Coupling of collective motions of the protein matrix to vibrations of the non-heme iron in bacterial photosynthetic reaction centers

Non-heme iron is a conservative component of type II photosynthetic reaction centers of unknown function. We found that in the reaction center from Rba. sphaeroides it exists in two forms, high and low spin ferrous states, whereas in Rsp. rubrum mostly in a low spin state, in line with our earlier finding of its low spin state in the algal photosystem II reaction center (Burda et al., 2003). The temperature dependence of the non-heme iron displacement studied by Mössbauer spectroscopy shows that the surrounding of the high spin iron is more flexible (Debye temperature ~ 165 K) than that of the low spin atom (~ 207 K). Nuclear inelastic scattering measurements of the collective motions in the Rba. sphaeroides reaction center show that the density of vibrational states, originating from non-heme iron, has well-separated modes between lower (4-17 meV) and higher (17-25 meV) energies while in the one from Rsp. rubrum its distribution is more uniform with only little contribution of low energy (~ 6 meV) vibrations. It is the first experimental evidence that the fluctuations of the protein matrix in type II reaction center are correlated to the spin state of non-heme iron. We propose a simple mechanism in which the spin state of non-heme iron directly determines the strength of coupling between the two quinone acceptors (Q_A and Q_B) and fast collective motions of protein matrix that play a crucial role in activation and regulation of the electron and proton transfer between these two quinones. We suggest that hydrogen bond network on the acceptor side of reaction center is responsible for stabilization of non-heme iron in different spin states.     

Spin States of Divalent Iron in Anhydrohemoglobin

Mössbauer spectra of anhydrohemoglobin establish the existence of two ferrous iron spin states in anhydrohemoglobin. Magnetic susceptibility measurements show that anhydrohemoglobin is paramagnetic and has an effective magnetic moment per iron atom of 3.5 ± 0.1 μ_B at low temperatures. In conjunction with the susceptibility results, the Mössbauer spectra indicate that the iron in anhydrohemoglobin is distributed between high and low spin states in roughly equal amounts.     

N-Alkylation of tripodal iron(III) imidazolate complexes: Reactivity and structure

The N-alkylation of iron(III) complexes of the tripodal imidazolate complexes derived from the Schiff base condensation of tris(2-aminoethyl)amine (tren) with three molar equivalents of 2-imidazolecarboxaldehyde (2ImH), 4-imidazolecarboxaldehyde (4ImH) or 4-methyl-5-imidazolecarboxaldehyde (5-Me4ImH) was investigated. While each complex possesses three nucleophilic imidazolate nitrogen atoms, only the complex derived from 2-imidazolecarboxaldehyde, Fetren(2Im)₃, was completely alkylated under the ambient conditions used in this work. Using methyl iodide as the alkylating agent, a correlation between spin state of the product and degree of methylation was observed. Low spin iron complexes were more nucleophilic than high spin systems. The structure reactivity relationship was exploited in the reaction of Fetren(2Im)₃ with methyl iodide and allyl iodide to give [Fetren(N-Me2Im)₃]²⁺ and [Fetren(N-allyl2Im)₃]²⁺. The products are iron(II) due to reduction of the iron(III) by iodide ion which builds up in the reaction mixture as the alkylation reaction proceeds. These complexes were characterized by a number of methods including EA, IR, ES-MS, Mössbauer spectroscopy, magnetic susceptibility and X-ray diffraction.     

X-ray and Fe Mössbauer study on a Fe(III) complex of 2-acetyl-1,3-indandione

Single-crystal X-ray structure of an iron(III) complex of 2-acetyl-1,3-indandione is resolved which reveals that a racemic mixture, composed of Δ-fac and Λ-fac stereoisomers, is formed. Both species have octahedral geometry, but slightly distinguishable. Detailed Mössbauer data indicate a high-spin electronic structure of the Fe(III) centres Fe1 and Fe2 with spin-spin magnetic relaxation process. Two different approaches for computer processing of the experimental Mössbauer spectra are considered.     

