Diferric Peroxo Research Articles

Nonheme Diiron Oxygenase Mimic That Generates a Diferric-Peroxo Intermediate Capable of Catalytic Olefin Epoxidation and Alkane Hydroxylation Including Cyclohexane.

Herein are described substrate oxidations with H2O2 catalyzed by [FeII(IndH)(CH3CN)3](ClO4)2 [IndH = 1,3-bis(2'-pyridylimino)isoindoline], involving a spectroscopically characterized (μ-oxo)(μ-1,2-peroxo)diiron(III) intermediate (2) that is capable of olefin epoxidation and alkane hydroxylation including cyclohexane. Species 2 also converts ketones to lactones with a decay rate dependent on [ketone], suggesting direct nucleophilic attack of the substrate carbonyl group by the peroxo species. In contrast, peroxo decay is unaffected by the addition of olefins or alkanes, but the label from H218O is incorporated into the the epoxide and alcohol products, implicating a high-valent iron-oxo oxidant that derives from O-O bond cleavage of the peroxo intermediate. These results demonstrate an ambiphilic diferric-peroxo intermediate that mimics the range of oxidative reactivities associated with O2-activating nonheme diiron enzymes.

Iron Binding in the Ferroxidase Site of Human Mitochondrial Ferritin.

Ferritins are nanocage proteins that store iron ions in their central cavity as hydrated ferric oxide biominerals. In mammals, further the L (light) and H (heavy) chains constituting cytoplasmic maxi-ferritins, an additional type of ferritin has been identified, the mitochondrial ferritin (MTF). Human MTF (hMTF) is a functional homopolymeric H-like ferritin performing the ferroxidase activity in its ferroxidase site (FS), in which Fe(II) is oxidized to Fe(III) in the presence of dioxygen. To better investigate its ferroxidase properties, here we performed time-lapse X-ray crystallography analysis of hMTF, providing structural evidence of how iron ions interact with hMTF and of their binding to the FS. Transient iron binding sites, populating the pathway along the cage from the iron entry channel to the catalytic center, were also identified. Furthermore, our kinetic data at variable iron loads indicate that the catalytic iron oxidation reaction occurs via a diferric peroxo intermediate followed by the formation of ferric-oxo species, with significant differences with respect to human H-type ferritin.

Impact of Phosphate on Iron Mineralization and Mobilization in Nonheme Bacterioferritin B from Mycobacterium tuberculosis.

Ferritins are supramolecular nanocage proteins, which synthesize hydrated ferric oxyhydroxide mineral via protein mediated rapid Fe2+ sequestration and ferroxidase reactions. Ferritin minerals are also associated with a significant amount of phosphate, which contribute toward their structure and reactivity. Like iron, phosphate also regulates the pathogenesis of Mycobacterium tuberculosis (Mtb), which expresses two types of ferritin: heme binding bacterioferritin A (BfrA) and nonheme binding bacterioferritin B (BfrB). Unlike Mtb BfrA, the rapid kinetics and mechanism of ferroxidase activity, and the mineral core formation/dissolution in Mtb BfrB are not well explored. Moreover, the effect of physiological levels of phosphate (0-10 mM) on the synthesis, structure, and reactivity of ferritin mineral core also require investigation in detail. Therefore, the stopped-flow rapid kinetics of ferroxidase activity (ΔA650/Δt) of Mtb BfrB was carried out, which detected a transient intermediate similar to diferric peroxo species as observed in frog and human ferritins. Increasing phosphate concentration increased the initial rate of iron mineralization (ΔA350/Δt) and dissolved O2 consumption (both ∼1.5-2-fold). Phosphate not only decreased the amount of iron loading and size of the BfrB mineral core (both up to 2-fold) but also decreased its crystallinity, resembling the variations observed in the core morphology of different native ferritins. In addition, phosphate inhibited the kinetics of reductive iron mobilization (∼6-8-fold) indicating its influence on the stability of the iron mineral core. Hence, the current work provides the kinetic/mechanistic insight toward the ferroxidase activity in Mtb BfrB, apart from demonstrating the role of phosphate toward the structure/reactivity of its iron mineral.

