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Overexpression of human H-chain ferritin (HuHF) is known to impart a degree of protection to cells against oxidative stress and the associated damage to DNA and other cellular components. However, whether this protective activity resides in the protein's ability to inhibit Fenton chemistry as found for Dps proteins has never been established. Such inhibition does not occur with the related mitochondrial ferritin which displays much of the same iron chemistry as HuHF, including an Fe(II)/H(2)O(2) oxidation stoichiometry of approximately 2:1. In the present study, the ability of HuHF to attenuate hydroxyl radical production by the Fenton reaction (Fe(2+) + H(2)O(2) --> Fe(3+) + OH(-) + *OH) was examined by electron paramagnetic resonance (EPR) spin-trapping methods. The data demonstrate that the presence of wild-type HuHF during Fe(2+) oxidation by H(2)O(2) greatly decreases the amount of .OH radical produced from Fenton chemistry whereas the ferroxidase site mutant 222 (H62K + H65G) and human L-chain ferritin (HuLF) lack this activity. HuHF catalyzes the pairwise oxidation of Fe(2+) by the detoxification reaction [2Fe(2+) + H(2)O(2) + 2H(2)O --> 2Fe(O)OH(core) + 4H(+)] that occurs at the ferroxidase site of the protein, thereby preventing the production of hydroxyl radical. The small amount of *OH radical that is produced in the presence of ferritin (

Good's zwitterionic buffers are widely used in biological and biochemical research in which hydrogen peroxide is a solution component. This study was undertaken to determine whether Good's buffers exhibit reactivity toward H(2)O(2). It is found that H(2)O(2) oxidizes both morpholine ring-containing buffers (e.g., Mops, Mes) and piperazine ring-containing zwitterionic buffers (e.g., Pipes, Hepes, and Epps) to produce their corresponding N-oxide forms. The percentage of oxidized buffer increases as the concentration of H(2)O(2) increases. However, the rate of oxidation is relatively slow. For example, no oxidized Mops was detected 2h after adding 0.1M H(2)O(2) to 0.1M Mops (pH 7.0), and only 5.7% was oxidized after 24h exposure to H(2)O(2). Thus, although all of these buffers can be oxidized by H(2)O(2), their slow reaction does not significantly perturb levels of H(2)O(2) in the time frame and at the concentrations of most biochemical studies. Therefore, the previously reported rapid loss of H(2)O(2) produced from the ferroxidase reaction of ferritin is unlikely due to reaction of H(2)O(2) with buffer, a conclusion supported by the fact that H(2)O(2) is also lost rapidly when the solution pH of the ferroxidase reaction is controlled by a pH stat apparatus in the absence of buffer.

The role of the ferroxidase center in iron uptake and hydrogen peroxide detoxification was investigated in Listeria innocua Dps by substituting the iron ligands His31, His43, and Asp58 with glycine or alanine residues either individually or in combination. The X-ray crystal structures of the variants reveal only small alterations in the ferroxidase center region compared to the native protein. Quenching of the protein fluorescence was exploited to assess stoichiometry and affinity of metal binding. Substitution of either His31 or His43 decreases Fe(II) affinity significantly with respect to wt L. innocua Dps (K approximately 10(5) vs approximately 10(7) M(-)(1)) but does not alter the binding stoichiometry [12 Fe(II)/dodecamer]. In the H31G-H43G and H31G-H43G-D58A variants, binding of Fe(II) does not take place with measurable affinity. Oxidation of protein-bound Fe(II) increases the binding stoichiometry to 24 Fe(III)/dodecamer. However, the extent of fluorescence quenching upon Fe(III) binding decreases, and the end point near 24 Fe(III)/dodecamer becomes less distinct with increase in the number of mutated residues. In the presence of dioxygen, the mutations have little or no effect on the kinetics of iron uptake and in the formation of micelles inside the protein shell. In contrast, in the presence of hydrogen peroxide, with increase in the number of substitutions the rate of iron oxidation and the capacity to inhibit Fenton chemistry, thereby protecting DNA from oxidative damage, appear increasingly compromised, a further indication of the role of ferroxidation in conferring peroxide tolerance to the bacterium.

Human ferritins sequester and store iron as a stable FeOOH((s)) mineral core within a protein shell assembled from 24 subunits of two types, H and L. Core mineralization in recombinant H- and L-subunit homopolymer and heteropolymer ferritins and several site-directed H-subunit variants was investigated to determine the iron oxidation/hydrolysis chemistry as a function of iron flux into the protein. Stopped-flow absorption spectrometry, UV spectrometry, and electrode oximetry revealed that the mineral core forms by at least three pathways, not two as previously thought. They correspond to the ferroxidase, mineral surface, and the Fe(II) + H2O2 detoxification reactions, respectively: [see reactions]. The H-subunit catalyzed ferroxidase reaction 1 occurs at all levels of iron loading of the protein but decreases with increasing iron added (48-800 Fe(II)/protein). Reaction 2 is the dominant reaction at 800 Fe(II)/protein, whereas reaction 3 occurs largely at intermediate iron loadings of 100-500 Fe(II)/protein. Some of the H2O2 produced in reaction 1 is consumed in the detoxification reaction 3; the 2/1 Fe(II)/H2O2 stoichiometry of reaction 3 minimizes hydroxyl radical production during mineralization. Human L-chain ferritin and H-chain variants lacking functional nucleation and/or ferroxidase sites deposit their iron largely through the mineral surface reaction 2. H2O2 is shown to be an intermediate product of dioxygen reduction in L-chain as well as in H-chain and H-chain variant ferritins.

