Crystal structure of bullfrog M ferritin at 2.8 Å resolution: analysis of subunit interactions and the binuclear metal center

Ferritins concentrate and store iron as a mineral in all bacterial, plant, and animal cells. The two ferritin subunit types, H or M (fast) and L (slow), differ in rates of iron uptake and mineralization and assemble in vivo to form heteropolymeric protein shells made up of 24 subunits; H/L subunit ratios reflect cell specificity of H and L subunit gene expression. A diferric peroxo species that is the initial reaction product of Fe(II) in H-type ferritins, as well as in ribonucleotide reductase (R2) and methane monooxygenase hydroxylase (MMOH), has recently been characterized, exploiting the relatively high accumulation of the peroxo intermediate in frog H-subunit type recombinant ferritin with the M sequence. The stability of the diferric reaction centers in R2 and MMOH contrasts with the instability of diferric centers in ferritin, which are precursors of the ferric mineral. We have determined the crystal structure of the homopolymer of recombinant frog M ferritin in two crystal forms: P4(1)2(1)2, a = b = 170.0 A and c = 481.5 A; and P3(1)21, a = b = 210.8 A and c = 328.1 A. The structural model for the trigonal form was refined to a crystallographic R value of 19.0% (Rfree = 19.4%); the two structures have an r.m.s.d. of approximately 0.22 A for all C alpha atoms. Comparison with the previously determined crystal structure of frog L ferritin indicates that the subunit interface at the molecular twofold axes is most variable, which may relate to the presence of the ferroxidase site in H-type ferritin subunits. Two metal ions (Mg) from the crystallization buffer were found in the ferroxidase site of the M ferritin crystals and interact with Glu23, Glu58, His61, Glu103, Gln137 and, unique to the M subunit, Asp140. The data suggest that Gln137 and Asp140 are a vestige of the second GluxxHis site, resulting from single nucleotide mutations of Glu and His codons and giving rise to Ala140 or Ser140 present in other eukaryotic H-type ferritins, by additional single nucleotide mutations. The observation of the Gln137xxAsp140 site in the frog M ferritin accounts for both the instability of the diferric oxy complexes in ferritin compared to MMOH and R2 and the observed kinetic variability of the diferric peroxo species in different H-type ferritin sequences.     

mu-1,2-peroxo diferric complex formation in horse spleen ferritin. A mixed H/L-subunit heteropolymer

Previous kinetics studies with homopolymer ferritins (bullfrog M-chain, human H-chain and Escherichia coli bacterial ferritins) have established that a mu-1,2-peroxo diferric intermediate is formed during Fe(II) oxidation by O2 at the ferroxidase site of the protein. The present study was undertaken to determine whether such an intermediate is formed also during iron oxidation in horse spleen ferritin (HoSF), a naturally occurring heteropolymer ferritin of H and L-subunits (approximately 3.3 H-chains/HoSF), and to assess its role in the formation of the mineral core. Multi-wavelength stopped-flow spectrophotometry of the oxidative deposition of iron in HoSF demonstrated that a transient peroxo complex (lambda(max) approximately 650 nm) is produced in this protein as for other ferritins. The peroxo complex in HoSF is formed about fourfold slower than in human H-chain (HuHF) and decays more slowly (approximately threefold) as well, at an iron level of two Fe(II)/H-chain. However, as found for HuHF, a second intermediate is formed in HoSF as a decay product of the peroxo complex. Only one-third of the expected peroxo complex forms at the ferroxidase centers of HoSF when two Fe(II)/H-subunits are added to the protein, dropping to only approximately 14% when 20 Fe(II)/H-chain are added, indicating a declining role of the peroxo complex in iron deposition. In contrast to HuHF, HoSF does not enzymatically regenerate the observable peroxo complex. The kinetics of mineralization in HoSF are modeled satisfactorily by a mechanism in which the ferroxidase site rapidly produces an incipient core from a single turnover of iron, upon which subsequent Fe(II) is oxidized autocatalytically to build the Fe(O)OH(s) mineral core. This model supports a role for the L-chain in iron mineralization and helps to explain the widespread occurrence of heteropolymer ferritins in tissues of vertebrates.     

