Protein dynamics: Mössbauer spectroscopy on deoxymyoglobin crystals

Mössbauer absorption experiments on ⁵⁷Fe of deoxygenated myoglobin crystals and on K₄⁵⁷Fe(CN)₆ dissolved in the water of metmyoglobin crystals were performed over a large temperature range. At low temperatures the mean square displacements, 〈x²〉, of the iron indicate solid-like behaviour of the whole system, whereas at higher temperatures protein-specific modes of motion contribute to 〈x²>. The protein dynamics are correlated with the mobility of the water within the protein crystals. A Brownian oscillator is used to model the protein-specific modes of motion measured at the ⁵⁷Fe nucleus. Three modes are necessary for understanding the Mössbauer spectrum. Two of them correspond to an extremely overdamped Brownian oscillator. The third mode can be understood as quasi-free diffusion. Whereas the protein molecule is frozen in conformational substates in the low temperature regime, it reaches transition states with a finite probability in the high temperature regime. The surface water mediates a possible trigger mechanism that switches on protein dynamics within a narrow temperature interval. Results from Mössbauer spectroscopy and from X-ray structure analysis are compared.     

Mössbauer spectroscopy of iron metabolism and iron intracellular distribution in liver of rats

Mössbauer spectroscopy was used to investigate the distribution of iron in rat organs and its localisation in liver subcellular fractions. A ⁵⁷Fe-sucrose complex solution was injected by 0.5 ml doses into tail veins of anmals every day, during a 6-day period. Mössbauer spectra were measured in spleen, blood, liver and liver subcellular fractions. The Mössbauer spectrum of a spleen sample has two symmetrical doublets, one with δ=0.6 mm/s and Δ=0.7 mm/s, and the other with δ=1.0 mm/s and Δ=2.35 mm/s. The Mössbauer spectrum of blood has parameters which are close to those for carboxyhemoglobin and oxyhemoglobin complexes. After the addition of sodium citrate, the proportion of the carboxyhemoglobin complexes increases. The Mössbauer spectrum of liver has a two-component pattern with two symmetrical doublets, the first with δ=0.6 mm/s and Δ=0.63 mm/s and the second with δ=1.4 mm/s and Δ=3.45 mm/s. The first component, which was identified as ferritin, is present in all subcellular fractions (800 × g_av sediment fraction, mitochondrial/lysosomal, microsomal and supernatant fractions), with its content in microsomal fraction. After the addition of NaBH₄ to mitochondrial/lysosomal fraction, about 20 % of the iron contained in ferritin was reduced. In the Mössbauer spectrum this is reflected by an appearance of a doublet with δ=0.85 mm/s and Δ=3.7 mm/s.     

Studies of the reactions of iron(II) ascorbate mixtures with molecular oxygen in solution

Mössbauer and electronic absorbance spectroscopic studies on the reactions of iron(II): ascorbic acid with molecular oxygen in aqueous and methanolic solutions are reported. Both spectroscopic techniques show that in the starting mixtures there are no iron(II): ascorbate complexes. On mixing the iron(II)/ascorbate solution with solutions containing molecular oxygen at pH 6–7 high spin iron(III) is observed in the Mössbauer spectrum. Coloured intermediates, the lifetimes of which are solvent dependent, are seen by stopped-flow spectrophotometry. We assign these coloured intermediates as iron(III) ascorbate complexes. The stoichiometry of the initial reaction between iron(II) and oxygen is shown to be 2Fe(II):O₂ by stopped-flow methods. A scheme for the overall reactions is discussed.     

A Mössbauer spectroscopic study of [phthalocyaninato tin(IV) tetracarbonyl iron(0)]

The action of Na₂Fe(CO)₄ and bis(chloro)phthalocyaninato tin(IV) yields phthalocyaninato tin(IV) tetracarbonyl iron(0). Mössbauer spectroscopic studies on the product indicate no change in formal oxidation state need be invoked to account for the formation of the metal-metal bond.     

