Catechol releases iron(III) from ferritin by direct chelation without iron(II) production

It has been traditionally considered that catechols release iron from ferritin by reduction to iron(II), which diffuses through the ferritin channels into the intracellular milieu where it participates in the Fenton reaction, producing highly toxic hydroxyl radicals. However, in the present work we have proved that the mechanism of the release of iron from ferritin by catechol does not take place by iron(II) reduction but by direct iron(III) chelation and therefore without iron(II) production. A possible extension of these findings to other catechols is discussed on the basis of the stability with respect to the internal redox reaction of the iron(III)-catechol complexes.     

Oxygen catalyzed mobilization of iron from ferritin by iron(III) chelate ligands

Tridentate chelate ligands of 2,6-bis[hydroxy(methyl)amino]-1,3,5-triazine family rapidly release iron from human recombinant ferritin in the presence of oxygen. The reaction is inhibited by superoxide dismutase, catalase, mannitol and urea. Suggested reaction mechanism involves reduction of the ferritin iron core by superoxide anion, diffusion of iron(II) cations outside the ferritin shell, and regeneration of superoxide anions through oxidation of iron(II) chelate complexes with molecular oxygen.     

Release of iron from ferritin by aceto- and benzohydroxamic acids

The release of iron from ferritin by aceto- and benzohydroxamic acids was studied at two different iron chelator concentrations (100 and 10 mM), at two pH values (7.4 and 5.2), and in the presence or absence of urea. Collectively, the results demonstrate that both aceto- and benzohydroxamic acids remove iron from ferritin. Aceto- and benzohydroxamic acids penetrate the ferritin shell and react directly with the iron core of the ferritin cavity probably forming mono(hydroxamate) iron(III) complexes which exit ferritin and react with the excess hydroxamate in the solution to produce bis(hydroxamate) iron(III) complexes. The sizes of both the benzohydroxamic acid and the mono(benzohydroxamate) iron(III) complex, 6 and 7 A, respectively, are larger than that of the ferritin channels which indicates the flexibility of the channels to allow the entry and exit of these molecules. The size of the hydroxamic acid influenced the effectiveness of the iron release from ferritin following the expected trend with smaller iron chelators showing greater effectiveness. Likewise, the percentage of iron removed from ferritin was pH-dependent; the percentage of iron removed at pH 5.2 was greater than that at pH 7.4. Finally, the presence of urea, capable of opening the ferritin channels, dramatically increased the effectiveness of the iron chelator in removing iron from ferritin, especially at pH 7.4.     

Hydrazone chelators for the treatment of iron overload disorders: iron coordination chemistry and biological activity

The potentially tridentate ligand 2-pyridinecarbaldehyde isonicotinoyl hydrazone (HPCIH) and its analogues are an emerging class of orally effective Fe chelators that show great promise for the treatment of Fe overload diseases. Herein, we present an extensive study of the Fe coordination chemistry of the HPCIH analogues including the first crystallographically characterised Fe(II) complex of these chelators. Unlike most other clinically effective Fe chelators, the HPCIH analogues bind Fe(II) and not Fe(III). In fact, these chelators form low-spin bis-ligand Fe(II) complexes, although NMR suggests that the complexes are close to the high-spin/low-spin crossover. All the Fe complexes show a high potential Fe(III/II) redox couple (> 500 mV vs. NHE) and cyclic voltammetry in aqueous or mixed aqueous/organic solvents is irreversible as a consequence of a rapid hydration reaction that occurs upon oxidation. A number of the HPCIH analogues show high activity at inducing Fe efflux from cells and also at preventing Fe uptake by cells from the serum Fe transport protein transferrin. As a class of ligands, these chelators are more effective at reducing Fe uptake from transferrin than inducing Fe mobilisation from cells. This may be related to their ability to intercept Fe(II) after its release from transferrin within the cell. Our studies indicate that their Fe chelation efficacy is due, at least in part, to the fact that these ligands and their Fe(II) complexes are neutral at physiological pH (7.4) and sufficiently lipophilic to permeate cell membranes.     