Mineralization in Ferritin: An Efficient Means of Iron Storage

Ferritins are a class of iron storage and mineralization proteins found throughout the animal, plant, and microbial kingdoms. Iron is stored within the protein shell of ferritin as a hydrous ferric oxide nanoparticle with a structure similar to that of the mineral "ferrihydrite." The eight hydrophilic channels that traverse the protein shell are thought to be the primary avenues by which iron gains entry to the interior of eukaryotic ferritins. Twenty-four subunits constitute the protein shell and, in mammalian ferritins, are of two types, H and L, which have complementary functions in iron uptake. The H chain contains a dinuclear ferroxidase site that is located within the four-helix bundle of the subunit; it catalyzes the oxidation of ferrous iron by O(2), producing H(2)O(2). The L subunit lacks this site but contains additional glutamate residues on the interior surface of the protein shell which produce a microenvironment that facilitates mineralization and the turnover of iron(III) at the H subunit ferroxidase site. Recent spectroscopic studies have shown that a di-Fe(III) peroxo intermediate is produced at the ferroxidase site followed by formation of a mu-oxobridged dimer, which then fragments and migrates to the nucleation sites to form incipient mineral core species. Once sufficient core has developed, iron oxidation and mineralization occur primarily on the surface of the growing crystallite, thus minimizing the production of potentially harmful H(2)O(2).     

M?ssbauer studies of the formation and reactivity of a quasi-stable peroxo intermediate of stearoyl-acyl carrier protein Delta 9-desaturase.

Stearoyl-ACP Delta(9)-desaturase (Delta 9D) is a diiron enzyme that catalyzes 18:0-ACP desaturation. Each subunit of homodimeric resting Delta 9D contains a diferric cluster, while chemical reduction by 4e(-) produces a diferrous cluster in each subunit. Reaction of 4e(-)-reduced Delta 9D with 18:0-ACP and O(2) yields a blue chromophore (lambda(max) approximately 700 nm) that exhibits a vibrational spectrum indicative of a micro-1,2-peroxo complex; this species has been designated peroxo Delta9D. In contrast to other enzymic peroxodiiron intermediates, peroxo Delta 9D is long-lived (t(1/2) approximately 30 min at 25 degrees C) and decays via an oxidase reaction without formation of either H(2)O(2) or product (18:1-ACP). In this work, optical, transient kinetic, and M?ssbauer techniques have been used to further investigate the origin and nature of this unusual peroxodiiron complex. Rapid mixing of 4e(-) Delta 9D with O(2)-equilibrated 18:0-ACP produced peroxo Delta 9D as revealed by a temperature-dependent, pseudo-first-order absorption increase at 700 nm (k = 46 s(-)(1) at 6 degrees C). The M?ssbauer spectrum of peroxo Delta 9D, accounting for 96% of the total iron, consists of two quadrupole doublets present in equal proportions: delta(1) = 0.68(1) mm/s, and Delta E(Q)(1) = 1.90(2) mm/s; delta(2) = 0.64(1) mm/s, and Delta E(Q)(2) = 1.06(2) mm/s. Decay of the 700 nm optical band (k = 0.004 min(-)(1) at 6 degrees C) correlates with the complete conversion of peroxo Delta 9D into a complex called peroxo-cycled Delta 9D, which exhibits two new doublets present in equal proportions: delta(1) = 0.57(2) mm/s, and Delta E(Q)(1) = 1. 91(3) mm/s; delta(2) = 0.52(2) mm/s, and Delta E(Q)(2) = 1.41(3) mm/s. Thus, peroxo Delta 9D contains two asymmetric diferric clusters and reacts to yield peroxo-cycled Delta 9D, also containing two asymmetric diferric clusters that most probably represent a substrate complex state. The clusters of both peroxo Delta 9D and peroxo-cycled Delta 9D have a diamagnetic ground state. Because peroxo Delta 9D and peroxo-cycled Delta 9D are observed only in the presence of 18:0-ACP, substrate binding appears to have introduced asymmetry into the Delta 9D diiron clusters. In situ photolysis of peroxo Delta 9D at 4.2 K in the M?ssbauer cryostat caused the release of O(2) and the reappearance of a diferrous Delta 9D.18:0-ACP complex with slightly changed parameters, suggesting a constrained cluster configuration was produced by the photolysis event. Annealing the photolyzed sample for 30 min at 77 K quantitatively restored the M?ssbauer spectrum of peroxo Delta 9D, showing that the released O(2) was effectively sequestered within the active site.     