Iron coordination geometry in full-length, truncated, and dehydrated forms of human tyrosine hydroxylase studied by Mössbauer and X-ray absorption spectroscopy

Full-length human tyrosine hydroxylase 1 (hTH1) and a truncated enzyme lacking the 150 N-terminal amino acids were expressed in Escherichia coli and purified either with or without (6×histidine) N-terminal tags. After reconstitution with ⁵⁷Fe(II), the Mössbauer and X-ray absorption spectra of the enzymes were compared before and after dehydration by lyophilization. Before dehydration, >90% of the iron in hTH1 had Mössbauer parameters typical for high-spin Fe(II) in a six-coordinate environment [isomer shift δ(1.8–77?K)=1.26–1.24?mm s^–1 and quadrupole splitting ΔE _Q=2.68?mm s^–1]. After dehydration, the Mössbauer spectrum changed and 63% of the area could be attributed to five-coordinate high-spin Fe(II) (δ=1.07?mm s^–1 and ΔE _Q=2.89?mm s^–1 at 77?K), whereas 28% of the iron remained as six-coordinate high-spin Fe(II) (δ=1.24?mm s^–1 and ΔE _Q=2.87?mm s^–1 at 77?K). Similar changes upon dehydration were observed for truncated TH either with or without the histidine tag. After rehydration of hTH1 the spectroscopic changes were completely reversed. The X-ray absorption spectra of hTH1 in solution and in lyophilized form, and for the truncated protein in solution, corroborate the findings derived from the Mössbauer spectra. The pre-edge peak intensity of the protein in solution indicates six-coordination of the iron, while that of the dehydrated protein is typical for a five-coordinate iron center. Thus, the active-site iron can exist in different coordination states, which can be interconverted depending on the hydration state of the protein, indicating the presence or absence of a water molecule as a coordinating ligand to the iron. The present study explains the difference in iron coordination determined by X-ray crystallography, which has shown a five-coordinate iron center in rat TH, and by our recent spectroscopic study of human TH in solution, which showed a six-coordinated iron center.     

Nature of iron deposits on the cardiac walls in β-thalassemia by Mössbauer spectroscopy

An identification of the nature and an estimation of the particle size distribution of the iron deposits on thalassemic heart tissue is carried out by variable temperature Mössbauer spectroscopy. Comparison of Mössbauer spectra obtained for the thalassemic heart tissue (I) with those of normal heart tissue (II) and of horse spleen ferritin (III) identifies the iron deposits to be small, superparamagnetic particles of ferritin and/or hemosiderin, two closely related iron storage proteins containing an iron core of (FeOOH)₈(FeO · OPO₃H₂). The dependence of the superparamagnetic relaxation time, τ₂, of magnetically ordered fine particles on their volume V via the magnetic anisotropy constant K of the material and the condition τ > τ_L, the Larmor precession time of the nuclear magnetic moment of ⁵⁷Fe about an effective magnetic field, for observation of hyperfine structure are used in analyzing the Mössbauer data to yield the particle size distribution. Particle diameters are estimated to be $\text{74 ± 12}\text{A}\text{?}$.     

Iron thioselenocarbamates

The synthesis of iron tris-thioselenocarbamates, Fe(R₂tsc)₃, is described. Magnetic properties and Mössbauer data of these new compounds are compared to those of the corresponding dithiocarbamate and diselenocarbamate compounds. Moreover the synthesis and Mössbauer data of the new mixed-ligand compounds Fe(R₂tsc)₂X (XCl, Br, I), CpFe(Et₂tsc)(CO) and Fe(Et₂tsc)₂(CO)₂ are reported.     

