The influence of conserved tyrosine 30 and tissue-dependent differences in sequence on ferritin function: use of blue and purple Fe(III) species as reporters of ferroxidation

Fetter, John; Cohen, Jonathan D.; Danger, Dana P.; Sanders-Loehr, Joann; Theil, Elizabeth C.

doi:10.1007/s007750050180

Cited by 47 publications

(73 citation statements)

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“…In the channel, a coordination site with different affinities for substrate ferrous (lower than the active site) and ferric product (higher than the active site) is needed for the migration of the diferric species. This mechanism is consistent with the reported tenfold reduction in iron oxidation rate upon substitution of Tyr with Phe at position 30 (33). When a second equivalent of iron(II) is added (2 irons/active site), NMR showed paramagnetic species penetrating further into the subunit channels.…”

Section: Discussionsupporting

confidence: 89%

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NMR reveals pathway for ferric mineral precursors to the central cavity of ferritin

Turano

Lalli

Felli

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

133

202

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Ferritin is a multimeric nanocage protein that directs the reversible biomineralization of iron. At the catalytic ferroxidase site two iron (II) ions react with dioxygen to form diferric species. In order to study the pathway of iron(III) from the ferroxidase site to the central cavity a new NMR strategy was developed to manage the investigation of a system composed of 24 monomers of 20 kDa each. The strategy is based on 13 C-13 C solution NOESY experiments combined with solid-state proton-driven 13 C-13 C spin diffusion and 3D coherence transfer experiments. In this way, 75% of amino acids were recognized and 35% sequence-specific assigned. Paramagnetic broadening, induced by iron(III) species in solution 13 C-13 C NOESY spectra, localized the iron within each subunit and traced the progression to the central cavity. Eight iron ions fill the 20-Å-long iron channel from the ferrous/dioxygen oxidoreductase site to the exit into the cavity, inside the four-helix bundle of each subunit, contrasting with short paths in models. Magnetic susceptibility data support the formation of ferric multimers in the iron channels. Multiple iron channel exits are near enough to facilitate high concentration of iron that can mineralize in the ferritin cavity, illustrating advantages of the multisubunit cage structure.¹³C direct detection | high molecular weight NMR | iron channels | solid-state NMR | paramagnetic NMR F erritins are a family of protein nanocages that concentrate iron in biominerals for controlled release and use in enzyme iron cofactors. A large cavity that occupies about 30% of the total protein volume at the center of the cage is the mineralization site of ferritin ferrihydrite. The importance of ferritin is illustrated by embryonic lethality of gene deletion in mammals (1) and resistance to oxidants in animals, plants, bacteria, and archea that reflects consumption of iron(II) and dioxygen or hydrogen peroxide (2-5). Some classes of nanomaterials use ferritin nanocages as templates (6). The protein nanocages self-assemble from fourhelix-bundle subunits into cages of two sizes: 12-subunit, hydrogen peroxide-consuming miniferritins of bacteria and archea, and 24-subunit maxiferritins of archea, bacteria, and higher organisms (3, 4, 7). Miniferritins may be the more primitive and are alternatively named DNA-binding proteins because some of them coat DNA.The first step in the conversion of iron(II) and dioxygen to ferric oxide mineral, in the 24-subunit maxiferritins, occurs at catalytic oxidoreductase (ferroxidase) sites in the center of each of the four-helix bundles, which have long axes oriented almost parallel to the protein surface (Fig. 1A). Each active site has residues contributed by each of the four helices (Fig. 1B) that create a diiron(II) site similar to the diiron cofactor sites in ribonucleotide reductases and stearoyl desaturases (8). When dioxygen, the second substrate, binds to the active site after iron(II) binding, diferric oxo products form via a diferric peroxo intermediate. The active site has...

