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Nanostructured materials are increasingly important for the construction of electrochemical energy storage devices that will meet the needs of portable nanodevices. Here we describe the development of a nanoenergy storage system based on inorganic mineral phases contained in ferritin proteins. The electrochemical cell consists of an anode containing ∼2000 iron atoms as Fe(OH)2 in the hollow protein interior of ferritin and a cathode containing ∼2000 of Co(OH)3 in a separate ferritin molecule. The achieved initial voltage output from a combination of Fe2+- and Co3+-ferritins adsorbed on gold electrodes was ∼500mV, while a combination of Fe2+- and Co3+-ferritins immobilized on gold produced a voltage of 350-405mV. When fully discharged, Fe(OH)3 and Co(OH)2 are the products of a single electron transfer per metal atom from anode to cathode. The spent components can be regenerated by chemical or electrochemical methods restoring battery function. The properties of ferritins are presented and their unique characteristics are described, which have led to the development of a functional bio-nanobattery. [ABSTRACT FROM AUTHOR]

Abstract: Large-molecule oxidants oxidize Fe(II) to form Fe(III) cores in the interior of ferritins at rates comparable to or faster than the iron deposition reaction using O2 as oxidant. Iron deposition into horse spleen ferritin (HoSF) occurs using ferricyanide ion, 2,6-dichlorophenol-indophenol, and several redox proteins: cytochrome c, stellacyanin, and ceruloplasmin. Cytochrome c also loads iron into recombinant human H-chain (rHF), human L-chain (rLF), and A. vinelandii bacterioferritin (AvBF). The enzymatic activities of ferritins were monitored anaerobically using stopped-flow kinetic spectrophotometry. The reactions exhibit saturation kinetics with respect to the large oxidant concentrations, giving apparent Michaelis constants for cytochrome c as oxidant: K m =39.6μM for HoSF and 6.9μM for AvBF. Comparison of the kinetic parameters with that of iron deposition by O2 shows that large oxidants load iron into HoSF and AvBF more effectively than O2 and may use a mechanism different than the ferroxidase center. Large oxidants did not deposit iron as efficiently with rHF and rLF. The results suggest that the heme groups in AvBF and the protein redox centers present in heteropolymers may assist in anaerobic iron deposition by large oxidants. The physiological relevance of iron deposition by large molecules, including protein oxidants is discussed. [Copyright &y& Elsevier]

Abstract: The electrochemical analysis was carried out by the electrochemical reduction of horse spleen ferritin (HoSF) using oxidized methyl viologen. The reduction and oxidation peaks of the methyl viologen and released Fe appeared on the cyclic voltammogram. The Fe was released on reduction of the HoSF by the methyl viologen. The effect of oxidized methyl viologen concentration and the ferritin solution pH on reduction of the HoSF were determined. The reduction of the ferritin procedure occurred by applying a potential that reduced the oxidized methyl viologen, and the HoSF was reduced by the reduced methyl viologen. [Copyright &y& Elsevier]

Ferritin catalyzes the oxidation of Fe2+ by O2 to form a reconstituted Fe3+ oxy-hydroxide mineral core, but extensive studies have shown that the Fe2+ to O2 stoichiometry changes with experimental conditions. At Fe2+ to horse spleen ferritin (HoSF) ratios greater than 200, an upper limit of Fe2+ to O2 of 4 is typically measured, indicating O2 is reduced to 2H2O. In contrast, a lower limit of Fe2+ to O2 of approximately 2 is measured at low Fe2+ to HoSF ratios, implicating H2O2 as a product of Fe2+ deposition. Stoichiometric amounts of H2O2 have not been measured, and H2O2 is proposed to react with an unknown system component. Evidence is presented that identifies this component as amine buffers, including 3- N-morpholinopropanesulfonic acid (MOPS), which is widely used in ferritin studies. In the presence of non-amine buffers, the Fe2+ to O2 stoichiometry was approximately 4.0, but at high concentrations of amine buffers (0.10 M) the Fe2+ to O2 stoichiometry is approximately 2.5 for iron loadings of eight to 30 Fe2+ per HoSF. Decreasing the concentration of amine buffer to zero resulted in an Fe2+ to O2 stoichiometry of approximately 4. Direct evidence for amine buffer modification during Fe2+ deposition was obtained by comparing authentic and modified buffers using mass spectrometry, NMR, and thin layer chromatography. Tris(hydroxymethyl)aminomethane, MOPS, and N-methylmorpholine (a MOPS analog) were all rapidly chemically modified during Fe2+ deposition to form N-oxides. Under identical conditions no modification was detected when amine buffer, H2O2, and O2 were combined with Fe2+ or ferritin separately. Thus, a short-lived ferritin intermediate is required for buffer modification by H2O2. Variation of the Fe2+ to O2 stoichiometry versus the Fe2+ to HoSF ratio and the amine buffer concentration are consistent with buffer modification. [ABSTRACT FROM AUTHOR]

