Journal of Structural Biology 126, 182–194 (1999)Article ID jsbi.1999.4118, available online at http://www.idealibrary.com on

Mineralization in Ferritin: An Efficient Means of Iron Storage

N. Dennis Chasteen* and Pauline M. Harrison†

*Department of Chemistry, University of New Hampshire, Durham, New Hampshire 03824; and †Department of Molecular Biology andBiotechnology, University of Sheffield, Western Bank, Sheffield, S10 2TN, United Kingdom

Received January 21, 1999

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Ferritins are a class of iron storage and mineraliza-ion proteins found throughout the animal, plant,nd microbial kingdoms. Iron is stored within therotein shell of ferritin as a hydrous ferric oxideanoparticle with a structure similar to that of theineral ‘‘ferrihydrite.’’ The eight hydrophilic chan-els that traverse the protein shell are thought to behe primary avenues by which iron gains entry tohe interior of eukaryotic ferritins. Twenty-fourubunits constitute the protein shell and, in mamma-ian ferritins, are of two types, H and L, which haveomplementary functions in iron uptake. The Hhain contains a dinuclear ferroxidase site that isocated within the four-helix bundle of the subunit;t catalyzes the oxidation of ferrous iron by O2,roducing H2O2. The L subunit lacks this site butontains additional glutamate residues on the inte-ior surface of the protein shell which produce aicroenvironment that facilitates mineralization

nd the turnover of iron(III) at the H subunit ferroxi-ase site. Recent spectroscopic studies have shownhat a di-Fe(III) peroxo intermediate is produced athe ferroxidase site followed by formation of a m-oxo-ridged dimer, which then fragments and migrateso the nucleation sites to form incipient mineralore species. Once sufficient core has developed,ron oxidation and mineralization occur primarilyn the surface of the growing crystallite, thus mini-izing the production of potentially harmful2O2. r 1999 Academic Press

Key Words: ferrihydrite; ferritin; ferritin struc-ures; iron core; iron mineralization; iron storage

I. INTRODUCTION

In bones and shells, biomineralization is used torovide strength or protection to an organic scaffoldnd the mineral is extracellular. In ferritin theituation is different: the mineral is sequesteredithin a single molecule, which has a protein shell ofefined size and form. The ferritin protein shell haseveral functions: it acquires iron(II), catalyses its

xidation, and induces mineralization within its h

182047-8477/99 $30.00opyright r 1999 by Academic Pressll rights of reproduction in any form reserved.

avity. Thus nonspecific iron(III) hydrolysis isvoided. Ferritin also influences the mineral form inhich iron(III) is stored within it, and its solublerotein coat prevents the uncontrolled growth andoalescence of the small mineral particles into largernsoluble aggregates. The protein shell also limitshe accessibility of cell constituents to the ironineral.Iron-free ferritin molecules (apoferritin) are hol-

ow spheres with an outer diameter of 12 nm, and annner diameter of 8 nm and have a molecular weightbout half a million (Lawson et al., 1991; Harrisonnd Arosio, 1996). They are composed of 24 proteinhains or subunits arranged in 432 symmetry (Fig.a). The three-dimensional structure is very wellonserved throughout the animal and indeed thelant and microbial kingdoms. The protein shell canouse up to 4500 iron atoms within its 8-nm-iameter cavity (Fig. 1b). Such a high Fe:proteinatio (200 times that in hemoglobin) is made possibley sequestering the iron as a compact mineral (Fig.). The fact that mineralization occurs within pre-ormed intact shells, which limit the size of theydrous ferric oxide particles, may also influence thevailability of the iron.Very little is known about the chemical nature of

he iron reaching ferritin in vivo. There is very littleree iron(II) within cells (about 1028 M (Williams,982) and an abundance of potential chelators suchs citrate. Nevertheless, controlling the iron mineral-zation of ferritin shells is accomplished through the

atching of protein concentration to cellular ironevels. This is achieved in both eukaryotes androkaryotes by the response of the protein’s biosyn-hetic apparatus to cellular iron (reviewed in Hentzend Kuhn, 1996).Here we discuss our current knowledge of the

tructure of the iron mineral and the ferritin proteinhell and our understanding of the steps leading toron mineralization. The emphasis will be on animalerritins, most of the information being derived from

uman, horse, and frog proteins. Bacterial ferritins

tsmopfi and (d) by T. Douglas.