High-resolution iron X-ray absorption spectroscopic and computational studies of non-heme diiron peroxo intermediates

Ferritin-like carboxylate-bridged non-heme diiron enzymes activate O2 for a variety of difficult reactions throughout nature. These reactions often begin by abstraction of hydrogen from strong CH bonds. The enzymes activate O2 at their diferrous cofactors to form canonical diferric peroxo intermediates, with a range of possible coordination modes. Herein, we explore the ability of high-energy resolution fluorescence detected X-ray absorption spectroscopy (HERFD XAS) to provide insight into the nature of peroxo level intermediates in non-heme diiron proteins. Freeze quenched (FQ) peroxo intermediates from p-aminobenzoate N-oxygenase (AurF), aldehyde-deformylating oxygenase (ADO), and the β subunit of class Ia ribonucleotide reductase from Escherichia coli (Ecβ) are investigated. All three intermediates are proposed to adopt different peroxo binding modes, and each exhibit different Fe Kα HERFD XAS pre-edge features and intensities. As these FQ-trapped samples consist of multiple species, deconvolution of HERFD XAS spectra based on speciation, as determined by Mössbauer spectroscopy, is also necessitated - yielding ‘pure’ diferric peroxo HERFD XAS spectra from dilute protein samples. Finally, the impact of a given peroxo coordination mode on the HERFD XAS pre-edge energy and intensity is evaluated through time-dependent density functional theory (TDDFT) calculations of the XAS spectra on a series of hypothetical model complexes, which span a full range of possible peroxo coordination modes to a diferric core. The utility of HERFD XAS for future studies of enzymatic intermediates is discussed.

Mechanism of the Dinuclear Iron Enzyme p‐Aminobenzoate N‐oxygenase from Density Functional Calculations

AbstractAurF is a diiron enzyme that utilizes two dioxygen molecules as the oxidant to catalyze the oxidation of p‐aminobenzoate to p‐nitrobenzoate. Density functional calculations were performed to elucidate the reaction mechanism of this enzyme. Two different models were considered, with the oxygenated intermediate being a diferric peroxo species or a diferric hydroperoxo species. The calculations strongly favor the model with a diferric peroxo species and support the mechanism proposed by Bollinger and co‐workers. The reaction starts with the binding of a dioxygen molecule to the diferrous center to generate a diferric peroxide complex. This is followed by the cleavage of the O−O bond, concertedly with the formation of the first N−O bond, which has a barrier of only 9.2 kcal/mol. Subsequently, the first‐shell ligand Glu227 abstracts a proton from the substrate. After the delivery of two electrons from the external reductant and two protons from solution, a water molecule and the experimentally suggested intermediate p‐hydroxylaminobenzoate are produced and the diferrous center is regenerated. The oxidation of the p‐hydroxylaminobenzoate intermediate requires the binding of a second dioxygen molecule to the diferrous center to generate the diferric peroxide complex. Similarly to the oxidation of p‐aminobenzoate, the O−O bond cleavage and the formation of the second N−O bond take place in a concerted step. The p‐nitrobenzoate product is formed after the release of two protons and two electrons from the substrate. The model with a hydroperoxo species gave a much high barrier of 28.7 kcal/mol for the substrate oxidation due to the large energy penalty for the generation of the active hydroperoxo species.

Fe(2+) substrate transport through ferritin protein cage ion channels influences enzyme activity and biomineralization.

Ferritins, complex protein nanocages, form internal iron-oxy minerals (Fe2O3·H2O), by moving cytoplasmic Fe(2+) through intracage ion channels to cage-embedded enzyme (2Fe(2+)/O2 oxidoreductase) sites where ferritin biomineralization is initiated. The products of ferritin enzyme activity are diferric oxy complexes that are mineral precursors. Conserved, carboxylate amino acid side chains of D127 from each of three cage subunits project into ferritin ion channels near the interior ion channel exits and, thus, could direct Fe(2+) movement to the internal enzyme sites. Ferritin D127E was designed and analyzed to probe properties of ion channel size and carboxylate crowding near the internal ion channel opening. Glu side chains are chemically equivalent to, but longer by one -CH2 than Asp, side chains. Ferritin D127E assembled into normal protein cages, but diferric peroxo formation (enzyme activity) was not observed, when measured at 650nm (DFP λ max). The caged biomineral formation, measured at 350nm in the middle of the broad, nonspecific Fe(3+)-O absorption band, was slower. Structural differences (protein X-ray crystallography), between ion channels in wild type and ferritin D127E, which correlate with the inhibition of ferritin D127E enzyme activity include: (1) narrower interior ion channel openings/pores; (2) increased numbers of ion channel protein-metal binding sites, and (3) a change in ion channel electrostatics due to carboxylate crowding. The contributions of ion channel size and structure to ferritin activity reflect metal ion transport in ion channels are precisely regulated both in ferritin protein nanocages and membranes of living cells.