Mössbauer spectroscopy and electron paramagnetic resonance studies of iron metabolites inPseudomonas aeruginosa: Fe2+ and Fe3+ ferritin in57ferripyoverdine incubated cells and57ferric citrate fed cells

Pseudomonas aeruginosa samples were studied using Mössbauer spectroscopy and electron paramagnetic resonance (EPR). Samples included whole cells, membranes, and soluble fractions from cells which had been grown with⁵⁷ferric chloride,⁵⁷ferric citrate or incubated with⁵⁷ferripyoverdine. These experiments show for the first time thatP. aeruginosa can accumulate iron in a bacterioferritin when grown under conditions of iron limitation and incubated with its cognate ferrisiderophore, ferripyoverdine. Soluble fraction fromP. aeruginosa cells which were grown iron starved and incubated with⁵⁷ferripyoverdine for 120 min showed the presence of both a ferric and ferrous complex whose Mössbauer spectra matched that of bacterioferritin extracted fromAzotobacter vinelandii and whose EPR spectra showed a characteristic ferritin-like resonance. A second soluble fraction sample from cells which had been grown with⁵⁷ferric citrate also showed the presence of a species with the same EPR and Mössbauer parameters. In addition Western blotting confirmed the presence of bacterioferritin in the soluble fraction of the cells which had been incubated with ferripyoverdine.     

The Crystal Structure of Ferritin from Helicobacter pylori Reveals Unusual Conformational Changes for Iron Uptake

The crystal structure of recombinant ferritin from Helicobacter pylori has been determined in its apo, low-iron-bound, intermediate, and high-iron-bound states. Similar to other members of the ferritin family, the bacterial ferritin assembles as a spherical protein shell of 24 subunits, each of which folds into a four-α-helix bundle. Significant conformational changes were observed at the BC loop and the entrance of the 4-fold symmetry channel in the intermediate and high-iron-bound states, whereas no change was found in the apo and low-iron-bound states. The imidazole rings of His149 at the channel entrance undergo conformational changes that bear resemblance to heme configuration and are directly coupled to axial translocation of Fe ions through the 4-fold channel. Our results provide the first structural evidence of the translocation of Fe ions through the 4-fold channel in prokaryotes and the transition from a protein-dominated process to a mineral-surface-dominated process during biomineralization.     

Molecular characterization of the iron binding protein ferritin in Eisenia andrei earthworms

Ferritin is a storage protein that plays a key role in iron metabolism. In this study, we report on the sequence characterization of a ferritin-coding cDNA in Eisenia andrei earthworms isolated by RT-PCR using degenerated primers, and we suggest the presence of a putative IRE in the 5′-UTR of ferritin mRNA. The obtained ferritin sequence was compared with those of other animals showing sequence and structure homology in consensus sites, including the iron-responsive element (IRE) and ferroxidase centers. Despite the sequence homology in the E. andrei mRNA of ferritin with the sequences of other animals in consensus IRE sites, the presented cytosine in the IRE of E. andrei ferritin in the expected position does not form a conventional bulge. The presence of ferritin in the coelomic fluid of E. andrei was proven by iron staining assay. Moreover, aconitase activity in the coelomic fluid was assessed by aconitase assay, suggesting the presence of an iron regulatory protein. Quantitative analysis revealed changes in the gene expression levels of ferritin in coelomocytes in response to bacterial challenge, reaching the maximum level 8 h after the stimulation with both Gram-positive and Gram-negative bacteria.     

Design of novel iron compounds as potential therapeutic agents against tuberculosis

In the search for new therapeutic tools against tuberculosis two novel iron complexes, [Fe(L-H)₃], with 3-aminoquinoxaline-2-carbonitrile N¹,N⁴-dioxide derivatives (L) as ligands, were synthesized, characterized by a combination of techniques, and in vitro evaluated. Results were compared with those previously reported for two analogous iron complexes of other ligands of the same family of quinoxaline derivatives. In addition, the complexes were studied by cyclic voltammetry and EPR spectroscopy. Cyclic voltammograms of the iron compounds showed several cathodic processes which were attributed to the reduction of the metal center (Fe(III)/Fe(II)) and the coordinated ligand. EPR signals were characteristic of magnetically isolated high-spin Fe(III) in a rhombic environment and arise from transitions between m_S = ± 1/2 (g_eff ~ 9) or m_S = ± 3/2 (g_eff ~ 4.3) states. Mössbauer experiments showed hyperfine parameters that are typical of high-spin Fe(III) ions in a not too distorted environment. The novel complexes showed in vitro growth inhibitory activity on Mycobacterium tuberculosis H₃₇Rv (ATCC 27294), together with very low unspecific cytotoxicity on eukaryotic cells (cultured murine cell line J774). Both complexes showed higher inhibitory effects on M. tuberculosis than the “second-line” therapeutic drugs.     