183MINERALIZATION IN FERRITIN

FIG. 1. Color graphic representations of the HuHF structure:race made with the MIDAS program of Ferrin et al. (1988); (b) Hatorage cavity. All atoms represented by solid spheres using conic pade as in (a). Helices labeled A to E; (d) view of a threefold channe

f the electrostatic potential, f (in kb T/e units) on the scale: f . 1ink; 24 . f . 26 darker pink; 26 . f, red. Electrostatic field lineeld has a ‘‘funnel’’ shape. Figures (a–c) provided by T. J. Stillman

(a) 24 subunits in 432 symmetry viewed down a fourfold axis. a-Carbonlf molecule viewed down a fourfold axis showing the 8-nm-diameter ironrogram of Huang et al. (1991); (c) a-carbon chain trace of a single subunitl (arrow) on the external surface of HuHF colored according to the values1, blue; 1/. f . O, light blue; O . f . 21, white; 21 . f . 24, light

s initiated from points near the molecular surface show the electrostatic

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184 CHASTEEN AND HARRISON

ill be touched on where they provide importantupplementary data.

II. THE MINERAL CORE

. Structure and Composition

Transmission electron micrographs of holoferritinhow well-defined nanoparticle crystallites encapsu-ated within the protein and having sizes whichpproach the interior 8-nm diameter of the proteinhell (Fig. 2) (Massover, 1993). Single or multiplerystallites and amorphous regions are observedithin a given protein shell. Early powder X-ray andlectron diffraction studies of ferritin cores showed aumber of d-spacings (Harrison et al., 1967) whichorrespond well with the values reported for theydrous ferric oxide mineral ferrihydrite (Towe andradley, 1967). Ferrihydrite has the approximate

ormula 5Fe2O3?9H2O and is a precursor to moretable minerals, such as a-Fe2O3 (hematite), formedpon loss of structural water (Towe and Bradley,967; Eggleton and Fitzpatrick, 1988). While thexact structure of ferrihydrite is uncertain, recentata suggest a model made up of double hexagonallylosest packed oxygen atoms in a stacked lattice,ith the majority of iron(III) in octahedrally coordi-ated sites but up to 1⁄3 of the iron in tetrahedral sites

Eggleton and Fitzpatrick, 1988), although this haseen disputed (Manceau et al., 1990). Sites are about0% occupied by iron, resulting in a partially disor-ered structure. Ligands to the Fe31 include O22 and

2

FIG. 2. (a) Low-magnification image of native horse spleen fer.5-nm native HoSF core showing regular lattice fringes (0.25 nmarrow) is also present in the core. Scale bar represents 3 nm. Elec

H ions and H2O. Fits of EXAFS data for the cores a

f horse spleen ferritin indicate that the iron(III) hasfirst coordination number in the range 5.0 6 0.3 to.6 6 0.5, with an Fe–O distance of 0.195 6 0.001 nme.g., Islam et al., 1989). A 1⁄3–2⁄3 mix of tetrahedralnd octahedral sites would give an average coordina-ion number of 5.3, thus allowing for the possibilityhat some iron in the core of ferritin is 4-coordinate.

The hydration and multiple lattice vacancies inhe ferrihydrite structure probably account for itsow stability relative to other common iron oxide

inerals such as goethite, maghemite, hematite,nd lepidocrocite and probably is important for rapidobilization of iron from ferritin (Powell, 1998). In

he absence of ferritin, the prevalent hydrous ironxide precipitated from aqueous solutions is lepido-rocite or goethite, g-FeO(OH) or a-FeO(OH), noterrihydrite (Macara et al., 1972). Therefore, therotein is not just a passive reservoir for iron butrovides a microenvironment within the proteinhell that influences the mineral phase that isormed. Because of the large surface to volume ratiof the mineral core, both the surface free energy andhe free energy of the bulk mineral phase contributeo the stability of the nanoparticle phase of ferritin.bout 40% of the Fe atoms in a core of 2100 Fe atomsre predicted to be at the surface (Frankel et al.,991).The properties of the cores of some representative

erritins from mammals, bacteria, plants, and mol-uscs are summarized in Table I. The cores of ferritin

cale bar represents 60 nm. (b) High-resolution lattice image of asponding to the (110) planes of ferrihydrite. Disordered materialicrograph provided by Professor S. Mann.

ritin. S) corretron m

re polydisperse in size, with approximate diameters

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185MINERALIZATION IN FERRITIN

anging from 2.5 to 9.0 nm and of variable geometrye.g., St. Pierre et al., 1990; Wade et al., 1993;

assover, 1993). A striking feature is their variablehosphate content, ranging from 44 Fe/Pi for ferritinrom the limpet Patella laticosata to 1.7 Fe/Pi forhat from the bacterium Pseuodmonas aeroginosaTable I). Mammalian ferritin cores have intermedi-te levels of phosphate. Cores containing low levelsf phosphate tend to be more crystalline and exhibitiffraction lines typical of ferrihydrite (e.g., Harrisont al., 1967; St. Pierre et al., 1990; Wade et al., 1993),hereas high concentrations of phosphate found inacterioferritins and phytoferritins produce amor-hous materials (e.g., Rohrer et al., 1990; Wade et al.,993). The small amounts of phosphate in mamma-ian ferritins appear to be largely surface adsorbed,hereas the large amount of phosphate in bacterio-