The iron oxidation and hydrolysis chemistry of Escherichia coli bacterioferritin

Bacterioferritins are members of a class of spherical shell-like iron storage proteins that catalyze the oxidation and hydrolysis of iron at specific sites inside the protein shell, resulting in formation of a mineral core of hydrated ferric oxide within the protein cavity. Electrode oximetry/pH stat was used to study iron oxidation and hydrolysis chemistry in E. coli bacterioferritin. Consistent with previous UV-visible absorbance measurements, three distinct kinetic phases were detected, and the stoichiometric equations corresponding to each have been determined. The rapid phase 1 reaction corresponds to pairwise binding of 2 Fe(2+) ions at a dinuclear site, called the ferroxidase site, located within each of the 24 subunits, viz., 2Fe(2+) + P(Z) --> [Fe(2)-P](Z) + 4H(+), where P(Z) is the apoprotein of net charge Z and [Fe(2)-P](Z) represents a diferrous ferroxidase complex. The slower phase 2 reaction corresponds to the oxidation of this complex by molecular oxygen according to the net equation: [Fe(2)-P](Z) + (1)/(2)O(2) --> [Fe(2)O-P](Z) where [Fe(2)O-P](Z) represents an oxidized diferric ferroxidase complex, probably a mu-oxo-bridged species as suggested by UV-visible and EPR spectrometric titration data. The third phase corresponds to mineral core formation according to the net reaction: 4Fe(2+) + O(2) + 6H(2)O --> 4FeO(OH)((core)) + 8H(+). Iron oxidation is inhibited by the presence of Zn(2+) ions. The patterns of phase 2 and phase 3 inhibition are different, though inhibition of both phases is complete at 48 Zn(2+)per 24mer, i.e., 2 Zn(2+) per ferroxidase center.     

Hydrogen peroxide dependent cis-dihydroxylation of benzoate by fully oxidized benzoate 1,2-dioxygenase

Rieske dioxygenases catalyze the reductive activation of O2 for the formation of cis-dihydrodiols from unactivated aromatic compounds. It is known that O2 is activated at a mononuclear non-heme iron site utilizing electrons supplied by a nearby Rieske iron sulfur cluster. However, it is controversial whether the reactive species is an Fe(III)-(hydro)peroxo or an Fe(II)-(hydro)peroxo (or electronically equivalent species formed by breaking the O-O bond). Here it is shown that benzoate 1,2 dioxygenase oxygenase component (BZDO) prepared in a form with the Rieske cluster oxidized and the mononuclear iron in the Fe(III) state can utilize H2O2 as a source of reduced oxygen to form the correct cis-dihydrodiol product from benzoate. The reaction approaches stoichiometric yield relative to the mononuclear Fe(III) concentration, being limited to a single turnover by inefficient product release from the Fe(III)-product complex. EPR and M?ssbauer studies show that the iron remains ferric throughout this single turnover "peroxide shunt" reaction. These results strongly support Fe(III)-(hydro)peroxo (or Fe(V)-oxo-hydroxo) as the reactive species because there is no source of additional reducing equivalents to form the Fe(II)-(hydro)peroxo state. This conclusion could be further tested in the case of BZDO because the peroxide shunt occurs very slowly compared with normal turnover, allowing the reactive intermediate to be trapped for spectroscopic analysis. We attribute the slow reaction rate to a forced change in the normally strict order of the substrate binding and enzyme reduction steps that regulate the catalytic cycle. The reactive intermediate is a high-spin ferric species exhibiting an unusual negative zero field splitting and other EPR and M?ssbauer spectroscopic properties reminiscent of previously characterized side-on-bound peroxide adducts of Fe(III) model complexes. If the species in BZDO is a similar adduct, its isomer shift is most consistent with an Fe(III)-hydroperoxo reactive state.     

M?ssbauer spectroscopic investigation of structure-function relations in ferritins.