Moving Fe2+ from ferritin ion channels to catalytic OH centers depends on conserved protein cage carboxylates.

Ferritin biominerals are protein-caged metabolic iron concentrates used for iron-protein cofactors and oxidant protection (Fe(2+) and O2 sequestration). Fe(2+) passage through ion channels in the protein cages, like membrane ion channels, required for ferritin biomineral synthesis, is followed by Fe(2+) substrate movement to ferritin enzyme (Fox) sites. Fe(2+) and O2 substrates are coupled via a diferric peroxo (DFP) intermediate, λmax 650 nm, which decays to [Fe(3+)-O-Fe(3+)] precursors of caged ferritin biominerals. Structural studies show multiple conformations for conserved, carboxylate residues E136 and E57, which are between ferritin ion channel exits and enzymatic sites, suggesting functional connections. Here we show that E136 and E57 are required for ferritin enzyme activity and thus are functional links between ferritin ion channels and enzymatic sites. DFP formation (Kcat and kcat/Km), DFP decay, and protein-caged hydrated ferric oxide accumulation decreased in ferritin E57A and E136A; saturation required higher Fe(2+) concentrations. Divalent cations (both ion channel and intracage binding) selectively inhibit ferritin enzyme activity (block Fe(2+) access), Mn(2+) << Co(2+) < Cu(2+) < Zn(2+), reflecting metal ion-protein binding stabilities. Fe(2+)-Cys126 binding in ferritin ion channels, observed as Cu(2+)-S-Cys126 charge-transfer bands in ferritin E130D UV-vis spectra and resistance to Cu(2+) inhibition in ferritin C126S, was unpredicted. Identifying E57 and E136 links in Fe(2+) movement from ferritin ion channels to ferritin enzyme sites completes a bucket brigade that moves external Fe(2+) into ferritin enzymatic sites. The results clarify Fe(2+) transport within ferritin and model molecular links between membrane ion channels and cytoplasmic destinations.

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 Fe(2+) 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 Fe(2+) 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. 2Fe(2+)/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 2Fe(2+)+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.

Ferritin Ion Channel Disorder Inhibits Fe(II)/O2 Reactivity at Distant Sites

Ferritins, a complex, mineralized, protein nanocage family essential for life, provide iron concentrates and oxidant protection. Protein-based ion channels and Fe(II)/O(2) catalysis initiate conversion of thousands of Fe atoms to caged, ferritin Fe(2)O(3)·H(2)O minerals. The ion channels consist of six helical segments, contributed by 3 of 12 or 24 polypeptide subunits, around the 3-fold cage axes. The channel structure guides entering Fe(II) ions toward multiple, catalytic, diiron sites buried inside ferritin protein helices, ~20 Å away from channel internal exits. The catalytic product, Fe(III)-O(H)-Fe(III), is a mineral precursor; mineral nucleation begins inside the protein cage with mineral growth in the central protein cavity (5-8 nm diameter). Amino acid substitutions that changed ionic or hydrophobic channel interactions R72D, D122R, and L134P increased ion channel structural disorder (protein crystallographic analyses) and increased Fe(II) exit [chelated Fe(II) after ferric mineral reduction/dissolution]. Since substitutions of some channel carboxylate residues diminished ferritin catalysis with no effect on Fe(II) exit, such as E130A and D127A, we investigated catalysis in ferritins with altered Fe(II) exit, R72D, D122R and L134P. The results indicate that simply changing the ionic properties of the channels, as in the R72D variant, need not change the forward catalytic rate. However, both D122R and L134P, which had dramatic effects on ferritin catalysis, also caused larger effects on channel structure and order, contrasting with R72D. All three amino acid substitutions, however, decreased the stability of the catalytic intermediate, diferric peroxo, even though overall ferritin cage structure is very stable, resisting 80 °C and 6 M urea. The localized structural changes in ferritin subdomains that affect ferritin function over long distances illustrate new properties of the protein cage in natural ferritin function and for applied ferritin uses.