erritins (and plant ferritins) penetrates throughouthe core and the mineral is more appropriatelyiewed as an amorphous hydrated iron phosphateMann et al., 1986; Wade et al., 1993). In highhosphate cores, an Fe–P distance of 0.326 nm isbserved; the absence of an 0.35-nm Fe–Fe distanceypical of mammalian ferritins indicates loss ofong-range order (Waldo et al., 1995; Rohrer et al.,990). Significantly, crystalline ferrihydrite-like coresan be formed in plant and bacterioferritins byeconstituting the ferritin from apoferritin (withe(II) plus O2) in the absence of phosphate (Rohrer etl., 1990; Wade et al., 1993; Waldo et al., 1995);onversely horse spleen ferritin reconstituted in theresence of phosphate produces an amorphous coreRohrer et al., 1990; St. Pierre et al., 1996). Thus theresence or the absence of phosphate in the core of aarticular ferritin reflects the composition of theedium from which it is formed and is not a property

f the protein itself. Hydrated iron phosphate coresre more thermodynamically stable than ferrihy-rite cores, as revealed by their more negativeeduction potentials (Watt et al., 1985, 1986) and by

TAProperties of Ferritin C

Ferritin sourceAverage no.

Fe atoms

uman thalassemia 2500orse spleen 2000acterium (Azotobacter vinelandii) 1000acterium (Pseudomonas aeruginosa) 800at hemosiderin —ollusc (Patella laticostata) 2500ollusc (Acanthopleura hirtosa) 1500–2500ea seed (Pisum sativum) 1800

a Data were taken from compilations in Treffry et al. (1987), St.

heir tendency to form in place of ferrihydrite when c

hosphate is present (Rohrer et al., 1990; St. Pierret al., 1996).Another form of iron storage in cells is hemosid-

rin, an insoluble poorly defined protein–mineralore complex that is thought to be derived from theysosomal degradation of the ferritin protein shellreviewed in Harrison and Arosio, 1996). The min-ral cores of hemosiderin are generally smaller thanhose of ferritins from the same tissue (Table I) butn normal individuals still have ferrihydrite-liketructures. Hemosiderin from some patients withhe iron overload disease secondary hemochromato-is (thalassemia) have cores more goethite-like intructure, whereas hemosiderin cores from patientsith primary hemochromatosis are more disorderednd ferrihydrite-like (St. Pierre et al., 1998; Andrewst al., 1988).Ferritin cores containing iron in the ferrous state

an be produced either by incomplete oxidation ofe(II) that has been added to the apoprotein or bylectrochemical reduction of the Fe(III) core of theoloprotein (Hilty et al., 1994; Rohrer et al., 1987;att et al., 1985). Whether such cores occur in vivo

r play some role in the biochemistry of iron isnknown. Other nanoparticle mineral cores of ferri-in have been synthesized, including amorphouserric sulfide, uranyl(VI) oxyhydroxide, mangane-e(III) oxyhydroxide (MnOOH), and crystalline mag-emite (g-Fe2O3) (Douglas et al., 1995, and refer-nces therein).

. Magnetic Properties

The magnetic properties of the ferritin core haveeen the subject of extensive investigation over theears but aspects of their interpretation remainontroversial (reviewed in Brooks et al., 1998). It isenerally agreed that the electron spin magneticoments of the individual Fe31 ions within the

erritin core are antiferromagnetically coupled (spinsaired). However, because of the small size of the

Ifrom Various Speciesa

n core(nm) Crystallinity Fe/Pi ratio 7TB8 (K)

–6.0 Good 21 40— Good 8 40— Amorphous 1.5–1.9 20–6.5 Amorphous 1.7 .3–6.7 Good 8 23–8.0 Limited 44 30–8.5 Limited 36 32–6.5 Amorphous 2.8 4.1,

,90

et al. (1989, 1990), Wade et al. (1993), and Andrews et al. (1988).

BLEores

Measize

5.5

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ore particle, cancellation of individual magnetic

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186 CHASTEEN AND HARRISON

oments is incomplete. The remaining net magneticoment becomes a property of the particle itself and

ives rise to the phenomenon known as superpara-agnetism. Mossbauer spectroscopy has been used

xtensively to study superparamagnetism of a vari-ty of ferritins having natural or synthetic cores (St.ierre et al., 1989, 1996; Treffry et al., 1987; Wade etl., 1993).Figure 3 illustrates the temperature dependence

f the Mossbauer spectrum of horse spleen ferritineconstituted with 1000 iron atoms at an Fe/Pi ratiof 7/1, typical of the native holoprotein (St. Pierre etl., 1996). The spectrum evolves from a magneticyperfine sextet at low temperature (4.2 K) to auadrupole doublet at high temperature (80 K). Theemperature at which the areas of the two types ofignals are equal is known as the mean blockingemperature 7TB8 (533 K in this instance), the aver-ge value for the ensemble of particles in the sample.igher blocking temperatures are predicted for larger