Ferritin plays an important role in iron metabolism and our aim is to understand the mechanisms by which iron is sequestered within its protein shell as the mineral ferrihydrite. We present M?ssbauer spectroscopic data on recombinant human and horse spleen ferritin from which we draw the following conclusions: (1) that apoferritin catalyses Fe(II) oxidation as a first step in ferrihydrite deposition, (2) that the catalysis of Fe(II) oxidation is associated with residues situated within H chains, at the postulated 'ferroxidase centre' and not in the 3-fold inter-subunit channels previously suggested as the initial Fe(II) binding and oxidation site; (3) that both isolated Fe(III) and Fe(III) mu-oxo-bridged dimers found previously by M?ssbauer spectroscopy to be intermediates in iron-core formation in horse spleen ferritin, are located on H chains; and (4) that these dimers form at ferroxidase centres. The importance of the ferroxidase centre is suggested by the conservation of its ligands in many ferritins from vertebrates, invertebrates and plants. Nevertheless iron-core formation does occur in those ferritins that lack ferroxidase centres even though the initial Fe(II) oxidation is relatively slow. We compare the early stages of core formation in such variants and in horse spleen ferritin in which only 10-15% of its chains are of the H type. We discuss our findings in relation to the physiological role of isoferritins in iron storage processes.     

Is hydrogen peroxide produced during iron(II) oxidation in mammalian apoferritins?

The ferritins are a class of iron storage and detoxification proteins that play a central role in the biological management of iron. These proteins have a catalytic site, "the ferroxidase site", located on the H-type subunit that facilitates the oxidation of Fe(II) to Fe(III) by O(2). Measurements during the past 10 years on a number of vertebrate ferritins have provided evidence that H(2)O(2) is produced at this diiron ferroxidase site. Recently reported experiments using three different analytical methods with horse spleen ferritin (HoSF) have failed to detect H(2)O(2) production in this protein [Lindsay, S., Brosnahan, D., and Watt, G. D. (2001) Biochemistry 40, 3340-3347]. These findings contrast with earlier results reporting H(2)O(2) production in HoSF [Xu, B., and Chasteen, N. D. (1991) J. Biol. Chem. 266, 19965-19970]. Here a sensitive fluorescence assay and an assay based on O(2) evolution in the presence of catalase were used to demonstrate that H(2)O(2) is produced in HoSF as previously reported. However, because of the relatively few H-chain ferroxidase sites in HoSF and the reaction of H(2)O(2) with the protein, H(2)O(2) is more difficult to detect in this ferritin than in recombinant human H-chain ferritin (HuHF). The proper sequence of addition of reagents is important for measurement of the total amount of H(2)O(2) produced during the ferroxidation reaction.     

Role of phosphate in initial iron deposition in apoferritin

Ferritins from microorganisms to man are known to contain varying amounts of phosphate which has a pronounced effect on the structural and magnetic properties of their iron mineral cores. The present study was undertaken to gain insight into the role of phosphate in the early stages of iron accumulation by ferritin. The influence of phosphate on the initial deposition of iron in apoferritin (12 Fe/protein) was investigated by EPR, 57Fe M?ssbauer spectroscopy, and equilibrium dialysis. The results indicate that phosphate has a significant influence on iron deposition. The presence of 1 mM phosphate during reconstitution of ferritin from apoferritin, Fe(II), and O2 accelerates the rate of oxidation of the iron 2-fold at pH 7.5. In the presence or absence of phosphate, the rate of oxidation at 0 degrees C follows simple first-order kinetics with respect to Fe(II) with half-lives of 1.5 +/- 0.3 or 2.8 +/- 0.2 min, respectively, consistent with a single pathway for iron oxidation when low levels of iron are added to the apoprotein. This pathway may involve a protein ferroxidase site where phosphate may bind iron(II), shifting its redox potential to a more negative value and thus facilitating its oxidation. Following oxidation, an intermediate mononuclear Fe(III)-protein complex is formed which exhibits a transient EPR signal at g' = 4.3. Phosphate accelerates the rate of decay of the signal by a factor of 3-4, producing EPR-silent oligonuclear or polynuclear Fe(III) clusters. In 0.5 mM Pi, the signal decays according to a single phase first-order process with a half-life near 1 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Iron(II) and hydrogen peroxide detoxification by human H-chain ferritin. An EPR spin-trapping study

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 (相似文献   

Kinetic studies of iron deposition catalyzed by recombinant human liver heavy and light ferritins and Azotobacter vinelandii bacterioferritin using O2 and H2O2 as oxidants