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 $\text{74 ± 12}\text{A}\text{?}$.     

Rapid reduction of iron in horse spleen ferritin by thioglycolic acid measured by dispersive X-ray absorption spectroscopy

Summary The release of iron from ferritin is important in the formation of iron proteins and for the management of diseases in both animals and plants associated with abnormal accumulations of ferritin iron. Much more iron can be released experimentally by reduction of the ferric hydrous oxide core than by chelation of Fe³⁺ which has led to the notion that reduction is also the major aspect of iron release in vivo. Variations in the kinetics of reduction of the mineral core of ferritin have been attributed to the redox potential of the reductant, redox properties of the iron core, the structure of the protein coat, the analytical method used to detect Fe²⁺ and reactions at the surface of the mineral. Direct measurements of the oxidation state of the iron during reduction has never been used to analyze the kinetics of reduction, although Mössbauer spectroscopy has been used to confirm the extent of reduction after electrochemical reduction using dispersive X-ray absorption spectroscopy (DXAS). We show that the near edge of X-ray absorption spectra (XANES) can be used to quantify the relative amounts of Fe²⁺ and Fe³⁺ in mixtures of the hydrated ions. Since the nearest neighbors of iron in the ferritin iron core do not change during reduction, XANES can be used to monitor directly the reduction of the ferritin iron core. Previous studies of iron core reduction which measured by Fe²⁺ · bipyridyl formation, or coulometric reduction with different mediators, suggested that rates depended mainly on the redox potential of the electron donor. When DXAS was used to measure the rate of reduction directly, the initial rate was faster than previously measured. Thus, previously measured differences in reduction rates appear to be influenced by the accessibility of Fe²⁺ to the complexing reagent or by the electrochemical mediator. In the later stages of ferritin iron core dissolution, reduction rates drop dramatically whether measured by DXAS or formation of Fe²⁺ complexes. Such results emphasize the heterogeneity of ferritin core structure.     

The sedimentation properties of ferritins. New insights and analysis of methods of nanoparticle preparation

Background

Ferritin exhibits complex behavior in the ultracentrifuge due to variability in iron core size among molecules. A comprehensive study was undertaken to develop procedures for obtaining more uniform cores and assessing their homogeneity.

Methods

Analytical ultracentrifugation was used to measure the mineral core size distributions obtained by adding iron under high- and low-flux conditions to horse spleen (apoHoSF) and human H-chain (apoHuHF) apoferritins.

Results

More uniform core sizes are obtained with the homopolymer human H-chain ferritin than with the heteropolymer horse spleen HoSF protein in which subpopulations of HoSF molecules with varying iron content are observed. A binomial probability distribution of H- and L-subunits among protein shells qualitatively accounts for the observed subpopulations. The addition of Fe²⁺ to apoHuHF produces iron core particle size diameters from 3.8 ± 0.3 to 6.2 ± 0.3 nm. Diameters from 3.4 ± 0.6 to 6.5 ± 0.6 nm are obtained with natural HoSF after sucrose gradient fractionation. The change in the sedimentation coefficient as iron accumulates in ferritin suggests that the protein shell contracts ∼ 10% to a more compact structure, a finding consistent with published electron micrographs. The physicochemical parameters for apoHoSF (15%/85% H/L subunits) are M = 484,120 g/mol, ν? = 0.735 mL/g, s_20,w = 17.0 S and D_20,w = 3.21 × 10⁻⁷ cm²/s; and for apoHuHF M = 506,266 g/mol, ν? = 0.724 mL/g, s_20,w = 18.3 S and D_20,w = 3.18 × 10⁻⁷ cm²/s.

Significance

The methods presented here should prove useful in the synthesis of size controlled nanoparticles of other minerals.     

Spin crossover behavior of a one-dimensional polymeric-chain compound {[Fe(abpt)2(μ-Ni(CN)4)]·xH2O}n (x = 0.5 → 0): Synthesis, spectral, thermal and magnetic properties

A one-dimensional polymeric-chain iron(II)-nickel(II) cyanido-bridged complex of the composition {[Fe(abpt)₂(μ-Ni(CN)₄)]·0.5H₂O}_n (1·0.5H₂O), where abpt = 4-amino-3,5-di-2-pyridyl-4H-1,2,4-triazole, was prepared and characterized by elemental and thermal analyses, FTIR and ⁵⁷Fe Mössbauer spectroscopies, and magnetic measurements. The incomplete spin crossover phenomenon was observed with approximately 12% of the frozen high-spin fraction at low temperatures and with the spin transition critical temperature above room temperature.     