olume particles and also for those with largerarrier anisotropy constants, which in turn reflectshe degree of crystallinity of the core. This expecta-ion is largely borne out by the data in Table I (seelso St. Pierre et al. (1996) and Frankel et al. (1991)).or example, reconstitution of horse spleen ferritinith 1000 Fe atoms at a 0.8 Fe/Pi ratio results in a

eduction in crystallinity and in the blocking tem-erature 7TB8 from 33 K (Fig. 3) to 17 K (St. Pierre etl., 1996).

II. THE FERRITIN PROTEIN SHELL AND ITS ROLE INIRON MINERALIZATION

. Shell Structure and Subunit Composition

Preparations of ferritins from animal tissues con-ain two different subunits, Mr , 20 000, known as Hnd L (Heavy and Light), which have only about 55%dentity in amino acid sequence (Arosio et al., 1978).he H:L subunit ratio depends on the tissue of originith the ferritins of heart and brain containingbout two thirds H subunit and those of liver andpleen (major iron storage organs) having up to 90%f L subunit (Arosio et al., 1978). Much work haseen devoted to establishing the structures of theifferent subunits and their roles in mineralizationrocesses (Harrison and Arosio, 1996). Determina-ion of the individual subunit structures was depen-ent on the production of recombinant H or Lubunit ‘‘homopolymers.’’

. Structures of Recombinant H and LHomopolymers

Three-dimensional structures analyzed at highesolution for human H (HuHF) and horse L (HoLF)nd also frog H (FrHF) and L ferritins (FrLF)Hempstead et al., 1997; Trikha et al., 1995; Harri-

on et al., 1998; Takagi et al., 1998) are highly

FIG. 3. Temperature dependence of the Mossbauer spectrumf horse spleen ferritin reconstituted with 1000 Fe/apoprotein at aatio 7 Fe:1 Pi. (Reprinted from Coord. Chem. Rev. 151, St. Pierrot al., Synthesis, structure and magnetic properties of ferritinores with varying composition and degrees of structural order—odels for iron oxide deposition in iron-over load diseases,

25–143, 1996, with permission from Elsevier Science.)

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187MINERALIZATION IN FERRITIN

imilar (a-carbon atoms superposing to within 0.5 Åms). In all ferritins analyzed, the subunits areolded into four-helix bundles which are over 5 nm inength each, with a long nonhelical loop connectingpposite ends of the bundle. A fifth shorter helix (E)s disposed at about 60° to the bundle (Fig. 1c).ubunits assemble spontaneously into symmetricalhells in which tetrads of E helices lie nearly parallelo each of the fourfold axes (Fig. 1a). A feature ofmportance to mineralization processes is the associa-ion of subunits into anti-parallel rhomb-shapedairs (Fig. 1a). Comparison of primary structures ofuHF and human L chain ferritin (HuLF) indicates

hat amino acids which participate in inter-subunitnteractions in HuHF are largely conserved (79%dentical) in HuLF (Hempstead et al., 1997). Thisxplains the ability of the two types of subunit tooassemble. Particularly striking is the conservationf the glutamates and aspartates directed towardhe threefold axes which are common to both subunitypes (see Fig. 6) and to many other animal ferritins,lthough not to bacterial ferritins. In contrast, aminocids buried within the subunit are less well con-erved in human H and L subunits, although oftenonservatively substituted. Residues on the outer

FIG. 4. Central region of subunits of (a) HuHF and (b) HoLFositions modeled on those found for Fe31 in EcFtnA; (b) shows thharge–charge interaction between K62 and E107. Drawing madecids in both ferritins are numbered according to the HuHF seque

olecular surface are poorly conserved (34%). A n

ost striking feature of the internal structure ofoth H and L subunits is a central hydrophilic regionurrounded by hydrophobic residues. However, therehe similarity ends: H subunits comprise a dinuclearetal binding site with highly conserved ligands but

n L subunits this feature is replaced by electrostaticnteractions between negatively and positivelyharged side chains (Lawson et al., 1991) (Fig. 4)).nner surface residues are also relatively poorlyonserved (43%) overall. For example, L subunitsave a cluster of four carboxylic acid residues (gluta-ates) on their inner surface, whereas there are only

wo in H subunits (Fig. 5). The complementaryunctions of H and L subunits depend on theirtructural differences: in heteropolymers the di-uclear centers of H subunits are responsible for the

erroxidase activity, whereas the negatively chargedesidues on the inner surfaces of L subunits promoteerrihydrite nucleation.