Ferritin‐specific diiron ligands control Fe/O2 chemistry in iron mineralization; diiron substrate vs diiron oxygenases

Catalytic coupling of Fe(II) and O2 at diiron sites in ferritin initiates iron mineralization. A diferric peroxo (DFP) intermediate is common to catalysis in ferritins and in diiron cofactor oxygenases. Two of six residues in the ferritin Fe2 site of the catalytic center differ from cofactor Fe2 sites. Conserved Gln137 and variable Asp/Ser/Ala140 are substituted to Glu and His in diiron oxygenases, respectively. Here, we report how mutation of ferritin‐specific diiron ligands alters ferroxidation activity. The substitution of Gln137 for Glu showed no detectable DFP but normal ferroxidation rates, indicating reaction mechanism alterations. Asp140 natural variants, Ser/Ala140 mutants, varied the formation/decay kinetics of DFP, but not mechanism. His140 ferritin formed no DFP and ferroxidation rates decreased 40‐fold. Protein structures (X‐ray crystallography with H‐L Ng and T Alber, LBNL) indicate that His140 can block Fe/O2/H2O access to diiron catalytic centers from the protein cavity. These new observations indicate that DFP formation depends on Fe2 site residues that use His to yield higher valent iron intermediates in the context of related diiron oxygenases, contrasting with His inhibition in the ferritin context where Asp/Ser/Ala are required for turnover with release of diferric oxo/hydroxo mineral precursors from the catalytic site. Part support; DK20251 (ECT, MRH and TT) and the JSPS (TT).

The ferritin Fe2 site at the diiron catalytic center controls the reaction with O2 in the rapid mineralization pathway.

Oxidoreduction in ferritin protein nanocages occurs at sites that bind two Fe(II) substrate ions and O(2), releasing Fe(III)(2)-O products, the biomineral precursors. Diferric peroxo intermediates form in ferritins and in the related diiron cofactor oxygenases. Cofactor iron is retained at diiron sites throughout catalysis, contrasting with ferritin. Four of the 6 active site residues are the same in ferritins and diiron oxygenases; ferritin-specific Gln(137) and variable Asp/Ser/Ala(140) substitute for Glu and His, respectively, in diiron cofactor active sites. To understand the selective functions of diiron substrate and diiron cofactor active site residues, we compared oxidoreductase activity in ferritin with diiron cofactor residues, Gln(137) --> Glu and Asp(140) --> His, to ferritin with natural diiron substrate site variations, Asp(140), Ser(140), or Ala(140). In Gln(137) --> Glu ferritin, diferric peroxo intermediates were undetectable; an altered Fe(III)-O product formed, DeltaA(350) = 50% of wild type. In Asp(140) --> His ferritin, diferric peroxo intermediates were also undetectable, and Fe(II) oxidation rates decreased 40-fold. Ferritin with Asp(140), Ser(140), or Ala(140) formed diferric peroxo intermediates with variable kinetic stabilities and rates: t(1/2) varied 1- to 10-fold; k(cat) varied approximately 2- to 3-fold. Thus, relatively small differences in diiron protein catalytic sites determine whether, and for how long, diferric peroxo intermediates form, and whether the Fe-active site bonds persist throughout the reaction cycle (diiron cofactors) or break to release Fe(III)(2)-O products (diiron substrates). The results and the coding similarities for cofactor and substrate site residues-e.g., Glu/Gln and His/Asp pairs share 2 of 3 nucleotides-illustrate the potential simplicity of evolving active sites for diiron cofactors or diiron substrates.

Paired Bacillus anthracis Dps (Mini-ferritin) Have Different Reactivities with Peroxide

