Spectroscopic Definition of the Ferroxidase Site in M Ferritin: Comparison of Binuclear Substrate vs Cofactor Active Sites

Schwartz, Jennifer K.; Liu, Xiaofeng S.; Tosha, Takehiko; Theil, Elizabeth C.; Solomon, Edward I.

doi:10.1021/ja801251q

Cited by 56 publications

(142 citation statements)

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“…Formation of ferryhydrite mineral through hydrolysis occurs in the protein cavity and is a well-studied inorganic reaction. In ferritin, water is coordinated to iron in diiron sites or to other metals bound at the active sites (4,8), but where or when the water participates in hydrolytic coupling among the ferric oxo mineral precursors is unknown. Moreover, little direct structural information is available about ferrous ions moving within the cage after entering at pores near the threefold axes (32) to the active sites buried in the center of four-helix bundles of each subunit or, until this study, how the diferric products leave the active site and move to the cavity.…”

Section: Discussionmentioning

confidence: 99%

“…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 been studied extensively, e.g., with ferrous ion substrate analogues in cocrystals, subtractive mutagenesis, and protein chimeras (4,(8)(9)(10)(11)(12), although little is known about where the ferroxidase products are until they appear in the mineral in the protein cavity. Simple inspection of the protein cage does not show an obvious path from the active site to the mineralization cavity.…”

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confidence: 99%

“…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.…”

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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.

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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: Discussionmentioning

confidence: 99%

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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.

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“…5, and more recently by active site amino acid substitutions (18), MCD/CD (19) and very high resolution x-ray crystallography (10). Sitedirected amino acid substitutions in frog-M ferritin protein cages were generated by PCR on the expression plasmid pET-3a frog-M, using the QuikChange II site-directed mutagenesis kit (Stratagene).…”

Section: Methodsmentioning

confidence: 99%

Moving Iron through Ferritin Protein Nanocages Depends on Residues throughout Each Four α-Helix Bundle Subunit

Haldar¹,

Bevers²,

Tosha³

et al. 2011

Journal of Biological Chemistry

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Ferritins are an ancient superfamily of protein nanocages that synthesize, reversibly, iron concentrates for cellular use, heme, FeS cluster, and Fe-protein synthesis and provide oxidant protection by consumption of dioxygen or hydrogen peroxide and ferrous iron during stress; the protein cavities containing the minerals are ϳ60% of the cage volume (1-4). Ferritins differ in cage size, location, and mechanism of catalytic sites, mineral size, and mineral crystallinity; the Fe 2ϩ /O 2 oxidoreductase sites are also called ferroxidase sites or FC (ferroxidase centers) sites (1-5). In eukaryotic H ferritins, Fe 2ϩ and dioxygen are substrates for oxidoreductase sites and are catalytically coupled at multiple protein sites to synthesize diferric oxo mineral precursors (Fig. 1). Consumption of both iron and oxygen by ferritins accounts for the antioxidant response and iron-controlled gene regulation of ferritins in eukaryotes (6). Many catalytic proteins use iron and oxygen to produce a variety of organic products. Such products include unsaturated fatty acids such as oleate stearoyl-CoA desaturase-1 (7), deoxyribose (ribonucleotide reductase), and prolyhydroxyl modification of oxygen-sensing DNA transcription factors, e.g. hypoxia-inducible factor-␣ (8). Only in ferritins are both substrates inorganic. The exclusive use of inorganic substrates may relate to the ancient origins of the ferritins, which are distributed in all kingdoms and in both anaerobes and aerobes; ferritin gene deletion is lethal early in mammalian embryogenesis (9).There are two types of protein channels that move iron into and through the 24 subunit cages during synthesis of [Fe 3ϩ O] n in eukaryotic ferritins. The two functional types of ferritin channels are: (i) ion entry channels around the 3-fold axes, for , where diferric oxo mineral precursors produced by oxidoreduction fuse to tetramers and larger multimers before exiting from the protein cage for mineral growth.Recent high resolution structural studies show that ferritin is a soluble ion channel protein with lines of multiple metal ions (10), much like K ϩ and other membrane ion channel proteins. The eight Fe 2ϩ entry channels (Fig. 1) suggest roles beyond just electrostatics for the conserved carboxylates, such as ion selectivity at the channel constriction or directing Fe 2ϩ substrate ions to active sites in three subunits that also form the entry channels (10). The Fe 3ϩ O nucleation channels were recently identified using 13 C-13 C solution NMR spectroscopy in the presence and absence of Fe 3ϩ , by the disappearance of resonances within 5 Å of Fe 3ϩ O moving away from the active sites (11). Nucleation channel exits into the cavity are clustered around the 4-fold axes of the cage, which facilitates ordered mineral growth. 4 The abbreviations used are: DFP, diferric peroxo; MCD, magnetic circular dichroism; bis-Tris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane.

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“…The molecular structure of the peroxodiferric species in MMO is still unknown 3 and Q is proposed to have a bis‐μ‐oxo diamond core 7. In contrast, it is generally believed that the peroxodiferric species in ferritin decays directly to Fe(III) products 4, 5, 8, 9 (Fig. 1D) and a high‐valent Fe(IV) species has never been reported.…”

Section: Figurementioning

confidence: 99%

Spectroscopic evidence for the presence of a high‐valent Fe(IV) species in the ferroxidase reaction of an archaeal ferritin

et al. 2017

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A high‐valent Fe(IV) species is proposed to be generated from the decay of a peroxodiferric intermediate in the catalytic cycle at the di‐iron cofactor center of dioxygen‐activating enzymes such as methane monooxygenase. However, it is believed that this intermediate is not formed in the di‐iron substrate site of ferritin, where oxidation of Fe(II) substrate to Fe(III) (the ferroxidase reaction) occurs also via a peroxodiferric intermediate. In opposition to this generally accepted view, here we present evidence for the occurrence of a high‐valent Fe(IV) in the ferroxidase reaction of an archaeal ferritin, which is based on trapped intermediates obtained with the freeze‐quench technique and combination of spectroscopic characterization. We hypothesize that a Fe(IV) intermediate catalyzes oxidation of excess Fe(II) nearby the ferroxidase center.

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Spectroscopic Definition of the Ferroxidase Site in M Ferritin: Comparison of Binuclear Substrate vs Cofactor Active Sites

Cited by 56 publications

References 59 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

Moving Iron through Ferritin Protein Nanocages Depends on Residues throughout Each Four α-Helix Bundle Subunit

Spectroscopic evidence for the presence of a high‐valent Fe(IV) species in the ferroxidase reaction of an archaeal ferritin

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