. Outline of Steps Leading to Iron Mineralizationwithin the Ferritin Protein Shell

Numerous in vitro experiments indicate that irontorage involves the uptake of iron(II) into therotein shell, its conversion to iron(III) at the di-

ows the dinuclear ferroxidase center of HuHF with two Fe31 atvalent region of HoLF in which the metal center is replaced by aMOLSCRIPT program of Kraulis (1991) by T. J. Stillman. Amino

. (a) She equiwith

uclear ‘ferroxidase’ centers, and the movement of

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188 CHASTEEN AND HARRISON

ron(III) into the cavity for deposition as ferrihydriteHarrison and Arosio, 1996; Chasteen, 1998; Harri-on et al., 1998; Harrison and Treffry, 1998). Therotein shell is exquisitely designed to enable andontrol these processes, as described in Section IV.

. Iron(II) Uptake into the Protein Shell: TheThreefold Channels

Iron(II) must find its way into the protein shellnd reach the ferroxidase centers. The X-ray crystal-ographic observation of metals (Cd21, Zn21, Ca21,nd Tb31) bound to the six conserved carboxylatesuggests the threefold channels as the likely route ofron entry into the protein shell in animal ferritinsLawson et al., 1991; Hempstead et al., 1997). Experi-

ental evidence consistent with this proposal in-ludes 113Cd NMR data which indicate that Fe21 ionsompete with Cd21 binding to HoSF in the channelsStefanini et al., 1989) and several studies whichhow that substitution of the threefold channelarboxylates by other amino acids (alanine, histi-ine, or leucine) inhibits iron incorporation intoerritin (Treffry et al., 1993; Levi et al., 1996). Entryy this route means that iron(II) must not only passhrough the channel (,1.2 nm) in length but also

FIG. 5. Detail of the inner surfaces of (a) HuHF and (b) HoLFxtensive cluster of carboxylate oxygens (black circles), which canormation in ferritins rich in L chains. Drawings made with the Mumbers are used for both ferritins.

raverse a distance of about 2 nm along a hydrophilic i

athway from the inside of the channel to theatalytic oxidation center (Treffry et al., 1993). Sup-ort for the threefold route has come from the recentalculations of electrostatic potentials in HuHFDouglas and Ripoli, 1998) which show that theegative outer entrance is surrounded by patches ofositive potential and this arrangement leads tolectrostatic fields directing cations toward the chan-el entrance (Fig. 1d). The region of negative poten-ial extends through the threefold channels to thenterior. The high degree of conservation of the threelutamates and three aspartates in both H and Lubunits enables these channels to maintain theirharacter in heteropolymer ferritins.

. Iron(II) Oxidation at the Dinuclear Centersof H Chains

Many kinetic experiments with recombinant HuHFnd also with HuLF and HoLF have pinpointed theinuclear center (Fig. 4a) as the catalytic (ferroxi-ase) center (Levi et al., 1988, 1989b, 1992; Treffry etl., 1993, 1995). Amino acid substitutions at theinuclear center in HuHF show greatly reducedxidative activity (e.g., E27A, E62K 1 H65G, E107A,nd Y34F, see Fig. 4) (Lawson et al., 1989; Baum-

ing parts of two subunits near a twofold axis. In HoLF the moremetal ligands, may account for the greater potential for iron coreIPT program of Kraulis (1991) by T. J. Stillman. HuHF sequence

showact as

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189MINERALIZATION IN FERRITIN

997; Sun et al., 1993). Binding of iron(II) at bothites A and B (Fig. 4a) is required for fast oxida-ion, which for HuHF is complete in well under 10 sith 48 Fe(II)/protein, pH 6.5 (Treffry et al., 1995,997).

. Putative Ferrihydrite Nucleation Sites

Several metal sites have been located on the innerurface of ferritin shells, some or all of which may beron-binding sites. Thus, in HoSF and HoLF twoites binding Tb31 or Cd21 were identified on theavity side of the B helix, one with ligands Glu53 andlu56 and the other ligated by Glu57 (61) and Glu60

64), the H subunit position numbers being given inarentheses (Harrison et al., 1989; Granier et al.,997). In HuHF, however, the first pair of glutamatess replaced by histidines and a single Tb31 is foundith ligands Glu61 and Glu64 equivalent to Glu57nd Glu60 in HoLF (Lawson et al., 1991).The negative patches of B helix glutamates di-

ected into the cavity are close to the twofold symme-ry axes relating subunits and, because of symmetry,xtend across these axes (Fig. 5). This is particularlybvious for the L subunit pair (Fig. 5b). The gluta-

FIG. 6. Detail of the HuHF structure near a threefold axis.esidues D131 and E134 from each of three chains point toward

he axis and attract Fe21 ions into the inter-subunit channel (seeig. 1d). D131 and E134 are conserved in HoLF. Drawing madeith the MOLSCRIPT program of Kraulis (1991) by T. J. Still-an.