Mössbauer studies of iron incorporation into ferritins

Iron(III) monomers, dimers and clusters have been identified by Mössbauer spectroscopy during the initial stages of iron incorporation into ferritins, following Fe(II) oxidation. Iron(III) monomers seem to arise from dimer dissociation. Some of the monomers are transferred from iron poor to iron rich ferritin molecules, where they join the iron core clusters. Horse spleen ferritin, several variants of human H chain ferritin andEscherichia coli ferritin (Ec-FTN) can all accept the iron from human H chain ferritin. The small iron cores of Ec-FTN are different from those of mammalian ferritins, which indicates that the structure of the iron core depends on the protein shell.     

Magnetic–fluorescent Langmuir–Blodgett films of fluorophore-labeled ferritin nanoparticles

We have covalently coupled fluorophore 4-(2-hydroxyethoxy)-7-nitro-2,1,3-benzoxadiazole (NBD) to the external ferritin shell through lysine residues. An increase in the luminescence quantum yield of the fluorescent ferritin particles and a blue shift in its emission peak compared to individual fluorophore were observed. The study of the particles by transmission electron microscopy showed that the native iron core ferritin is intact and that no degradation occurs during chemical functionalization of the protein shell. The NBD-labeled ferritin particles are water soluble, which allowed their controlled deposition by the Langmuir–Blodgett (LB) technique. Superparamagnetic and fluorescent properties of the particles are preserved within the LB film.     

Iron chelating agents for the treatment of iron overload

The importance of iron chelators in medicine has significantly increased in recent years. Iron is essential for life but it is also potentially more toxic than other trace elements. This is because we lack effective means to protect human cells against iron overload and because of the role of iron in the generation of free radicals. In order to protect patients from the consequences of iron toxicity, iron chelating agents have been introduced in clinical practice. Unfortunately, the ideal chelator for treating iron overload in humans has not been identified yet. In this paper we examine a few characteristics of iron chelators, with some emphasis on the effects of redox cycling, on absorption mechanisms and on some properties of the pFe. A brief summary is then made of the chelators recently proposed or in development for the treatment of iron overload.     

Direct determination of iron in ferritin using the polarographic catalytic current

A new method for the determination of iron in ferritin using the polarographic catalytic current has been developed. Iron is released in NaOH and triethanolamine (TEA) solution and subsequently determined based on the polarographic catalytic wave in the presence of bromate with the peak potential of –1.0 V (vs.SCE). The 2.5-derivative peak height of iron is linearly proportional to its concentration in the range of 8.0 × 10^–8–1.0 × 10^–6 g/ml with a correlation coefficient of 0.9991. The method has been applied to the determination of iron in human fetal liver and horse-spleen ferritin and the recovery is 95–112%.     

Computational study of iron(II) and -(III) complexes with a simple model human H ferritin ferroxidase center

Interaction of iron ions with a six-amino acid model of the ferroxidase center of human H chain ferritin has been examined in density functional theory calculations. The model, based on experimental studies of oxidation of Fe2+ at the center, consists of Glu27, Glu62, His65, Glu107, Gln141, and Ala144. Reasonable structures are obtained in a survey of types of iron complexes inferred to occur in the ferroxidase reaction. Structures of complexes of the model center with one and two Fe2+ ions, with diiron(III) bridged by peroxide and bridged by oxide-peroxide combinations, have been optimized. Calculations on diiron(III) complexes confirm that stable peroxide-bridged complexes can form and that the Fe-Fe distance in at least one is consistent with the experimental Fe-Fe distance observed in the blue peroxodiferric complex of ferritin.     

Nanophase iron phosphate, iron arsenate, iron vanadate, and iron molybdate minerals synthesized within the protein cage of ferritin

Nanoparticles of iron phosphate, iron arsenate, iron molybdate, and iron vanadate were synthesized within the 8 nm interior of ferritin. The synthesis involved reacting Fe(II) with ferritin in a buffered solution at pH 7.4 in the presence of phosphate, arsenate, vanadate, or molybdate. O2 was used as the oxidant to deposit the Fe(III) mineral inside ferritin. The rate of iron incorporation into ferritin was stimulated when oxo-anions were present. The simultaneous deposition of both iron and the oxo-anion was confirmed by elemental analysis and energy-dispersive X-ray analysis. The ferritin samples containing iron and one of the oxo-anions possessed different UV/vis spectra depending on the anion used during mineral formation. TEM analysis showed mineral cores with approximately 8 nm mineral particles consistent with the formation of mineral phases inside ferritin.     