Mössbauer and spectroscopic studies on substituted tetraphenylporphyrinato iron(III) complexes in aqueous solutions and the formation of the μ-oxo-bridged species

Electronic and ⁵⁷Fe Mössbauer spectra are reported for two new water-soluble porphyrinato iron(III) complexes. Equilibrium constants for μ-oxo bishaem formation are calculated assuming two protons are released.Comparisons are made of the data with other porphyrinato iron(III) systems and it is shown that, in the absence of well-defined fifth ligands, the mononuclear species in acidic solution probably contain two axial water ligands. The μ-oxo bishaems do not contain water or hydroxide coordinated to iron but may hold water by hydrogen-bonding to the oxygen bridge or possibly by aquation of the porphyrin ligands.μ-Oxo bridge formation is controlled by the acid strength of the water coordinated to the iron in the mononuclear species, low pK_a values assisting oxo-bridge formation. Such low pK_a values are assisted by electron-attracting substituents on the porphyrin periphery. It is noted that this same property assists the stabilisation of iron(II) complexes. Steric inhibition of oxo-bridge formation requires large substituents, unsubstituted phenyl groups being apparently not large enough.     

Towards an understanding of the quadrupole splittings found in the Mössbauer spectroscopic data of a number of substituted tetraphenylporphyriniron(III) halides

Mononuclear and μ-oxo-dimers of a series of unsymmetrical substituted tetraphenylporphyriniron(III) complexes have been prepared and studied using Mössbauer spectroscopy. The Mössbauer data are discussed and compared to the other known data for similar high spin Fe(III) porphyrinsA good correlation between Hammet σ_R° (the mesomeric values) of the porphyrin substituent and the Mössbauer quadrupole splitting (Δ) has been found.     

Fe3O4 precipitation in magnetotactic bacteria

Using Mössbauer resonance spectroscopy of ⁵⁷Fe, we have determined the nature and distribution of major iron compounds in the magnetotactic bacterium Aquaspirillum magnetotacticum. In addition to magnetite (Fe₃O₄), cells contained a low-density hydrous ferric oxide, a high-density hydrous ferric oxide (ferrihydrite), and ferrous iron. Analysis at different temperatures of whole cells harvested early and late in growth, of mutant cells unable to synthesize magnetite, and of cell fractions enriched in ⁵⁷Fe indicated that Fe₃O₄ precipitation resulted from partial reduction of the high-density hydrous ferric oxide precursor.     

Investigation of iron pools in cucumber roots by Mössbauer spectroscopy: direct evidence for the Strategy I iron uptake mechanism

Distinct chemical species of iron were investigated by Mössbauer spectroscopy during iron uptake into cucumber roots grown in unbuffered nutrient solution with or without ⁵⁷Fe-citrate. Mössbauer spectra of iron deficient roots supplied with 10–500 μM ⁵⁷Fe-citrate for 30–180 min and 24 h and iron-sufficient ones, were recorded. The roots were analysed for Fe concentration and Fe reductase activity. The Mössbauer parameters in the case of iron-sufficient roots revealed high-spin iron(III) components suggesting the presence of Fe^III-carboxylate complexes, hydrous ferric oxides and sulfate–hydroxide containing species. No Fe^II was detected in these roots. However, iron-deficient roots supplied with 0.5 mM ⁵⁷Fe^III-citrate for 30 min contained significant amount of Fe^II in a hexaaqua complex form. This is a direct evidence for the Strategy I iron uptake mechanism. Correlation was found between the decrease in Fe reductase activity and the ratio of Fe^II–Fe^III components as the time of iron supply was increased. The data may refer to a higher iron reduction rate as compared to its uptake/reoxidation in the cytoplasm in accordance with the increased reduction rate in iron deficient Strategy I plants.     

Complexes of chromium(II) and iron(II) with hexamethylphosphoramide

The chromium(II) complexes CrX₂(HMPA)₂, in which X = Cl or Br and HMPA is hexamethylphosphoramide, and Cr(HMPA)₄(BF₄)₂ have been prepared. The effective magnetic moments show little deviation from the value expected for high spin chromium(II) from room temperature to liquid nitrogen temperature. The diffuse reflectance spectra suggest that the chromium ions are in a strongly distorted six coordinate environment. The iron(II) complexes, FeX₂(HMPA)₂, X = Br or I, and [Fe(HMPA)_4](BF₄)₂, from their magnetic behaviour and Mössbauer and electronic spectra, contain tetrahedral iron(II). The isomer shift of the last complex is the most positive so far reported for a tetrahedral iron(II) complex.     