ates may be involved in ferrihydrite nucleation, d

ith L chains being more efficient in promotingucleation than H chains (Levi et al., 1989b, 1992;awson et al., 1991; Wade et al., 1991; Bauminger etl., 1991a; Crichton et al., 1996; Santambrogio et al.,996). Amino acid substitutions at these residuesead to diminished core formation (Levi et al., 1994b;antambrogio et al., 1996). In H chains, the gluta-ates lie quite close to the ferroxidase center andlu61 can act alternately as a ligand to the ferroxi-ase and inner surface sites, suggesting a means byhich Fe(III) is moved into the cavity to form an

ncipient core (Lawson et al., 1991; Harrison andreffry, 1998). Glu61 is shown in its inner surfaceosition in Fig. 5a.

. The Complementary Roles of H and L Chains

There is now considerable evidence supporting theypothesis that H and L subunits have complemen-ary functions, ferroxidation, and mineralization,espectively. For mineralization within the ferritinhell the protein must efficiently compete with non-pecific iron(III) hydrolysis and precipitation in bulkolution (Wade et al., 1991). HuLF takes up iron(II)t pH 7.0, converting it slowly to ferrihydrite in itsavity, but is unable to do so at pH 5.5. However,hen HuLF and HuHF proteins are mixed at pH 5.5,

ome of the iron oxidized by HuHF is acquired andetained by HuLF (Levi et al., 1992). In heteropoly-er proteins, only 18–30% H subunit is required foraximum iron incorporation and mineralization

Levi et al., 1994b). More slowly incorporating L-richerritins yield iron core particles of greater averageize, crystallinity, and magnetic ordering than the-rich ferritins (Wade et al., 1991; Bauminger et al.,991a). Modification of both ferroxidase center andavity carboxylate residues leads to ferritins that doot oxidize or incorporate iron (Wade et al., 1991;un et al., 1993).

IV. IRON STORAGE MECHANISMS: OXIDATION ANDMINERALIZATION

. Overall Reaction Paths of Iron Oxidation andHydrolysis

Since iron(II) oxidation and ferrihydrite nucle-tion occur, respectively, within the four-helix bundlef the protein shell and on the inner surface of theavity, iron(III) must be able to move between thesewo locations. The oxidation step is controlled by theigand geometry of the ferroxidase center, whereaserrihydrite initiation and growth are likely to in-olve a series of hydrolytic steps in which iron(III)toms are added progressively and protons are re-eased. Although the first few iron(III) atoms form-ng ferrihydrite nuclei may be protein-bound, theulk of the iron(III) is bound (through O22 or OH2)

irectly to the growing mineral nucleus (Sections IV

Bos

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190 CHASTEEN AND HARRISON

and C). Once the iron core nuclei have reached 100r more iron(III) they are also able to performurface oxidation of iron(III) (Section IV F).The stoichiometries of oxygen consumption and

ron(III) hydrolysis have been recently determinedy Yang et al. (1998), who used a combined oximeternd pH stat to measure oxygen consumed androtons released and also followed the progress ofhe reactions spectrophotometrically. The consump-ion of two iron(II) per O2 for the protein-basedeaction implied that H2O2 is the product of theerroxidase reaction in both HuHF and HoSF, aesult confirmed by the change in measured stoichi-metry in the presence of catalase. One proton waseleased per iron(II). The overall reaction at theerroxidase center postulated by Yang et al. (1998) is

rotein 1 2Fe21 1 O2

1 3H2O = Protein2[Fe2O(OH)2]21

(ferroxidase complex)

1 H2O2 1 2H1.

(1)

hey further suggested that the net reaction for theransfer of iron(III) to the core is

rotein2[Fe2O(OH)2]21 1 H2O = Protein

1 2FeOOH(core) 1 2H1. (2)

ydrolysis produces one more proton per iron atomnd the ferroxidase center is vacated. Once sufficientore is developed (.100 Fe atoms), the alternativeineral surface iron(II) oxidation and hydrolysis on

he surface of the growing core may now proceed:

4Fe21 1 O2 1 6H2O = 4FeOOH(core) 1 8H1. (3)