Magnetic Langmuir-Blodgett films of ferritin with different iron contents

Magnetic Langmuir-Blodgett films of four ferritin derivatives with different iron contents containing 4220, 3062, 2200, and 1200 iron atoms, respectively, have been prepared by using the adsorption properties of a 6/1 mixed monolayer of methyl stearate (SME) and dioctadecyldimethylammonium bromide (DODA). The molecular organization of the mixed SME/DODA monolayer is strongly affected by the presence of the water-soluble protein in the subphase as shown by pi-A isotherms, BAM images, and imaging ellipsometry at the water-air interface. BAM images reveal the heterogeneity of this mixed monolayer at the air-water interface. We propose that the ferritin is located under the mixed matrix in those regions where the reflectivity is higher whereas the dark regions correspond to the matrix. Ellipsometric angle measurements performed in zones of different brightness of the mixed monolayer confirm such a heterogeneous distribution of the protein under the lipid matrix. Transfer of the monolayer onto different substrates allowed the preparation of multilayer LB films of ferritin. Both infrared and UV-vis spectroscopy indicate that ferritin molecules are incorporated within the LB films. AFM measurements show that the heterogeneous distribution of the ferritin at the water-air interface is maintained when it is transferred onto solid substrates. Magnetic measurements show that the superparamagnetic properties of these molecules are preserved. Thus, marked hysteresis loops of magnetization are obtained below 20 K with coercive fields that depend on the number of iron atoms of the ferritin derivative.     

Origin of the unusual kinetics of iron deposition in human H-chain ferritin

From microorganisms to humans, ferritin plays a central role in the biological management of iron. The ferritins function as iron storage and detoxification proteins by oxidatively depositing iron as a hydrous ferric hydroxide mineral core within their shell-like structures. The mechanism by which the mineral core is formed has been the subject of intense investigation for many years. A diiron ferroxidase site located on the H-chain subunit of vertebrate ferritins catalyzes the oxidation of Fe(II) to Fe(III) by molecular oxygen. A previous stopped-flow kinetics study of a transient mu-peroxodiFe(III) intermediate formed at this site revealed very unusual kinetics curves, the shape of which depended markedly on the amount of iron presented to the protein. In the present work, a mathematical model for catalysis is developed that explains the observed kinetics. The model consists of two sequential mechanisms. In the first mechanism, turnover of iron at the ferroxidase site is rapid, resulting in steady-state production of the peroxo intermediate with continual formation of the mineral core until the available Fe(II) in solution is consumed. At this point, the second mechanism comes into play whereby the peroxo intermediate decays and the ferroxidase site is postulated to vacate its complement of iron. The kinetic data reveal for the first time that Fe(II) in excess of that required to saturate the ferroxidase site promotes rapid turnover of Fe(III) at this site and that the ferroxidase site plays a role in catalysis at all levels of iron loading of the protein (48-800 Fe/protein). The data also provide evidence for a second intermediate, a putative hydroperoxodiFe(III) complex, that is a decay product of the peroxo intermediate.     

Mass spectrometric characteristics and kinetics of iron release in visceral mass of Saccostrea cucullata

Ferritins with electrophoretic homogeneity were prepared from the visceral mass of Saccostrea cucullata in batch. The native PAGE approach showed similar electrophoretic mobility among pig pancreatic ferritin, liver ferritin of Dasyatis akajei, and visceral mass ferritin of Saccostrea cucullata. SDS-PAGE indicated that the Saccostrea cucullata visceral ferritin (SCVF) consisted of a single subunit type and had a molecular weight (MW) of approximately 20 kDa, suggesting that the protein shell in SCVF was composed of a single subunit. In addition, peptide mass fingerprinting and transmission electron microscopy were used to identify SCVF further, and to observe its molecular structure. We found that the molecular structure in SCVF was similar to that of most mammalian ferritins, which are composed of a protein shell and an iron core. The results of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry under the assistance of an acidic matrix, sinapic acid, also showed that SCVF was composed of a single subunit type and its subunit MW was calculated to be 19871.042 Da in the absence of heme. Kinetics analysis revealed that the complete process of iron release fitted the law of a first-order reaction, which is similar to that of most ferritins in mammals. Similar to bacterial ferritin, studies indicated that the shell consisted of a single subunit type and showed similar kinetics of iron release, suggesting that this subunit plays two important roles in iron release and storage, and that it shows different stability and intensity of interaction in carrying out its physiological functions in SCVF.     