Identification and characterization of the iron compounds in bone marrow by means of Mössbauer spectrometry

In order to determine and to demonstrate the cellular iron molecular states in hematopoietic bone marrow, direct investigations were performed by means of different and complementary spectroscopic techniques: optical absorption, electron spin resonance and Mössbauer spectrometry. In fact, the latter appears to have been the most informative. In addition to the hemoglobin forms, five- and six-coordination ligand protoporphyrins IX (monomeric and polymeric stacking, respectively) were observed. A small amount of non-hemic high-spin iron III storage component (ferritin) was measured. No diferric transferrin was detected. A ferrous compound was also observed and attributed to the mitochondrial iron pool.     

Mössbauer and susceptibility experiments on different compounds of Fe3+-myoglobin

The static magnetic susceptibilities of different ferric high spin and low spin compounds of myoglobin (Mb(H₂O), Mb(H₂O) frozen under high pressure, MbF, MbCN) were measured in the temperature region between 4.2 K and 130 K. Mössbauer absorption experiments on Mb(H₂O) and MbF were perormed at different temperatures between 4.2 K and 180 K and in small magnetizing fields H1 kOe. The evaluation of our experimental data was performed with a Hamiltonian describing the 3d ⁵-configuration of the ferric iron by taking into account the Coulomb repulsion of the five electrons within the 3d-shell, the crystal electric field of -symmetry, and the spin-orbit coupling. The Hamiltonian contains the splitting energies of the five antibonding d-orbitals (d _xy , d _xz , d _yz , d _x 2_–y 2, d _z 2) as parameters. The values of these energies were obtained by a least squares fitting procedure using our magnetic susceptibility data together with the g-factors taken from the literature. In the case of MbF the energy difference between the two lowest Kramers doublets was also determined from present Mössbauer data. The results of the susceptibility and the Mössbauer data are in good agreement.The splitting energies of the 3d-orbitals can be correlated to the distances between the iron and its nearest neighbours. The different positions of the iron in the compounds investigated are discussed.     

Ferredoxin from Halobacterium of the Dead Sea — Mössbauer and EPR spectra and comparison with mössbauer spectrum of whole cells

Recoil-free measurements were carried out on a 2 Fe-ferredoxin, which was isolated and purified from an extreme halophile, Halobacterium of the Dead Sea. The spectrum of this ferredoxin in the oxidized state at 82 K is a superposition of two quadrupole doublets, representing two non-equivalent Fe³⁺ sites of equal intensity. The spectrum of the reduced ferredoxin is consistent with the presence of two pure classes of iron atoms, ferric (lower isomer shift) and ferrous (higher isomer shift). Interpretations of the recoil-free spectra are discussed. Mössbauer measurements were also carried out on frozen whole bacterial cells and the resulting spectrum was found to be quite different from that observed in the isolated ferredoxin. Tentative conclusions are reached concerning the localization of this ferredoxin in the cytosol of the Halobacteria.The EPR spectrum of the reduced ferredoxin obtained at 24 K exhibits rhombic symmetry with the following g values: 1.894, 1.984 and 2.07. These values are similar to those obtained with 2 Fe-ferredoxins of the plant type, except that the g _y and g _z values are somewhat higher. Both from the EPR and Mössbauer data, it is deduced that the spin relaxation times in reduced halophilic ferredoxins are faster than in the reduced plant ferredoxins.     

Oxido-reduction is not the only mechanism allowing ions to traverse the ferritin protein shell

Background

Most models for ferritin iron release are based on reduction and chelation of iron. However, newer models showing direct Fe(III) chelation from ferritin have been proposed. Fe(III) chelation reactions are facilitated by gated pores that regulate the opening and closing of the channels.

Scope of review

Results suggest that iron core reduction releases hydroxide and phosphate ions that exit the ferritin interior to compensate for the negative charge of the incoming electrons. Additionally, chloride ions are pumped into ferritin during the reduction process as part of a charge balance reaction. The mechanism of anion import or export is not known but is a natural process because phosphate is a native component of the iron mineral core and non-native anions have been incorporated into ferritin in vitro. Anion transfer across the ferritin protein shell conflicts with spin probe studies showing that anions are not easily incorporated into ferritin. To accommodate both of these observations, ferritin must possess a mechanism that selects specific anions for transport into or out of ferritin. Recently, a gated pore mechanism to open the 3-fold channels was proposed and might explain how anions and chelators can penetrate the protein shell for binding or for direct chelation of iron.