. Mechanisms of Iron Oxidation and Iron CoreBuilding Up: Observed Intermediates

Considerable data have accumulated which allowome of the steps to be analyzed further. The construc-ion of the dinuclear iron site (Lawson et al., 1991;reffry et al., 1992) and the stoichiometry of 2e(II)/O2 (Yang et al., 1998) suggest the transfer of

wo electrons, one from each Fe(II), to an O2 moleculeound at the same site. Maximal oxidation ratesere also obtained with 2 Fe(II)/H subunit in stopped-ow analyses (Treffry et al., 1995, 1997). An initiallue species which forms and decays within a feweconds after the addition of 48 Fe(II) atoms/poferritin molecule is now thought to be a diferric-eroxo intermediate (Zhao et al., 1997; Harrison andreffry, 1998). Its UV-visible absorbance spectrummaximum at 650 nm in HuHF) resembles those ofnown diferric-peroxo model complexes. Moreover,

he first species to be observed at 25 ms after rapid t

reeze–quench of samples of FrMF (a frog ferritinunctionally similar to FrHF), to which 36 Fe(II)olecules had been added aerobically, gave Moss-

auer parameters (QS 5 1.08 mm/s, IS 5 0.62 mm/s)ypical of diferric-peroxo complexes (Pereira et al.,998).A second intermediate, appearing in both HuHF

nd HoSF within 30–60 s after Fe(II) addition, butfter the blue complex, gave absorbance bands at05–310 and 340–350 nm typical of µ-oxobridgede(III) dimers and with a stoichiometry of 2Fe/Hubunit, confirming the assignment based on Moss-auer parameters (Bauminger et al., 1991a,b, 1993;reffry et al., 1992; Yang et al., 1998). Such a dimerould be expected if µ-oxobridge formation occurredt the ferroxidase center after the initial oxidationtep (Eq. 1).The first reaction at the ferroxidase centers may

ow be postulated as addition of dioxygen to thei-Fe(II) center (either at a terminal position (as inreffry et al., 1992) or bridging) and leading to theormation of µ-oxobridged Fe(III) dimer plus perox-de. One such pathway is

Dioxygen binding:

Protein2[Fe2]41 1 O2 = Protein2[Fe241O2]41.

(dioxygen complex) (4)

ron(II) oxidation:

Protein2[Fe241O2]41 = Protein2[Fe2

61O222]41.

(peroxo intermediate) (5)

ron(III) hydrolysis:

rotein2[Fe261O2

22]41

1 H2O = Protein2[Fe2O]41 1 H2O2.(µ-oxobridged complex)

(6)

n the scheme of Yang et al. (1998) further hydrolysisnvolving two H2O is proposed to give a Protein-Fe2O(OH)2]21 species (Eq. 1) at the ferroxidaseenter followed by another hydrolysis step duringransfer of this iron to the core (Eq. 2). It may beoted that the postulated dihydroxy species is notharge-neutral (the iron pair has four glutamateigands) and is thus inherently unstable.

Studies with HuHF and HoSF using both UVifference and Mossbauer spectroscopy indicatedhat the µ-oxobridged dimers split prior to theirransfer to the cavity according to the sequence:imer = monomer = cluster (Bauminger et al.,991a). There was a marked increase in the propor-ion of clusters and a decrease in monomers as theumber of Fe atoms/molecule was increased from 4

o 40 (Bauminger et al., 1991a). This probably re-

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191MINERALIZATION IN FERRITIN

ects the stabilization of the ferrihydrite core as itncreases in size, a consequence of the decrease inurface free energy of the nanoparticle. Splitting ofhe oxobridge may occur through addition of a waterolecule to give two monomeric [Fe(OH)2]1 ions

ollowed by migration to the cavity to form core, as inquation 2.Core initiation could be enhanced by proximal

inding of two Fe31 at the putative cavity ‘‘hetero-ucleation’’ center (Hempstead et al., 1997; Graniert al., 1997; Gallois et al., 1997) to give a µ-oxo-ridged dimer similar to that proposed by Yang et al.1998) for the ferroxidase site. Hydrolytic addition ofurther Fe31 produced at the ferroxidase site in theavity would then allow the buildup of linear andhree-dimensional polymers of increasing stability.

. Movement of Iron from Protein Sites toFerrihydrite Cores in the Same or Other FerritinMolecules

Of the multiple Fe(III)-binding sites on both therotein and the ferrihydrite core, the former areonstant in number, whereas the number of sites onhe core particle grows with the surface area (Macarat al., 1972). This feature together with the greatertability of the core sites increases their ability toompete for Fe(III). An ‘‘all-or-none’’ distribution wasoticed when newly formed ferritin was analyzed inn ultracentrifuge (Macara et al., 1972) and Moss-auer spectroscopy has revealed the presence ofagnetically ordered cores when only 10 Fe atoms/olecule were added (Yang et al., 1987; Bauminger

t al., 1991b). A magnetic ordering would not bexpected for particles containing less than about 70e(III) atoms (Bauminger et al., 1991b). Contribut-

ng to this uneven distribution of core size is thebility of ferritin molecules containing mature coreso acquire iron from other ferritin molecules, asemonstrated by Mossbauer spectroscopy (Baum-nger et al., 1991b). These observations and thending that iron oxidized by ferritin can be taken upy iron-free transferrin (Bakker and Boyer, 1986)ndicate that Fe(III) transfer does not require apecific structural relationship between donor andcceptor molecules.