Abstract: The kinetics of the change in mass related to the release from and deposition onto the cavities of a ferritin in the SWCNT nanocomposite by electrochemical redox reactions, and the effects of the SWCNT on the kinetics of the variation in mass of the ferritin nanocomposite were characterized using an electrochemical quartz crystal microbalance. The change in mass of reconstituted ferritin in the SWCNT nanocomposite shows reversible variation and stability of the ferritin/SWCNT nanocomposite on redox reactions was confirmed by using a coreless apoferritin and a Fe2+ chelating agent. The ferritin/SWCNT nanocomposite is a good candidate for applications based on electron transfer, such as biosensor, biobatteries and electrodes for biofuel cell. [Copyright &y& Elsevier]

Examines the formation of hydrogen peroxide during iron deposition in horse spleen ferritin (HoSF) using oxygen as oxidant. Deposition of Fe(OH)[sub 3] in HoSF interior; Methods used to assess hydrogen peroxide formation; Oxidation of iron under constant air-saturation conditions; Mechanism of iron II oxidation reactions.

Abstract: When the iron core of equine spleen ferritin is reduced, anions in solution cross the protein shell and enter the ferritin interior as part of a charge balancing reaction. Anion sequestration inside ferritin during iron core reduction was monitored using ion selective electrodes, inductively coupled plasma emission, and energy-dispersive X-ray spectroscopy. The requirement for anion translocation to the ferritin interior occurs because upon iron core reduction, two OH− ions per iron are released or neutralized inside ferritin leaving a net positive charge. Halides and oxoanions were tested as anionic substrates for this reaction. A general trend for the halides showed that the smaller halides accumulated inside ferritin in greater abundance than larger halides, presumably because the protein channels restrict the transfer of the larger anionic species. In contrast, oxoanion accumulation inside ferritin did not show selectivity based on size or charge. Vanadate and molybdate accumulated to the highest concentrations and nitrate, phosphate and tungstate showed poor accumulation inside ferritin. Fe(II) remains stably sequestered inside ferritin, as shown by electron microscopy and by column chromatography. Upon oxidation of the iron core, the anions are expelled from ferritin, and OH− ions coordinate to the Fe(III) to form the original Fe(O)OH mineral. Anion transport across the ferritin protein shell represents an important mechanism by which ferritin maintains proper charge balance inside the protein cavity. [Copyright &y& Elsevier]

Abstract: The rate of Fe3+ release from horse spleen ferritin (HoSF) was measured using the Fe3+-specific chelator desferoxamine (DES). The reaction consists of two kinetic phases. The first is a rapid non-linear reaction followed by a slower linear reaction. The overall two-phase reaction was resolved into three kinetic events: 1) a rapid first-order reaction in HoSF (k 1); 2) a second slower first-order reaction in HoSF (k 2); and 3) a zero-order slow reaction in HoSF (k 3). The zero-order reaction was independent of DES concentration. The two first-order reactions had a near zero-order dependence on DES concentration and were independent of pH from 6.8 to 8.2. The two first-order reactions accounted for 6–9 rapidly reacting Fe3+ ions. Activation energies of 10.5±0.8, 13.5±2.0 and 62.4±2.1kJ/mol were calculated for the kinetic events associated with k 1, k 2, and k 3, respectively. Iron release occurs by: 1) a slow zero-order rate-limiting reaction governed by k 3 and corresponding to the dissociation of Fe3+ ions from the FeOOH core that bind to an Fe3+ binding site designated as site 1 (proposed to be within the 3-fold channel); 2) transfer of Fe3+ from site 1 to site 2 (a second binding site in the 3-fold channel) (k 2); and 3) rapid iron loss from site 2 to DES (k 1). [ABSTRACT FROM AUTHOR]

Abstract: Site-specific reconstituted nanoparticles were fabricated via electrochemically controlled biomineralization through the immobilization of biomolecules. The work reported herein includes the immobilization of ferritin with various surface modifications, the electrochemical biomineralization of ferritins with different inorganic cores, and the electrocatalytic reduction of oxygen on the reconstituted Pt-cored ferritins. Protein immobilization on the substrate is achieved by anchoring ferritins with dithiobis-N-succinimidyl propionate (DTSP). A reconstitution process of site-specific electrochemical biomineralization with a protein cage loads ferritins with different core materials. The ferritin acts as a nano-scale template, a biocompatible cage, and a separator between the nanoparticles. This first demonstration of electrochemically controlled site-specific reconstitution of biomolecules provides a new tool for biomineralization and opens the way to produce the bio-templated nanoparticles by electrochemical control. The nanosized platinum-cored ferritins on gold displayed good catalytic activity for the electrochemical reduction of oxygen, which is applicable to biofuel cell applications. This results in a smaller catalyst loading on the electrodes for fuel cells or other bioelectronic devices. [Copyright &y& Elsevier]