The discrepancy between predicted and measured H(2)O(2) formation during iron deposition with recombinant heavy human liver ferritin (rHF) was attributed to reaction with the iron protein complex [Biochemistry 40 (2001) 10832-10838]. This proposal was examined by stopped-flow kinetic studies and analysis for H(2)O(2) production using (1) rHF, and Azotobacter vinelandii bacterial ferritin (AvBF), each containing 24 identical subunits with ferroxidase centers; (2) site-altered rHF mutants with functional and dysfunctional ferroxidase centers; and (3) recombinant human liver light ferritin (rLF), containing no ferroxidase center. For rHF, nearly identical pseudo-first-order rate constants of 0.18 s(-1) at pH 7.5 were measured for Fe(2+) oxidation by both O(2) and H(2)O(2), but for rLF, the rate with O(2) was 200-fold slower than that for H(2)O(2) (k = 0.22 s(-1)). A Fe(2+)/O(2) stoichiometry near 2.4 was measured for rHF and its site altered forms, suggesting formation of H(2)O(2). Direct measurements revealed no H(2)O(2) free in solution 0.5-10 min after all Fe(2+) was oxidized at pH 6.5 or 7.5. These results are consistent with initial H(2)O(2) formation, which rapidly reacts in a secondary reaction with unidentified solution components. Using measured rate constants for rHF, simulations showed that steady-state H(2)O(2) concentrations peaked at 14 muM at approximately 600 ms and decreased to zero at 10-30 s. rLF did not produce measurable H(2)O(2) but apparently conducted the secondary reaction with H(2)O(2). Fe(2+)/O(2) values of 4.0 were measured for AvBF. Stopped-flow measurements with AvBF showed that both H(2)O(2) and O(2) react at the same rate (k = 0.34 s(-1)), that is faster than the reactions with rHF. Simulations suggest that AvBF reduces O(2) directly to H(2)O without intermediate H(2)O(2) formation.     

Multiple pathways for mineral core formation in mammalian apoferritin. The role of hydrogen peroxide

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.     

Iron oxidation chemistry in ferritin. Increasing Fe/O2 stoichiometry during core formation

The origin of previously observed variations in stoichiometry of iron oxidation during the oxidative deposition of iron in ferritin has been poorly understood. Knowledge of the stoichiometry of Fe(II) oxidation by O2 is essential to establishing the mechanism of iron core formation. In the present work, the amount of Fe(II) oxidized was measured by M?ssbauer spectrometry and the O2 consumed by mass spectrometry. The number of protons produced in the reaction was measured by "pH stat" titration and hydrogen peroxide production by the effect of the enzyme catalase on the measured stoichiometry. For protein samples containing low levels of iron (24 Fe(II)/protein) the stoichiometry was found to be 1.95 +/- 0.18 Fe(II)/O2 with H2O2 being a product, viz. Equation 1. 2Fe2+ + O2 + 4H2O----2FeOOH + H2O2 + 4H+ (1) EPR spin trapping experiments showed no evidence of superoxide radical formation. The stoichiometry markedly increased with additional iron (240-960 Fe/protein), to a value of 4 Fe(II)/O2 as in Equation 2. 4Fe2+ + O2 + 6H2O----4FeOOH + 8H+ (2) As the iron core is progressively laid down, the mechanism of iron oxidation changes from a protein dominated process with H2O2 being the primary product of O2 reduction to a mineral surface dominated process where H2O is the primary product. These results emphasize the importance of the apoferritin shell in facilitating iron oxidation in the early stage of iron deposition prior to significant development of the polynuclear iron core.     

Mechanistic studies of reactions of peroxodiiron(III) intermediates in T201 variants of toluene/o-xylene monooxygenase hydroxylase

Site-directed mutagenesis studies of a strictly conserved T201 residue in the active site of toluene/o-xylene monooxygenase hydroxylase (ToMOH) revealed that a single mutation can facilitate kinetic isolation of two distinctive peroxodiiron(III) species, designated T201(peroxo) and ToMOH(peroxo), during dioxygen activation. Previously, we characterized both oxygenated intermediates by UV-vis and Mo?ssbauer spectroscopy, proposed structures from DFT and QM/MM computational studies, and elucidated chemical steps involved in dioxygen activation through the kinetic studies of T201(peroxo) formation. In this study, we investigate the kinetics of T201(peroxo) decay to explore the reaction mechanism of the oxygenated intermediates following O(2) activation. The decay rates of T201(peroxo) were monitored in the absence and presence of external (phenol) or internal (tryptophan residue in an I100W variant) substrates under pre-steady-state conditions. Three possible reaction models for the formation and decay of T201(peroxo) were evaluated, and the results demonstrate that this species is on the pathway of arene oxidation and appears to be in equilibrium with ToMOH(peroxo).     

