are four iron atoms in the 'Fe2+, doublet, two iron atoms in the 'S' doublet, twelve iron atoms in the 'D' doublet and sixteen iron atoms in the 'M' doublet. Therefore we propose that the MoFe-cofactor contains one molybdenum and eight iron atoms ('M'). We also argue that none of the previous M?ssbauer spectroscopic studies have been performed on the highest-activity preparation now obtainable, nor has there been any study to prove that the M?ssbauer spectra are independent of activity. We consider that the M?ssbauer spectroscopic studies of the MoFe-protein of nitrogenase are a re-opened and unsolved problem.     

Investigation of two deoxygenated haemoglobin-Haptoglobin complexes by Mössbauer spectroscopy

Haemoglobin Haptoglobin complexes formed when [Hp⁺]/[Hb] = 1/1 and [Hp]/[Hb] = 2/1 were investigated by ⁵⁷Fe Mössbauer spectroscopy. Both samples gave a spectrum consisting of a single quadrupole doublet. The temperature dependence of the quadrupole splitting was also identical for both samples. This proves that in both samples the nearest neighbour environment of the iron atom must be the same. A comparison with earlier investigations on myoglobin and haemoglobin indicates that the electronic structure of iron in the HbHp-complexes is similar to that in myoglobin.This work was supported by the Deutsche Forschungsgemeinschaft...     

Mössbauer spectroscopic studies on protoporphyrin IX iron(III) cyanide complexes

The Mössbauer and other spectral data of a dicyanoprotoporphyrin IX iron(III) complex are reported. The low quadrupole splitting is discussed in relation to known low-spin iron(III) porphyrin structures and their Mössbauer parameters.     

M?ssbauer spectroscopic study of the initial stages of iron-core formation in horse spleen apoferritin: evidence for both isolated Fe(III) atoms and oxo-bridged Fe(III) dimers as early intermediates

Ferritin stores iron within a hollow protein shell as a polynuclear Fe(III) hydrous oxide core. Although iron uptake into ferritin has been studied previously, the early stages in the creation of the core need to be clarified. These are dealt with in this paper by using M?ssbauer spectroscopy, a technique that enables several types of Fe(II) and Fe(III) to be distinguished. Systematic M?ssbauer studies were performed on samples prepared by adding 57Fe(II) atoms to apoferritin as a function of pH (5.6-7.0), n [the number of Fe/molecule (4-480)], and tf (the time the samples were held at room temperature before freezing). The measurements made at 4.1 and 90 K showed that for samples with n less than or equal to 40 at pH greater than or equal to 6.25 all iron was trivalent at tf = 3 min. Four different Fe(III) species were identified: solitary Fe(III) atoms giving relaxation spectra, which can be identified with the species observed before by EPR and UV difference spectroscopy; oxo-bridged dimers giving doublet spectra with large splitting, observed for the first time in ferritin; small Fe(III) clusters giving doublets of smaller splitting and larger antiferromagnetically coupled Fe(III) clusters, similar to those found previously in larger ferritin iron cores, which, for samples with n greater than or equal to 40, gave magnetically split spectra at 4.1 K. Both solitary Fe(III) and dimers diminished with time, suggesting that they are intermediates in the formation of the iron core. Two kinds of divalent iron were distinguished for n = 480, which may correspond to bound and free Fe(II).     

Mössbauer studies of the non-heme iron and cytochrome b559 in a Chlamydomonas reinhardtii PSI- mutant and their interactions with alpha-tocopherol quinone

Spin and valence states of the non-heme iron and the heme iron of cytochrome b559, as well as their interactions with alpha-tocopherol quinone (alpha-TQ) in photosystem II (PSII) thylakoid membranes prepared from the Chlamydomonas reinhardtii PSI- mutant have been studied using M?ssbauer spectroscopy. Both of the iron atoms are in low spin ferrous states. The Debye temperature of the non-heme is 194 K and of the heme iron is 182 K. The treatment of alpha-TQ does not change the spin and the valence states of the non-heme iron but enhances the covalence of its bonds. alpha-TQ oxidizes the heme iron into the high spin Fe3+ state. A possible role of the non-heme iron and alpha-TQ in electron flow through the PSII is discussed.     

Chloroperoxidase compound I: Electron paramagnetic resonance and M?ssbauer studies

The green primary compound of chloroperoxidase was prepared by freeze-quenching the enzyme after rapid mixing with a 5-fold excess of peracetic acid. The electron paramagnetic resonance (EPR) spectra of these preparations consisted of at least three distinct signals that could be assigned to native enzyme, a free radical, and the green compound I as reported earlier. The absorption spectrum of compound I was obtained through subtraction of EPR signals measured under passage conditions. The signal is well approximated by an effective spin Seff = 1/2 model with g = 1.64, 1.73, 2.00 and a highly anisotropic line width. M?ssbauer difference spectra of compound I samples minus native enzyme showed well-resolved magnetic splitting at 4.2 K, an isomer shift delta Fe = 0.15 mm/s, and quadrupole splitting delta EQ = 1.02 mm/s. All data are consistent with the model of an exchange-coupled spin S = 1 ferryl iron and a spin S' = 1/2 porphyrin radical. As a result of the large zero field splitting, D, of the ferryl iron and of intermediate antiferromagnetic exchange, S.J.S'.J approximately 1.02 D, the system consists of three Kramers doublets that are widely separated in energy. The model relates the EPR and M?ssbauer spectra of the ground doublet to the intrinsic parameters of the ferryl iron, D/k = 52 K, E/D congruent to 0.035, and A perpendicular (gn beta n) = 20 T, and the porphyrin radical.(ABSTRACT TRUNCATED AT 250 WORDS)     