Azotobacter vinelandii bacterioferritin (AvBF) containing 800-1500 Co or Mn atoms as Co-(III) and Mn(III) oxyhydroxide cores (Co-AvBF, Mn-AvBF) was synthesized by the same procedure used previously for horse spleen ferritin (H0SF). The kinetics of reduction of Co-AvBF and Mn-AvBF by ascorbic acid are first-order in each reactant. The rate constant for the reduction of Mn-AvBF (8.52 M-1 min-1) is ~12 times larger than that for Co-AvBF (0.72 M-1 min-1), which is consistent with a previous observation that Mn-HoSF is reduced ~10-fold faster than Co-HoSF [Zhang, B. et al. (2005) inorg. Chem. 44, 3738-3745]. The rates of reduction of M-AvBF (M = Co and Mn) are more than twice that for the reduction of the corresponding M-HoSF. HoSF containing reduced Fe(II) cores (Fe(II)-HoSF), prepared by methyl viologen and CO, also reduces M-HoSF and M-AvBF species, with both cores remaining within ferritin, suggesting that electrons transfer through the ferritin shell. Electron transfer from Fe(II)-HoSF to Co-AvBF occurs at a rate ~3 times faster than that to Co-HoSF, indicating that the Co cores in AvBF are more accessible to reduction than the Co cores in HoSF. The presence of nonconductive (SiO2) or conductive (gold) surfaces known to bind ferritins enhances the rate of electron transfer. A more than ~4-fold increase in the apparent reaction rate is observed in the presence of gold. Although both surfaces (SiO2 and gold) enhance reaction by providing binding sites for molecular interaction, results show that ferritins with different mineral cores bound to a gold surface transfer electrons through the gold substrate so that direct contact of the reacting molecules is not required. [ABSTRACT FROM AUTHOR]

Abstract: Iron (2+ and 3+) is believed to transfer through the three-fold channels in the ferritin shell during iron deposition and release in animal ferritins. However, the rate of iron transit in and out through these channels has not been reported. The recent synthesis of [Fe(CN)6]3−, Prussian Blue (PB) and desferrioxamine (DES) all trapped within the horse spleen ferritin (HoSF) interior makes these measurements feasible. We report the rate of Fe2+ penetrating into the ferritin interior by adding external Fe2+ to [Fe(CN)6]3− encapsulated in the HoSF interior and measuring the rate of formation of the resulting encapsulated PB. The rate at which Fe2+ reacts with [Fe(CN)6]3− in the HoSF interior is much slower than the formation of free PB in solution and is proceeded by a lag period. We assume this lag period and the difference in rate represent the transfer of Fe2+ through the HoSF protein shell. The calculated diffusion coefficient, D ∼5.8×10−20 m2/s corresponds to the measured lag time of 10–20 s before PB forms within the HoSF interior. The activation energy for Fe2+ transfer from the outside solution through the protein shell was determined to be 52.9 kJ/mol by conducting the reactions at 10∼40 °C. The reaction of Fe3+ with encapsulated [Fe(CN)6]4− also readily forms PB in the HoSF interior, but the rate is faster than the corresponding Fe2+ reaction. The rate for Fe3+ transfer through the ferritin shell was confirmed by measuring the rate of the formation of Fe-DES inside HoSF and an activation energy of 58.4 kJ/mol was determined. An attempt was made to determine the rate of iron (2+ and 3+) transit out from the ferritin interior by adding excess bipyridine or DES to PB trapped within the HoSF interior. However, the reactions are slow and occur at almost identical rates for free and HoSF-encapsulated PB, indicating that the transfer of iron from the interior through the protein shell is faster than the rate-limiting step of PB dissociation. The method described in this work presents a novel way of determining the rate of transfer of iron and possibly other small molecules through the ferritin shell. [Copyright &y& Elsevier]