Dps (DNA protection during starvation) proteins, mini-ferritins in the ferritin superfamily, catalyze Fe(2+)/H(2)O(2)/O(2) reactions and make minerals inside protein nanocages to minimize radical oxygen-chemistry (metal/osmotic/temperature/nutrient/oxidant) and sometimes to confer virulence. Paired Dps proteins in Bacillus, rare in other bacteria, have 60% sequence identity. To explore functional differences in paired Bacilli Dps protein, we measured ferroxidase activity and DNA protection (hydroxyl radical) for Dps protein dodecamers from Bacillus anthracis (Ba) since crystal structures and iron mineralization (iron-stain) were known. The self-assembled (200 kDa) Ba Dps1 (Dlp-1) and Ba Dps2 (Dlp-2) proteins had similar Fe(2+)/O(2) kinetics, with space for minerals of 500 iron atoms/protein, and protected DNA. The reactions with Fe(2+) were novel in several ways: 1) Ba Dps2 reactions (Fe(2+)/H(2)O(2)) proceeded via an A(650 nm) intermediate, with similar rates to maxi-ferritins (Fe(2+)/O(2)), indicating a new Dps protein reaction pathway, 2) Ba Dps2 reactions (Fe(2+)/O(2) versus Fe(2+)/O(2) + H(2)O(2)) differed 3-fold contrasting with Escherichia coli Dps reactions, with 100-fold differences, and 3) Ba Dps1, inert in Fe(2+)/H(2)O(2) catalysis, inhibited protein-independent Fe(2+)/H(2)O(2) reactions. Sequence similarities between Ba Dps1 and Bacillus subtilis DpsA (Dps1), which is regulated by general stress factor (SigmaB) and Fur, and between Ba Dps2 and B. subtilis MrgA, which is regulated by H(2)O(2) (PerR), suggest the function of Ba Dps1 is iron sequestration and the function of Ba Dps2 is H(2)O(2) destruction, important in host/pathogen interactions. Destruction of H(2)O(2) by Ba Dps2 proceeds via an unknown mechanism with an intermediate similar spectrally (A(650 nm)) and kinetically to the maxi-ferritin diferric peroxo complex.

Ferritin reactions: direct identification of the site for the diferric peroxide reaction intermediate.

Ferritins managing iron-oxygen biochemistry in animals, plants, and microorganisms belong to the diiron carboxylate protein family and concentrate iron as ferric oxide approximately 10(14) times above the ferric K(s). Ferritin iron (up to 4,500 atoms), used for iron cofactors and heme, or to trap DNA-damaging oxidants in microorganisms, is concentrated in the protein nanocage cavity (5-8 nm) formed during assembly of polypeptide subunits, 24 in maxiferritins and 12 in miniferritins/DNA protection during starvation proteins. Direct identification of ferritin ferroxidase (F(ox)) sites, complicated by multiple types of iron-ferritin interactions, is now achieved with chimeric proteins where putative F(ox) site residues were introduced singly and cumulatively into an inactive host, an L maxiferritin. A dimagnesium ferritin cocrystal model guided site design and the diferric peroxo F(ox) intermediates (A at 650 nm) monitored activity. Diferric peroxo formation in chimeric and WT proteins had similar K(app) values and Hill coefficients. Catalytic activity required cooperative ferrous substrate binding to two sites A (E, EXXH) and B (E, QXXD). The weaker B sites in ferritin contrast with stronger B sites (E, EXXH) in diiron carboxylate oxygenases, explaining diferric oxo/hydroxo product release in ferritin vs. diiron cofactor retention in oxygenases. Codons for Q/H and D/E differ by single nucleotides, suggesting simple DNA mutations relate site B diiron substrate sites and diiron cofactor sites in proteins. The smaller k(cat) values in chimeras indicate the absence of second-shell residues important for ferritin substrate-product channeling that, when identified, will outline the entire iron path from ferritin pores through the F(ox) site to the mineral cavity.

A Diferric Peroxo Complex with an Unprecedented Spin Configuration: An S=2 System Arising from an S=5/2, 1/2 Pair

In a spin: Transient peroxo iron complexes have been characterized by the means of Mössbauer and HF-EPR spectroscopies. Complex 1 has an unexpected S=2 ground state resulting from the coupling between the S=5/2 and S=1/2 iron(III) ions.

A Diferric Peroxo Complex with an Unprecedented Spin Configuration: AnS=2 System Arising from anS=5/2, 1/2 Pair

A Diferric Peroxo Complex with an Unprecedented Spin Configuration: AnS=2 System Arising from anS=5/2, 1/2 Pair

A Diferric Peroxo Complex with an Unprecedented Spin Configuration: AnS=2 System Arising from anS=5/2, 1/2 Pair

In a spin: Transient peroxo iron complexes have been characterized by the means of Mössbauer and HF-EPR spectroscopies. Complex 1 has an unexpected S=2 ground state resulting from the coupling between the S=5/2 and S=1/2 iron(III) ions.