M?ssbauer effect in Scenedesmus and spinach ferredoxins. The mechanism of electron transfer in plant-type iron–sulphur proteins

1. The Mössbauer spectra of Scenedesmus ferredoxin enriched in ⁵⁷Fe were measured and found to be identical with those of two other plant-type ferredoxins (from spinach and Euglena) that had been previously measured. Better resolved Mössbauer spectra of spinach ferredoxin are also reported from protein enriched in ⁵⁷Fe. All these iron–sulphur proteins are known to contain two iron atoms in a molecule that takes up one electron on reduction. 2. The Mössbauer spectra at 195°K have electric hyperfine structure only and show that on reduction the electron goes to one of the iron atoms, the other appearing to remain unchanged. 3. In the oxidized state, both iron atoms are in a similar chemical state, which appears from the chemical shift and quadrupole splitting to be high-spin Fe³⁺, but they are in slightly different environments. In the reduced state the iron atoms are different and the molecule appears to contain one high-spin Fe²⁺ and one high-spin Fe³⁺ atom. 4. At lower temperatures (77 and 4.2°K) the spectra of both iron atoms in the reduced proteins show magnetic hyperfine structure which suggests that the iron in the oxidized state also has unpaired electrons. This provides experimental evidence for earlier suggestions that in the oxidized state there is antiferromagnetic exchange coupling, which would result in a low value for the magnetic susceptibility. 5. In a small magnetic field the spectrum of the reduced ferredoxin shows a Zeeman splitting with hyperfine field (H_n) of 180kG at the nuclei. On application of a strong magnetic field H the spectrum splits into two spectra with effective fields H_n±H, thus confirming the presence of the two antiferromagnetically coupled iron atoms. 6. These results are in agreement with the model proposed by Gibson, Hall, Thornley & Whatley (1966); in the oxidized state there are two Fe³⁺ atoms (high spin) antiferromagnetically coupled and on reduction of the ferredoxin by one electron one of the ferric atoms becomes Fe²⁺ (high spin).     

Biophysical investigation of the ironome of human jurkat cells and mitochondria

The speciation of iron in intact human Jurkat leukemic cells and their isolated mitochondria was assessed using biophysical methods. Large-scale cultures were grown in medium enriched with (57)Fe citrate. Mitochondria were isolated anaerobically to prevent oxidation of iron centers. 5 K M?ssbauer spectra of cells were dominated by a sextet due to ferritin. They also exhibited an intense central quadrupole doublet due to S = 0 [Fe(4)S(4)](2+) clusters and low-spin (LS) Fe(II) heme centers. Spectra of isolated mitochondria were largely devoid of ferritin but contained the central doublet and features arising from what appear to be Fe(III) oxyhydroxide (phosphate) nanoparticles. Spectra from both cells and mitochondria contained a low-intensity doublet from non-heme high-spin (NHHS) Fe(II) species. A portion of these species may constitute the "labile iron pool" (LIP) proposed in cellular Fe trafficking. Such species might engage in Fenton chemistry to generate reactive oxygen species. Electron paramagnetic resonance spectra of cells and mitochondria exhibited signals from reduced Fe/S clusters, and HS Fe(III) heme and non-heme species. The basal heme redox state of mitochondria within cells was reduced; this redox poise was unaltered during the anaerobic isolation of the organelle. Contributions from heme a, b, and c centers were quantified using electronic absorption spectroscopy. Metal concentrations in cells and mitochondria were measured using inductively coupled plasma mass spectrometry. Results were collectively assessed to estimate the concentrations of various Fe-containing species in mitochondria and whole cells - the first "ironome" profile of a human cell.     

Solution studies on tetra(p-carboxyphenyl)porphyrin iron(II) using Mössbauer spectroscopy and electronic absorption spectroscopies

Mössbauer (78 K) and electronic absorption spectra (298 K) of tetra(p-carboxyphenyl)porphyrin iron(II) solutions are reported and discussed. Evidence for only two iron(II) complexes, the first an intermediate spin and the second a high spin complex, is found in the Mössbauer spectra. Electronic absorption spectra show a low spin complex is present at very low concentrations. It is observed from these results that the carboxy groups on the phenyl rings of this porphyrin greatly influence the chemistry. From the difference in the quadrupole splitting for the intermediate spin complex compared to that found in the tetra(p-sulphophenyl)porphyrin iron(II) system, the substituent on the phenyl ring clearly changes the electron density on the pyrrole nitrogen atoms.     

Heme iron state in various oxyhemoglobins probed using Mössbauer spectroscopy with a high velocity resolution

A comparative study of oxyhemoglobins from pig, rabbit, normal human and patients with blood system malignant diseases was performed using Mössbauer spectroscopy with a high velocity resolution at 90 K. Mössbauer spectra were fitted with the help of two models: using one quadrupole doublet (model of equivalent iron electronic structure in α- and β-subunits of hemoglobins) and superposition of two quadrupole doublets (model of non-equivalent iron electronic structure in α- and β-subunits of hemoglobins). The results obtained using both models demonstrated small variations of hyperfine parameters that were related to the heme iron state variation in different hemoglobins. These results were compared with structural and functional differences of the hemoglobins investigated.