Iron detoxification properties of Escherichia coli bacterioferritin. Attenuation of oxyradical chemistry

Bacterioferritin (EcBFR) of Escherichia coli is an iron-mineralizing hemoprotein composed of 24 identical subunits, each containing a dinuclear metal-binding site known as the "ferroxidase center." The chemistry of Fe(II) binding and oxidation and Fe(III) hydrolysis using H(2)O(2) as oxidant was studied by electrode oximetry, pH-stat, UV-visible spectrophotometry, and electron paramagnetic resonance spin trapping experiments. Absorption spectroscopy data demonstrate the oxidation of two Fe(II) per H(2)O(2) at the ferroxidase center, thus avoiding hydroxyl radical production via Fenton chemistry. The oxidation reaction with H(2)O(2) corresponds to [Fe(II)(2)-P](Z) + H(2)O(2) --> [Fe(III)(2)O-P](Z) + H(2)O, where [Fe(II)(2)-P](Z) represents a diferrous ferroxidase center complex of the protein P with net charge Z and [Fe(III)(2)O-P](Z) a micro-oxo-bridged diferric ferroxidase complex. The mineralization reaction is given by 2Fe(2+) + H(2)O(2) + 2H(2)O --> 2FeOOH((core)) + 4H(+), where two Fe(II) are again oxidized by one H(2)O(2). Hydrogen peroxide is shown to be an intermediate product of dioxygen reduction when O(2) is used as the oxidant in both the ferroxidation and mineralization reactions. Most of the H(2)O(2) produced from O(2) is rapidly consumed in a subsequent ferroxidase reaction with Fe(II) to produce H(2)O. EPR spin trapping experiments show that the presence of EcBFR greatly attenuates the production of hydroxyl radical during Fe(II) oxidation by H(2)O(2), consistent with the ability of the bacterioferritin to facilitate the pairwise oxidation of Fe(II) by H(2)O(2), thus avoiding odd electron reduction products of oxygen and therefore oxidative damage to the protein and cellular components through oxygen radical chemistry.     

Coordinating subdomains of ferritin protein cages with catalysis and biomineralization viewed from the C 4 cage axes

Integrated ferritin protein cage function is the reversible synthesis of protein-caged, solid Fe₂O₃·H₂O minerals from Fe²⁺ for metabolic iron concentrates and oxidant protection; biomineral order differs in different ferritin proteins. The conserved 432 geometric symmetry of ferritin protein cages parallels the subunit dimer, trimer, and tetramer interfaces, and coincides with function at several cage axes. Multiple subdomains distributed in the self-assembling ferritin nanocages have functional relationships to cage symmetry such as Fe²⁺ transport though ion channels (threefold symmetry), biomineral nucleation/order (fourfold symmetry), and mineral dissolution (threefold symmetry) studied in ferritin variants. On the basis of the effects of natural or synthetic subunit dimer cross-links, cage subunit dimers (twofold symmetry) influence iron oxidation and mineral dissolution. 2Fe²⁺/O₂ catalysis in ferritin occurs in single subunits, but with cooperativity (n = 3) that is possibly related to the structure/function of the ion channels, which are constructed from segments of three subunits. Here, we study 2Fe²⁺ + O₂ protein catalysis (diferric peroxo formation) and dissolution of ferritin Fe₂O₃·H₂O biominerals in variants with altered subunit interfaces for trimers (ion channels), E130I, and external dimer surfaces (E88A) as controls, and altered tetramer subunit interfaces (L165I and H169F). The results extend observations on the functional importance of structure at ferritin protein twofold and threefold cage axes to show function at ferritin fourfold cage axes. Here, conserved amino acids facilitate dissolution of ferritin-protein-caged iron biominerals. Biological and nanotechnological uses of ferritin protein cage fourfold symmetry and solid-state mineral properties remain largely unexplored.     