The ferritin iron entry and exit problem

The entry and exit of Fe(II) ions in ferritin are endpoints in the process which concentrates iron as a solid (hydrated ferric oxide) to be used by living cells. Ferritin is a response to the trillion fold mismatch between the solubility of iron in neutral, aqueous, aerated solutions and the requirements for protein biosynthesis. The supramolecular structure of 24 polypeptides (subunits) joined by non-covalent bonds in a highly symmetrical (4, 3, 2), large (12 nm diameter) protein with a cavity (8 nm diameter) is found in plants, animals and microorganisms. Of the five types of iron sites which can be defined, iron entry (site 1) is likely at the junction of three subunits. A ferroxidase site (site 2), present in H-type ferritins, binds Fe(II) which reacts with dioxygen to form an initial, μ-1,2 diferric peroxo complex. The peroxo complex decays into hydrogen peroxide and the multiple diferric oxo complexes that are mineral precursors (sites 3a, 3b, 3x) in transit across the protein to the cavity; the ferroxidase site is sensitive to both natural and engineered variations in Fe ligands and ‘second shell amino’ acids such as tyrosine 30 and leucine 134. Studies of ferritin with subunits which lack the ferroxidase site (L-type) show that the mineral anchor sites of clustered R-COOH (site 4) are sensitive to RCH 3 and RSH replacements. Iron exit (site 5), studied by adding NADH/FMN as a trigger, is at or near the entry site. Iron exit can be enhanced by localized unfolding of the polypeptide at the junction of three subunits, suggesting a regulation-sensitive biological signal for iron exit. The entry and exit of iron to and from the mineral, unique to ferritin has steps which parallel those in channel ion transport, and ion transport to and from biomineral such as tooth and bone. Iron entry involves oxidation at non-heme iron catalytic centers, similar to ribonucleotide reductase and methane monoxygenase, but diverging in the role of iron as a substrate rather than a cofactor. Sorting out the evolutionary and mechanistic relationships of ferritin and other proteins which use metal ions, as well as fully characterizing all five types of the functional iron sites are challenges which will keep biological inorganic chemists occupied for some time to come.

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.

A kinetic study of the formation of a model high-valent diiron non-heme complex, [FeIIIFeIV(μ-O)2(tpa)2]3+ (tpa = tris(2-pyridylmethyl)amine), by cryogenic stopped-flow techniques †

[Fe2(µ-O)(tpa)2(OH)(H2O)]3+ reacts with H2O2 in acetonitrile at –40 °C forming a transient diferric peroxo intermediate (λmax = 700 nm, e = 1800 dm3 mol–1 cm–1), which then reacts with a second H2O2 molecule to give [FeIIIFeIV(µ-O)2(tpa)2]3+.

Diiron-cluster intermediates in biological oxygen activation reactions

The activation of molecular oxygen is a fundamental process that is important in metabolic reactions in biological systems. In addition to the well-studied role of hemoproteins in participating in oxygen activation, there is increasing evidence that dinuclear iron centers in proteins also function in this role. Recent work on the interaction of the dinuclear iron centers found in ribonucleotide reductase and in methane monooxygenase with molecular oxygen is reviewed. Available spectroscopic data on reaction intermediates trapped by the rapid freeze quench kinetic technique allow insights into their structures and on the oxidation level of each Fe ion in the complex. These data suggest the initial intermediate formed on reaction of the diferrous center with oxygen is the formation of a diferric peroxo complex which undergoes further internal electron reorganization to form a diferryl-oxo complex termed intermediate Q. It is suggested that these intermediates are formed as initial steps in both methane monooxygenase and in ribonucleotide reductase activation of molecular oxygen. Further reaction steps of Q in each enzyme are distinguished by the source of the subsequent reducing equivalent. In the case of methane monooxygenase, the hydrocarbon substrate is the source of the required reducing equivalent to form a mixed valent ferryl/ferric complex which is proposed to donate a hydroxyl group to the hydrocarbon radical with resulting formation of a diferric ‘resting’ state and product. An external electron source supplies the single electron to the difrrryl center in ribonucleotide reductase to form a mixed valent complex that is capable of oxidizing a proximal tyrosyl residue (122) to its stable radical form with formation of the ‘resting’ diferric form of the enzyme. This ‘unified’ scheme for the activation of molecular oxygen by dinuclear iron centers in these two enzymes may have applicability to other enzymes containing these centers.