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.     

Complexes of diphosphaferrocenes with Lewis acids and Cu(I), Ag(I) salts

Complexes of diphosphaferrocenes have been prepared using (a) Lewis acids BF₃·Et₂O, A1C1₃ and (b) Cu(I) and Ag(I) salts. The ³¹P spectra showed that BF₃·Et₂O complexes at phosphorus (characterised by a large downfield shift of 103 ppm) whereas all the other adducts involved binding at iron (characterised by large upfield shifts of 36–94 ppm). Deep purple adducts were isolated and characterised for the reaction of 2, 2′, 5, 5′-tetraphenyldipho sphaferrocene with silver trifluoroacetate and triflate and also with copper(I) iodide. Similar complexes were isolated from 3, 3′, 4, 4′-tetramethyldiphosphaferrocene. ⁵⁷Fe Mössbauer spectroscopy of the adducts showed that unlike the ferrocene system, quadrupole splittings generally decreased on iron complexation. The results are discussed in terms of current MO theory of ferrocene and diphosphaferrocene.     

Model complexes for the high spin iron(II) state in the catalytic cycle of cytochrome P450

Studies using ⁵⁷Fe Mössbauer spectroscopy on frozen concentrated solutions of two iron(II) porphyrins in the presence of a large excess of thiophenol or 2-mercaptoethanol are reported. The iron(II) porphyrins used were photoporphyrin IX iron(II) (PPIXFe(II)) and tetra(p-sulphophenyl)porphinato-iron(II) (TTPPSFe(II)). Evidence for high-spin five-coordinate iron(II) complexes were found for both iron(II) porphyrins with thiophenol, but no reaction was found to occur with 2-mecaptoethanol. In contrast to these findings in solutions dilute in TPPSFe(II) evidence was found (from electronic absorption spectroscopy and spectrophotometric titrations) for both thiophenol and 2-mercaptoethanol acting as axial ligands in high-spin five-coordinate TPPSFe(II) species. Mössbauer data for frozen solutions containing TPPSFe(II), carbon monoxide (CO) and either thiol, are consistent with the presence of only low-spin six-coordinate iron(II) complexes. These latter complexes are deduced to contain both a thiol and a CO molecule as axial ligands> These results are discussed in relation to earlier work on PPIXFe(II)-thiol solutions and also in relation to the high-spin iron(II) state in the catalytic cycle of cytochrome P450.The reactions of TPPSFe(II) with excess of either 2-mercaptoethanol or ethyl 2-mercaptoacetate in air are also reported and their complex nature discussed.     

High-spin iron(II) complexes of ortho-functionalized paraquinones as models for quinone binding sites in reaction centres of photosynthetic bacteria

In an attempt to mimic the iron–quinone couple of bacterial reaction center, spectral, magnetic and Mössbauer characterization of seven high-spin iron(II) complexes of the ortho-functionalized paraquinones is reported where the quinones are coordinated in their fully oxidized, monoanionic form.     

Preparation and properties of Fe3+-amino acid complexes: Crystalline complexes with aliphatic amino acids

Crystalline complexes of Fe³⁺ and several aliphatic amino acids have been prepared. All have a basic molecular constitution [Fe[AA]₂H₂O]₃O (ClO₄)₇, as determined by optical, magnetic, and Mössbauer measurements. The physical properties of these compounds display a marked similarity to those of ferritin.     