The DNA-binding proteins from starved cells (Dps) are a family of proteins induced in microorganisms by oxidative or nutritional stress. Escherichia coli Dps, a structural analog of the 12-subunit Listeria innocua ferritin, binds and protects DNA against oxidative damage mediated by H(2)O(2). Dps is shown to be a Fe-binding and storage protein where Fe(II) oxidation is most effectively accomplished by H(2)O(2) rather than by O(2) as in ferritins. Two Fe(2+) ions bind at each of the 12 putative dinuclear ferroxidase sites (P(Z)) in the protein according to the equation, 2Fe(2+) + P(Z) --> [(Fe(II)(2)-P](FS)(Z+2) + 2H(+). The ferroxidase site (FS) bound iron is then oxidized according to the equation, [(Fe(II)(2)-P](FS)(Z+2) + H(2)O(2) + H(2)O --> [Fe(III)(2)O(2)(OH)-P](FS)(Z-1) + 3H(+), where two Fe(II) are oxidized per H(2)O(2) reduced, thus avoiding hydroxyl radical production through Fenton chemistry. Dps acquires a ferric core of approximately 500 Fe(III) according to the mineralization equation, 2Fe(2+) + H(2)O(2) + 2H(2)O --> 2Fe(III)OOH((core)) + 4H(+), again with a 2 Fe(II)/H(2)O(2) stoichiometry. The protein forms a similar ferric core with O(2) as the oxidant, albeit at a slower rate. In the absence of H(2)O(2) and O(2), Dps forms a ferrous core of approximately 400 Fe(II) by the reaction Fe(2+) + H(2)O + Cl(-) --> Fe(II)OHCl((core)) + H(+). The ferrous core also undergoes oxidation with a stoichiometry of 2 Fe(II)/H(2)O(2). Spin trapping experiments demonstrate that Dps greatly attenuates hydroxyl radical production during Fe(II) oxidation by H(2)O(2). These results and in vitro DNA damage assays indicate that the protective effect of Dps on DNA most likely is exerted through a dual action, the physical association with DNA and the ability to nullify the toxic combination of Fe(II) and H(2)O(2). In the latter process a hydrous ferric oxide mineral core is produced within the protein, thus avoiding oxidative damage mediated by Fenton chemistry.

Biomineralization of the ferritin iron core involves a complex series of events in which H(2)O(2) is produced during iron oxidation by O(2) at a dinuclear centre, the 'ferroxidase site', located on the H-subunit of mammalian proteins. Rapid-freeze quench Mössbauer spectroscopy was used to probe the early events of iron oxidation and mineralization in recombinant human ferritin containing 24 H-subunits. The spectra reveal that a mu-1,2-peroxodiFe(III) intermediate (species P) with Mössbauer parameters delta (isomer shift)=0.58 mm/s and DeltaE(Q) (quadrupole splitting)=1.07 mm/s at 4.2 K is formed within 50 ms of mixing Fe(II) with the apoprotein. This intermediate accounts for almost all of the iron in the sample at 160 ms. It subsequently decays within 10 s to form a mu-oxodiFe(III)-protein complex (species D), which partially vacates the ferroxidase sites of the protein to generate Fe(III) clusters (species C) at a reaction time of 10 min. The intermediate peroxodiFe(III) complex does not decay under O(2)-limiting conditions, an observation suggesting inhibition of decay by unreacted Fe(II), or a possible role for O(2) in ferritin biomineralization in addition to that of direct oxidation of iron(II).

Abstract: Ferritins are ubiquitous iron storage and detoxification proteins distributed throughout the plant and animal kingdoms. Mammalian ferritins oxidize and accumulate iron as a ferrihydrite mineral within a shell-like protein cavity. Iron deposition utilizes both O2 and H2O2 as oxidants for Fe2+ where oxidation can occur either at protein ferroxidase centers or directly on the surface of the growing mineral core. The present study was undertaken to determine whether the nature of the mineral core formed depends on the protein ferroxidase center versus mineral surface mechanism and on H2O2 versus O2 as the oxidant. The data reveal that similar cores are produced in all instances, suggesting that the structure of the core is thermodynamically, not kinetically controlled. Cores averaging 500 Fe/protein shell and diameter ∼2.6 nm were prepared and exhibited superparamagnetic blocking temperatures of 19 and 22 K for the H2O2 and O2 oxidized samples, respectively. The observed blocking temperatures are consistent with the unexpectedly large effective anisotropy constant K eff =312 kJ/m3 recently reported for ferrihydrite nanoparticles formed in reverse micelles [E.L. Duarte, R. Itri, E. Lima Jr., M.S. Batista, T.S. Berquó and G.F. Goya, Large Magnetic Anisotropy in ferrihydrite nanoparticles synthesized from reverse micelles, Nanotechnology 17 (2006) 5549–5555.]. All ferritin samples exhibited two magnetic phases present in nearly equal amounts and ascribed to iron spins at the surface and in the interior of the nanoparticle. At 4.2 K, the surface spins exhibit hyperfine fields, H hf, of 436 and 445 kOe for the H2O2 and O2 samples, respectively. As expected, the spins in the interior of the core exhibit larger H hf values, i.e. 478 and 486 kOe for the H2O2 and O2 samples, respectively. The slightly smaller hyperfine field distribution DHhf for both surface (78 kOe vs. 92 kOe) and interior spins (45 kOe vs. 54 kOe) of the O2 sample compared to the H2O2 samples implies that the former is somewhat more crystalline. [Copyright &y& Elsevier]

Examines the production of hydrogen peroxide (H[sub 2]O[sub 2]) during iron (II) oxidation in Mammalian apoferritins. Description on ferritins; Evidence on the production of H[sub 2]O[sub 2] in ferroxidase site; Importance on proper sequence of reagents in measuring total amount of H[sub 2]O[sub 2] production.