Electron transfer of horse spleen ferritin at gold electrodes modified by self-assembled monolayers

Electron transfer is known to be an important step in the sequestering of iron by cellular ferritin. In this work, direct electron transfer between ferritin and a gold electrode was performed in order to probe its electron transfer kinetics. Gold electrodes were modified by the formation of self-assembled monolayers of 3-mercapto-propionic acid on the gold surface. Cyclic voltammetry using these electrodes shows that ferritin exhibits slow electron transfer kinetics at low potentials, yet fairly well-defined current—potential curves. In addition, the voltammetry indicates that adsorption of ferritin precedes the electron transfer step. Controlled potential electrolysis measurements yielded an n-value of 1910 electrons transferred per mole of ferritin. Cyclic voltammetry of a solution containing ferritin as well as nitrilotriacetate yields no electrolytic currents at potentials where the iron—nitrilotriacetate complex undergoes redox reactions, indicating that the currents observed in the voltammetry of ferritin were not due to free iron in the ferritin sample. In addition, the voltammetry of iron-free ferritin (apoferritin) did not yield appreciable currents, providing additional support to the suggestion that the observed voltammetric currents were due to the redox reactions of ferritin iron. Self-assembled monolayers containing carboxylate end groups effectively promoted the direct electron transfer of ferritin at a gold electrode, thus demonstrating that the electron transfer mechanisms of ferritin can now be probed electrochemically.     

Structure and function of ferritin

Ferritin is the major iron storage protein of mammals and consists of up to 4500 atoms of ferric iron surrounded by a shell of protein subunits. The protein component, apoferritin, consists of 24 identical polypeptide chains each of molecular weight 18500. The function of ferritin is to store iron in a soluble form from which it can be readily mobilized. Recent results concerning the structure of the protein are reported, and progress in the elucidation of the mechanisms whereby iron is introduced into apoferritin and released from ferritin is reviewed.     

The Electrode Behavior and the Storing Capacity from Liver Ferritin of Dasynatis Akajei

Ferritin, a metallic protein, is a special biological large molecule that plays two central physiological functions in living cell simultaneously^[1]. In addition, the ferritin is able to express two functions having activity of H₂-uptake and exhibiting electrode behavior that picks the electrons up from a bare platinum electrode directly under anaerobic condition^[2-3]. In this paper, a direct electrochemical technology under aerobic conditions is employed to study kinetics of iron release from liver ferritin of Dasyatis akajei (DALF). Using aerobic condition, an electron tunnel across the ferritin shell plays still a critical role in taking the electrons from a bare platinum electrode for iron release without any mediator aid. Moreover, using similar condition, two reduction potentials of the ferritin core with -135 mV and -416 mV vs. NHE are measured by the technology. Unlike other processes of iron release, a kinetic study for complete iron release is confirmed to show a triphasic behavior defined as zero-order reaction. DALF is known to heme bind to generate a hemeoprotein, named DALF_h The heme function has been investigated to accelerate rate of iron release. Using similar studies, the kinetic datum indicates that the rate of iron release depends tightly on not only the reduction potential of the electrode, but also the regulation capacity of protein shell itself.     

Comparative structural and chemical studies of ferritin cores with gradual removal of their iron contents

Transmission Electron Microscopy (TEM), X-ray Absorption Near Edge Spectroscopy (XANES), Electron Energy-Loss Spectroscopy (EELS), Small-Angle X-ray Scattering (SAXS), and SQUID magnetic studies were performed in a batch of horse spleen ferritins from which iron had been gradually removed, yielding samples containing 2200, 1200, 500, and 200 iron atoms. Taken together, findings obtained demonstrate that the ferritin iron core consists of a polyphasic structure (ferrihydrite, magnetite, hematite) and that the proportion of phases is modified by iron removal. Thus, the relative amount of magnetite in ferritin containing 2200 to 200 iron atoms rose steadily from approximately 20% to approximately 70% whereas the percentage of ferrihydrite fell from approximately 60% to approximately 20%. These results indicate a ferrihydrite-magnetite core-shell structure. It was also found that the magnetite in the ferritin iron core is not a source of free toxic ferrous iron, as previously believed. Therefore, the presence of magnetite in the ferritin cores of patients with Alzheimer's disease is not a cause of their increased brain iron(II) concentration.     