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Section: Discussionsupporting

confidence: 89%

“…Frog (R. catesbieana) M ferritin protein was expressed from a pET3a plasmid (33). Uniformly 13 C, 15 N-labeled protein for solid-state and 2 H, 13 C, 15 N-labeled protein (deuteration > 90%) for solution studies were expressed in minimal medium with label algal hydrolysate as described in (13).…”

Section: Methodsmentioning

confidence: 99%

NMR reveals pathway for ferric mineral precursors to the central cavity of ferritin

Turano

Lalli

Felli

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

133

202

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“…The potential for fewer amino acid substitutions to create an active guest F ox site minimizes the possibility of insoluble protein, as encountered in an earlier experiment with human L ferritin (32). In addition DFP has been mainly characterized in F ox active frog ferritins (14,16,28,34,39), and both active and inactive frog ferritins have been crystallized (10,12,27).…”

Section: Resultsmentioning

confidence: 99%

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

Liu

Theil

2004

Proc. Natl. Acad. Sci. U.S.A.

125

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Ferritins managing iron-oxygen biochemistry in animals, plants, and microorganisms belong to the diiron carboxylate protein family and concentrate iron as ferric oxide Ϸ10 14 times above the ferric Ks. 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 miniferritins͞DNA protection during starvation proteins. I ron and oxygen are integral to life, but their chemistry requires many proteins with iron cofactors to harness them for respiration, photosynthesis, and DNA synthesis, while minimizing radical side reactions. Ferritins, cage-like nanoproteins, concentrate iron for cofactor synthesis, as a mineral (hydrated ferric oxide), in large, central cavities (5-8 nm). Ferritin nanocavities form during the spontaneous assembly of ellipsoidal, polypeptide subunits (1-3), 24 in maxiferritins (Ϸ480 kDa) and 12 in miniferritins (240 kDa), also called DNA protection during starvation proteins. The biological importance of ferritin is emphasized by the lethality of ferritin gene deletion in mammals (4), defects in the CNS from mutations in humans (5), and oxidant sensitivity after deletions of the multiple ferritins in bacteria (6)(7)(8). Reactions of iron and oxygen catalyzed by maxiferritins, in cells of higher plants, animals including humans, and microorganisms, stabilize iron with oxygen at 10 14 times above the K s to match cellular iron concentrations, whereas miniferritins protect DNA by trapping oxidants with iron in the hydrated ferric oxide mineral.Catalytic ferroxidase (F ox ) sites in ferritin catalyze the first in the series of reactions converting soluble ferrous ions to solid, concentrated ferric biomineral inside ferritin. The location of F ox sites in ferritin has been inferred to be in the center of each active subunit for maxiferritins and at junctions of subunit dimers in miniferritins, based on models of protein cocrystals with Fe 2ϩ analogues such as Mg 2ϩ or Ca 2ϩ (7-13). Such models are reasonable for the ferrous substrate, but not for ferric intermediates such as the diferric peroxo (DFP) complex or products such as diferric oxo͞hydroxo mineral precursors. There are at least four types of metal-protein sites in ferritin (iron entry, exit, nucleation, and F ox substrate). Thus, a number of assumptions are required if a crystal-metal site is assigned to one of the multiple, iron-dependent ferritin functions. Even when two Mg 2ϩ or two Ca 2ϩ ions are relatively near each other in ferritin cocrystals, the metal-metal distances in the models were much longer than the Fe 3ϩ -Fe 3ϩ distance in the functional DFP intermediate, determined by extended x-ray absorption fine structure analysis in solution (11,12,14). F ox site models have also relied on analogies to iron sites of other diiron carboxylate family members that form DFP intermediates (15-17), even when iron is a cofactor and contrasts with ferriti...