Conclusions and general significance

These proposed mechanisms are used to evaluate three in vivo iron release models based on (1) equilibrium between ferritin iron and cytosolic iron, (2) iron release by degradation of ferritin in the lysosome, and (3) metallo-chaperone mediated iron release from ferritin.     

Mössbauer effect study of gamma-irradiated human oxyhemoglobin

Summary Preliminary results of the Mössbauer effect study of human adult oxyhemoglobin in erythrocytes exposed to gamma-irradiation with doses of 100, 300 and 600 kGy are presented. Mössbauer spectra measured at 87 K have been analyzed in two ways. At first, to fit these spectra we used the four components oxyhemoglobin, deoxyhemoglobin, hemochromes and nonheme Fe(III) compound which had been obtained earlier from Mössbauer spectra of X-irradiated oxyhemoglobin by Chevalier et al. (1983). However, this approximation was not satisfactory. Then a new model of spectral fitting with five components was used. These were oxyhemoglobin, deoxyhemoglobin and components marked1, 2 and3. Using Mössbauer hyperfine parameters of each component the valence/spin states of iron ions were determined and possible complexes were considered. The most probable compounds for components1, 2 and3 were hematin and/orµ-oxodimers, methemoglobin hydroxide and/or hemichromes, and the high spin Fe(III) complex, respectively. Changes of the relative areas of Mössbauer subspectra of all components (its content in samples) versus doses were evaluated and the presence of the high and low spin aquomethemoglobin was indicated.     

Synthesis and Characterization of Tripodal Iron(II) Complexes Prepared from 2-Pyridinecarboxaldehyde and 1-Methyl-2-Imidazolecarboxaldehyde. Stabilization of Iron(II) Cations with N(6) Donor Sets

Reaction in air of iron(III) salts with tripodal ligands formed from the condensation of tris-(2-aminoethyl)amine (tren) with three equivalents of 2-pyridinecarboxaldehyde (py) or 1-methyl-2-imidazolecarboxaldehyde (NCH₃Im) yielded exclusively the iron(II) complexes, [Fetren(py)₃]X₂ (X=ClO₄ ⁻ or PF₆ ⁻) or [Fetren(NCH₃Im)₃](ClO₄)₂. The complexes were characterized by EA, IR, UV, Mössbauer, and mass spectroscopy. The structure of [Fetren(py)₃](ClO₄)₂ was determined at 100 and 290 K. The structures are essentially the same and feature an octahedral iron with facial coordination of pyridine and imine nitrogen atoms with average bond distances of 1.9747 and 1.9523 Å, respectively, at 290 K. The short Fe-N bond distances and lack of variation with temperature support a low spin, ¹A, assignment for the iron atom. The center nitrogen atom of the tren is essentially planar and is outside of bonding interaction with the iron, 3.45 Å. The low spin assignment is supported by Mössbauer spectroscopy, which reveals the presence of two low spin forms that are not in thermal equilibrium. In contrast, the Mössbauer spectrum of [Fetren(NCH₃Im)₃](ClO₄)₂ reveals the presence of both high spin, ⁵T, and low spin, ¹A, forms at room temperature, which on cooling to 77 K simplify to the low spin form.     

Characterization of iron compounds in tumour tissue from temporal lobe epilepsy patients using low temperature magnetic methods

Excess iron accumulation in the brain has been shown to be related to a variety of neurodegenerative diseases. However, identification and characterization of iron compounds in human tissue is difficult because concentrations are very low. For the first time, a combination of low temperature magnetic methods was used to characterize iron compounds in tumour tissue from patients with mesial temporal lobe epilepsy (MTLE). Induced magnetization as a function of temperature was measured between 2 and 140 K after cooling in zero-field and after cooling in a 50 mT field. These curves reveal an average blocking temperature for ferritin of 10 K and an anomaly due to magnetite at 48 K. Hysteresis measurements at 5 K show a high coercivity phase that is unsaturated at 7 T, which is typical for ferritin. Magnetite concentration was determined from the saturation remanent magnetization at 77 K. Hysteresis measurements at various temperatures were used to examine the magnetic blocking of magnetite and ferritin. Our results demonstrate that low temperature magnetic measurements provide a useful and sensitive tool for the characterisation of magnetic iron compounds in human tissue.Published online: March 2005