Horse spleen ferritin (HoSF) containing 800–1500 cobalt or 250–1200 manganese atoms as Co(O)OH and Mn-(O)OH mineral cores within the HoSF interior (Co-HoSF and Mn-HoSF) was synthesized, and the chemical reactivity, kinetics of reduction, and the reduction potentials were measured. Microcoulometric and chemical reduction of HoSF containing the M(O)OH mineral core (M = Co or Mn) was rapid and quantitative with a reduction stoichiometry of 1.05 ± 0.10 e/M forming a stable M(OH)2 mineral core. At pH 9.0, ascorbic acid (AH2), a two-electron reductant, effectively reduced the mineral cores; however, the reaction was incomplete and rapidly reached equilibrium. The addition of excess AH2 shifted the reaction to completion with a M3+/AH2 stoichiometry of 1.9–2.1, consistent with a single electron per metal atom reduction. The rate of reaction between M(O)OH and excess AH2 was measured by monitoring the decrease in mineral core absorbance with time. The reaction was first order in each reactant with second-order rate constants of 0.53 and 4.74 M-1 min-1 respectively, for Co- and Mn-HoSF at pH 9.0. From the variation of absorbance with increasing AH2 concentration, equilibrium constants at pH 9.0 of 5.0 ± 1.9 for Co-HoSF and 2.9 ± 0.9 for Mn-HoSF were calculated for 2M(O)OH + AH2 = 2M(OH)2 + D, where AH2 and D are ascorbic acid and dehydroascorbic acid, respectively. Consistent with these equilibrium constants, the standard potential for the reduction of Co(III)-HoSF is 42 mV more positive than that of the ascorbic acid reaction, while the standard potential of Mn(III)-HoSF is 27 mV positive relative to AH2. Fe2+ in solution with Co- and Mn-HoSF under anaerobic conditions was oxidized to form Fe(O)OH within the HoSF interior, resulting in partial displacement of the Co or Mn by iron. [ABSTRACT FROM AUTHOR]

The reaction of horse spleen ferritin (HoSF) with Fe2+ at pH 6.5 and 7.5 using O2, H2O2 and 1:1 a mixture of both showed that the iron deposition reaction using H2O2 is ∼20- to 50-fold faster than the reaction with O2 alone. When H2O2 was added during the iron deposition reaction initiated with O2 as oxidant, Fe2+ was preferentially oxidized by H2O2, consistent with the above kinetic measurements. Both the O2 and H2O2 reactions were well defined from 15 to 40 °C from which activation parameters were determined. The iron deposition reaction was also studied using O2 as oxidant in the presence and absence of catalase using both stopped-flow and pumped-flow measurements. The presence of catalase decreased the rate of iron deposition by ∼1.5-fold, and gave slightly smaller absorbance changes than in its absence. From the rate constants for the O2 (0.044 s-1) and H2O2 (0.67 s-1) iron-deposition reactions at pH 7.5, simulations of steady-state H2O2 concentrations were computed to be 0.45 μM. This low value and reported Fe2+/O2 values of 2.0–2.5 are consistent with H2O2 rapidly reacting by an alternate but unidentified pathway involving a system component such as the protein shell or the mineral core as previously postulated [Biochemistry 22 (1983) 876; Biochemistry 40 (2001) 10832]. [Copyright &y& Elsevier]

An optical flow cell provided a means to conveniently measure the rate of successive Fe2+ oxidation reactions catalyzed by horse spleen ferritin (HoSF) to determine if both ferroxidase and mineral core Fe2+ oxidation reactions occur. The oxygen concentration and pH were held constant and multiple additions of Fe2+/HoSF ratios of 1, 10, 100, 150, 250 and 400 were conducted, creating core sizes ranging from 12 to 2800. During these oxidations, the absence of nonspecific Fe(OH)3 formation and the presence (>95%) of Fe(OH)3 deposited within the core of HoSF demonstrated the validity of monitoring iron deposition into HoSF by this procedure. Initial rates for oxidation of 5–50 Fe2+/HoSF established that the reaction is overall first order in Fe2+ concentration. However, when full progress curves were analyzed at a variety of Fe2+/HoSF ratios, two first-order reactions (k1∼0.035 s−1 and k2∼0.007 s−1) were found to contribute to the overall Fe2+ oxidation reaction. The proportion of the fast reaction increased with increasing Fe2+/HoSF ratio until at ∼400, it was the dominant reaction. For the Fe2+/HoSF ratios examined, the overall rate of iron deposition is independent of the size of the mineral core, a result suggesting that an increasing mineral core size does not enhance the rate of Fe2+ oxidation. Comparison of successive additions of 1.0 Fe2+/HoSF showed that oxidation of the first 8–10 Fe2+ produced a Fe(III) species with a lower molar absorptivity per Fe(III) than that of the bulk core. Measurement of the H+/Fe2+ ratio confirmed this difference in behavior by giving an H+/Fe2+ ratio of ∼1.0 below and 2.0 for ratios >30 Fe2+/HoSF. The faster reaction was attributed to ferroxidase catalysis and the slow reaction to nonspecific ferroxidase activity of the HoSF protein shell. [Copyright &y& Elsevier]