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“…In ribonucleotide reductase, this residue is observed to form a functionally essential tyrosyl free radical (43). Mutation of the corresponding tyrosine residue in ferritin does slow the rate of iron oxidation; however, it is not essential for the activity of the ferroxidase center (44)(45)(46). Thus, the exact role of this residue in bacterioferritin and SsDPSL is not clear.…”

Section: Conserved Tyrosine Residuesmentioning

confidence: 99%

Structure of the DPS-Like Protein from Sulfolobus solfataricus Reveals a Bacterioferritin-Like Dimetal Binding Site within a DPS-Like Dodecameric Assembly^,

et al. 2006

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The superfamily of ferritin-like proteins has recently expanded to include a phylogenetically distinct class of proteins termed DPS-like (DPSL) proteins. Despite their distinct genetic signatures, members of this subclass share considerable similarity to previously recognized DPS proteins. Like DPS, these proteins are expressed in response to oxidative stress, form dodecameric cage-like particles, preferentially utilize H 2 O 2 in the controlled oxidation of Fe 2+ , and possess a short N-terminal extension implicated in stabilizing cellular DNA. Given these extensive similarities, the functional properties responsible for the preservation of the DPSL signature in the genomes of diverse prokaryotes have been unclear. Here, we describe the crystal structure of a DPSL protein from the thermoacidophilic archaeon Sulfolobus solfataricus. Although the overall fold of the polypeptide chain and the oligomeric state of this protein are indistinguishable from those of authentic DPS proteins, several important differences are observed. First, rather than a ferroxidase site at the subunit interface, as is observed in all other DPS proteins, the ferroxidase site in SsDPSL is buried within the four-helix bundle, similar to bacterioferritin. Second, the structure reveals a channel leading from the exterior surface of SsDPSL to the bacterioferritin-like dimetal binding site, possibly allowing divalent cations and/or H 2 O 2 to access the active site. Third, a pair of cysteine residues unique to DPSL proteins is found adjacent to the dimetal binding site juxtaposed between the exterior surface of the protein and the active site channel. The cysteine residues in this thioferritin motif may play a redox active role, possibly serving to recycle iron at the ferroxidase center.Oxidative stress, in which reactive oxygen species (ROS 1 ) react indiscriminately with DNA, proteins and lipids, is a universal phenomenon experienced by organisms in all domains of life. † This work was supported by the National Institutes of Health (DK57776) and the National Aeronautics and Space Administration NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptCumulative damage from ROS is now thought to contribute to numerous disease states and a multitude of reports in the primary literature describe the role of oxidative stress in human diseases. Thus, understanding molecular responses to oxidative stress is of significant medical importance. Although much work has been done to characterize the management of oxidative stress in eukaryotes and bacteria, the oxidative stress response in archaea is poorly understood. However, elucidation of the protective mechanisms utilized in archaea is certain to provide insight into the diversity of molecular responses to oxidative stress across all domains of life.Numerous mechanisms have evolved to minimize and repair the damaging effects of ROS. These include enzymes such as superoxide dismutase and superoxide reductase, which convert the superoxide ion into molecular oxygen (O 2 ) and/...

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The influence of conserved tyrosine 30 and tissue-dependent differences in sequence on ferritin function: use of blue and purple Fe(III) species as reporters of ferroxidation

Cited by 47 publications

References 0 publications

NMR reveals pathway for ferric mineral precursors to the central cavity of ferritin

NMR reveals pathway for ferric mineral precursors to the central cavity of ferritin

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

Structure of the DPS-Like Protein from Sulfolobus solfataricus Reveals a Bacterioferritin-Like Dimetal Binding Site within a DPS-Like Dodecameric Assembly^,

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The influence of conserved tyrosine 30 and tissue-dependent differences in sequence on ferritin function: use of blue and purple Fe(III) species as reporters of ferroxidation

Cited by 47 publications

References 0 publications

NMR reveals pathway for ferric mineral precursors to the central cavity of ferritin

NMR reveals pathway for ferric mineral precursors to the central cavity of ferritin

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

Structure of the DPS-Like Protein from Sulfolobus solfataricus Reveals a Bacterioferritin-Like Dimetal Binding Site within a DPS-Like Dodecameric Assembly,

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Structure of the DPS-Like Protein from Sulfolobus solfataricus Reveals a Bacterioferritin-Like Dimetal Binding Site within a DPS-Like Dodecameric Assembly^,