The putative "nucleation site" in human H-chain ferritin is not required for mineralization of the iron core

It is widely believed that the putative nucleation site (Glu61, Glu64, and Glu67) in mammalian H-chain ferritin plays an important role in mineral core formation in this protein. Studies of nucleation site variant A2 (E61A/E64A/E67A) of H-chain ferritin have traditionally shown impaired iron oxidation activity and mineralization. However, recent measurements have suggested that the previously observed impairment may be due to disruption of the ferroxidase site of the protein since Glu61 is a shared ligand of the ferroxidase and nucleation sites of the protein. This study employed a new nucleation site variant A1 (E64A/E67A) which retains the ferroxidase site ligand Glu61. The data (O(2) uptake, iron binding, and conventional and stopped-flow kinetics measurements) show that variant A1 retains a completely functional ferroxidase site and has iron oxidation and mineralization properties similar to those of the wild-type human H-chain protein. Thus, in contrast to previously published literature, this study demonstrates that the putative "nucleation site" does not play an important role in iron uptake or mineralization in H-chain ferritin.     

Oxidation of Good's buffers by hydrogen peroxide

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.     

Ferroxidase kinetics of horse spleen apoferritin.

Protein ferroxidase site(s), which catalyze the reaction between ferrous ion and dioxygen, have long been thought to play a role in core formation in ferritin; however, the mechanism of the reaction has never been studied in detail. In the present work, the enzymatic activity of ferritin was examined using oximetry, the net Fe2+ oxidation reaction being as follows. [formula: see text] The reaction exhibits saturation kinetics with respect to both Fe2+ and O2 (apparent Michaelis constants: Km,Fe = 0.35 +/- 0.01 mM and Km,O2 = 0.14 +/- 0.03 mM). The enzyme has a turnover number kcat = 80 +/- 3 min-1 at 20 degrees C with maximal activity at pH 7. The kinetics are discussed in terms of two mechanisms, one involving monomeric and the other dimeric iron protein complexes. In both instances Fe(II) oxidation occurs in 1-electron steps. Zinc(II) is a competitive inhibitor of iron(II) oxidation at Zn2+/apoprotein ratios > or = 6 (inhibitor constant KI,Zn = 0.067 +/- 0.011 mM) but appears to be a noncompetitive inhibitor at lower ratios (< or = 2), indicating the presence of more than one type of zinc binding site on the protein. At increments of 50 Fe2+/protein or less, all of the iron is oxidized via the protein ferroxidase site(s), independent of the amount of core already present. However, when larger increments are employed, some iron oxidation appears to occur on the surface of the mineral core. The results of these studies emphasize the role of the protein shell in all phases of core growth and confirm the presence of a functionally important catalytic site in ferritin in addition to other binding sites on the protein for iron.     

The ferroxidase reaction of ferritin reveals a diferric mu-1,2 bridging peroxide intermediate in common with other O2-activating non-heme diiron proteins

Ferritins are ubiquitous proteins that concentrate, store, and detoxify intracellular iron through oxidation of Fe2+ (ferroxidation), followed by translocation and hydrolysis to form a large inorganic mineral core. A series of mutagenesis, kinetics, and spectroscopic studies of ferritin led to the proposal that the oxidation/translocation path involves a diiron protein site. Recent stopped-flow absorption and rapid freeze-quench M?ssbauer studies have identified a single peroxodiferric species as the initial transient intermediate formed in recombinant frog M ferritin during rapid ferroxidation [Pereira, S. A., Small, W., Krebs, C., Tavares, P., Edmondson, D. E., Theil, E. C., and Huynh, B. H. (1998) Biochemistry 37, 9871-9876]. To further characterize this transient intermediate and to establish unambiguously the peroxodiferric assignment, rapid freeze-quenching was used to trap the initial intermediate for resonance Raman investigation. Discrete vibrational modes are observed for this intermediate, indicating a single chromophore in a homogeneous state, in agreement with the M?ssbauer conclusions. The frequency at 851 cm-1 is assigned as nu(O-O) of the bound peroxide, and the pair of frequencies at 485 and 499 cm-1 is attributed, respectively, to nus and nuas of Fe-O2-Fe. Identification of the chromophore as a micro-1,2 bridged diferric peroxide is provided by the isotope sensitivity of these Raman bands. Similar peroxodiferric intermediates have been detected in a mutant of the R2 subunit of ribonucleotide reductase from Escherichia coli and chemically reduced Delta9 stearoyl-acyl carrier protein desaturase (Delta9D), but in contrast, the ferritin intermediate is trapped from the true reaction pathway of the native protein. Differences in the Raman signatures of these peroxide species are assigned to variations in Fe-O-O-Fe angles and may relate to whether the iron is retained in the catalytic center or released as an oxidized product.