Synthesis,characterization and antitumor activity of some metal complexes of 3- and 5-substituted salicylaldehyde o-hydroxybenzoylhydrazones

Complexes of Mn(II), Fe(III), Fe(II), Co(II), Ni(II), Cu(II), Zn(II) and Pt(II) with 3- and 5-substituted salicylaldehyde o-hydroxybenzoylhydrazones (XSBH, X = H, 3-NO₂, 3-CH₃O, 5-Cl, 5-Br, 5-CH₃ or 5-NO₂) have been prepared and characterized by elemental analysis, conductance measurements, magnetic susceptibilities (from room temperature down to liquid nitrogen temperature) and spectral studies. These studies indicate the following structures: monomeric, high-spin, distorted octahedral for Mn(XSBH)₂; monomeric, high-spin, five-coordinate for Fe(XSBH)SO₄·H₂O; dimeric, high-spin phenoxide bridged, five-coordinate for Fe(XSBH)Cl; dimeric, high-spin five-coordinate for Co(XSBH)Cl·2H₂O; dimeric low-spin, five-coordinate for Ni(XSBH)Cl·2H₂O; dimeric, four-coordinate for Zn(XSBH); and a square-planar structure for M(XSBH)Cl·H₂O (M = Cu(II) or Pt(II).Intermolecular antiferromagnetic exchange interactions are present in Fe(III) complexes, where the exchange parameter (J) is ca. −8.0 cm⁻¹ for these complexes. The Fe(III) complexes exhibit asymmetric quadrupole split doublets in their ⁵⁷Fe Mössbauer spectra. The asymmetry is found to be temperature dependent with relatively symmetrical doublets seen at low temperature. The polycrystalline ESR spectra of Cu(II) complexes are isotropic and indicate a d_x²−y² ground state in square-planar stereo-chemistry. All these metal complexes have been screened for their antitumor activity against the P 388 lymphocytic leukaemia test system in mice and enhanced antitumor activity relative to the free ligand was found but no significant activity at the dosages used.     

Investigations of intercalation in inorganic solids with layered structures: iron-57 mössbauer spectroscopy studies of size fractionated and iron-exchanged montmorillonite clays

⁵⁷Fe mössbauer spectroscopy has been used to examine some naturally occurring layer silicates in which cations located in exchange sites in the interlayer regions can be replaced by other species. The ⁵⁷Fe Mössbauer spectra recorded from differing size fractions of two types of non-exchanged and sodium-exchanged montmorillonite clays were found to be independent of the fraction size. The spectra have been interpreted in terms of the occupation by iron(III) of a heterogeneity of similar sites within the montmorillonite lattice. No justification has been obtained for computer analysis of the data in terms of more than one characterisable lattice site and no evidence has been found for the association of any iron oxyhydroxide impurity with the montmorillonite fractions.The ⁵⁷Fe Mössbauer parameters recorded from iron(III)-exchanged montmorillonite, in which iron(III) species are intercalated within the layers, show that the process is best performed at fairly low pH using low concentrations of iron(III). Failure to control such conditions can result in the formation of iron oxyhydroxides or hydrolysed iron(III) species. The preparation of iron(II)-exchanged montmorillonite was accompanied by partial oxidation of the iron(II) to iron(III).     

Some μ-oxo compounds of iron-hemiporphyrazine. Synthesis and properties

Details of the conditions necessary for preparing the recently reported μ-oxo-ironhemiporphyrazinate compounds (Fe(IV)hpO)_n and [(Fe(III)hp)₂O]H₂O are given. Two further homologous μ-oxo Fe(III)hemiporphyrazinates are described: an amorphous μ-oxo dimer and a μ-oxo dimer HCl adduct. The thermal and spectroscopic (UV-Vis, IR, Mössbauer, XPS) properties and magnetic susceptibilities of these compounds have been investigated. The reducibility of these species in solution, in the presence and absence of O₂, has been monitored. The results do not indicate a change in oxidation state of the iron in (FehpO)_n. Attempts to obtain the hemiporphyrazinato Fe(III) chloride are described.