A new approach to the ferritin iron core growth: influence of the H/L ratio on the core shape

An electron microscopy study, in combination with modeling and image simulation, of four different reconstituted ferritin samples: recombinant human H and L homopolymers, and H and L heteropolymers of native L-subunit-rich horse spleen and H-subunit-rich human heart ferritins, points out the existence of a correlation between iron core shape and protein shell.     

Composition and structure of iron oxidation surface layers produced in weak acidic solutions

Although oxidation/passivation of iron in basic solution has been extensively investigated, there is very little information on iron corrosion in weak acidic solutions. In this work, iron surface composition and structure, produced in aerobic aqueous solutions ranging from pH 2 to 5, were determined in detail by the use of infrared external reflection spectroscopy, X-ray photoelectron spectroscopy and scanning electron microscopy. The most striking observation is that at pH 2 and 3 almost all oxidized iron is dissolved in solution, whereas at pH 4 and 5 the product of iron oxidation is deposited on the iron surface in the form of lepidocrocite, gamma-FeOOH. Detailed iron surface and solution analyses allow the proposition of the following overall oxidation reactions: [EQUATION: SEE TEXT]. At pH 2 and 3, only a very thin surface layer consisting of FeO and Fe(OH)2 with polymeric structure is observed on the iron surface. The amounts of these surface species remain almost constant (2-5 nm) from the first minutes to a few hours of reaction, if pH is kept constant. Nevertheless, with time the akaganeite-like, beta-FeOOH structure is also detected. At pH 4 and 5, the amount of lepidocrocite deposited on the iron surface increases with reaction time. Detailed quantitative evaluation of the lepidocrocite produced at pH 5 and its surface distribution on iron was performed based on the comparison of infrared spectroscopic data with spectral simulation results of assumed surface structures. At pH 4 and 5 and a temperature of 40-50 degrees C, in addition to a very large amount of lepidocrocite other oxy-hydroxide surface species such as goethite (alpha-FeOOH) and feroxyhite (delta-FeOOH), were identified. Addition of Cl- ions to solution at 10(-3) M concentration at pH 5 increases the oxidation rate of iron by about 50%, and lepidocrocite remains the only oxidation product. Similarly, an addition of Fe2+ ions to solution at pH 5 very strongly enhances lepidocrocite formation as well as its conductivity. The latter finding is important for the possible application of metallic iron as a catalyst in redox reactions, for example, for decomposition of difficult-to-biodegrade water pollutants.     

Xanthene-modified and hangman iron corroles

Iron corroles modified with a xanthene scaffold are delivered from easily available starting materials in abbreviated reaction times. These new iron corroles have been spectroscopically examined with particular emphasis on defining the oxidation state of the metal center. Investigation of their electronic structure using (57)Fe Mo?ssbauer spectroscopy in conjunction with density functional theory (DFT) calculations reveals the non-innocence of the corrole ligand. Although these iron corroles contain a formal Fe(IV) center, the deprotonated corrole macrocycle ligand is one electron oxidized. The electronic ground state of these complexes is best described as an intermediate spin S = 3/2 Fe(III) site strongly antiferromagnetically coupled to the S = 1/2 of the monoradical dianion corrole [Fe(III)Cl-corrole(+?)]. We show here that iron corroles as well as xanthene-modified and hangman xanthene iron corroles are redox active and catalyze the disproportionation of hydrogen peroxide via the catalase reaction, and that this activity scales with the oxidation potential. The meso position of corrole macrocycle is susceptible toward nucleophilic attack during catalase turnover. The reactivity of peroxide within the hangman cleft reported here adds to the emerging theme that corroles are good at catalyzing two-electron activation of the oxygen-oxygen bond in a variety of substrates.