. Loss of Protons or Gain of Phosphate IonsMaintains Charge Neutrality of the Core

Core formation during Fe(III) hydrolysis produceslectrically neutral ferrihydrite but releases protons.f not lost from the cavity or their charges neutral-zed by incoming anions, these protons would desta-ilize both the protein and the core. In most ferritinolecules some of the OH2 ions of the core are

eplaced by orthophosphate ions, the majority ofhese residing on core surfaces (Treffry and Harri-

on, 1978; Huang et al., 1993). Electrostatic poten- T

ial calculations indicate that the expulsion of pro-ons or uptake of H2PO4

2 (or other anions such as Cl2)ould be facilitated by the direction of electric fields

hrough the fourfold channels in HuHF (Douglasnd Ripoli, 1998). This pathway may also be theoute taken by Fe31 or Fe21 ions leaving the mol-cule.

. Iron(II) Oxidation on the Mineral SurfaceProduces Water and Avoids Fenton Chemistry

Fe(II) oxidation at HuHF ferroxidase centers yields2O2 and, if this meets free Fe(II), dangerous Fenton

eactions may ensue, with the production of hy-roxyl radicals which cause damage to nucleic acids,ipids, and protein:

Fe21 1 H2O2 = Fe31 1 OH2 1 ·OH. (7)

uch radical reactions have been observed withoSF (Chen-Barrett et al., 1995). However, Fenton

eactions are minimized in HoSF and HuHF whene(II) is added in ratios of about 200 atoms/moleculer more, since the oxidation stoichiometry ap-roaches 4 Fe(II)/O2 with the production of waternstead of peroxide, as in the mineral surface reac-ion 3 (Sun et al., 1993; Yang et al., 1998). In EcFTNenton reactions are avoided because 3–4 Fe(II) arexidized per subunit, 2 at the ferroxidase center and3rd at a neighboring position (Treffry et al., 1998).

. Reutilization of Ferroxidase Centers

Movement of iron from ferroxidase center to corehould regenerate ferroxidase activity on vacatedenters. However, at iron loadings of 12 or 38 Fetoms/molecule µ-oxobridged dimers persisted forong periods in HuHF (50 or 28%, respectively at 24). In contrast all the iron was in clusters in HoSF athese loadings (Bauminger et al., 1991a). Stopped-ow studies of initial rates of Fe(II) oxidation in bothrHF and HuHF indicated that regain of activityith a second Fe(II) addition required several hours

Waldo and Theil, 1993; Treffry et al., 1995). Yang etl. (1998) have found that with repeated additions of

Fe(II)/subunit made at 37°C, pH 7.04, and atntervals of 10 min or more, most of the iron wasrocessed by the ferroxidase center, but at shorterntervals the mineral surface pathway was increas-ngly utilized. Activity regeneration measured byximetry was faster with HoSF or if moleculesontaining performed iron cores were added to HuHFTreffry et al., 1995). Thus, L subunits not onlyromote the growth of a high-affinity sink for Fe(III)the iron cores) and mineral surfaces for oxidationhey promote reutilization of H subunit sites as well.

he unanswered question inevitably arises: to what

ev

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192 CHASTEEN AND HARRISON

xtent are both types of oxidation reaction utilized inivo?

V. CONCLUSION

Our understanding of how ferritin sequesters ironnd converts it into a mineral form has advancedreatly since its isolation some 60 years ago (Lauf-erger, 1937). The structures of several ferritinsave now been determined and much of their ironhemistry has been defined. However, a number ofutstanding questions remain. One of these is theole of the inner surface carboxylates in promotingineralization. Do these residues provide specific

ron-binding and mineral nucleation sites, as sometudies have indicated, or do they simply create aicroenvironment favorable to mineralization? The

ack of a specific structural relationship betweenineral and protein in HoSF (Harrison et al., 1967),

he wide chemical composition of inorganic com-lexes that can be sequestered within the ferritinavity, and the absence from bacterial ferritins of thelutamate cluster of L ferritins all suggest that theriving force for mineralization is not a highlytructured nucleation site. Moreover, the concentra-ion of a single Fe31 ion within the ferritin cavity isbout 14 orders of magnitude greater than theolubility of free Fe31, so that the thermodynamicorce for hydrolytic polymerization as iron(III) isequestered by ferritin must be enormous.The ferroxidase site of ferritin plays a central role

n the harvesting of iron from the environment of therotein by accelerating the internal oxidation ofron(II) so that hydrolysis and polymerization canhen ensue, resulting in growth of the nanoparticleron hydroxide oxide phase contained within therotein shell. Once a core of sufficient size is ob-ained, subsequent iron(II) oxidation and mineraliza-ion then takes place directly on the growing mineralurface until the protein acquires its full comple-ent of iron. Ferritin has evolved a molecular design

hat limits this chemistry within its interior, thusvoiding nonspecific iron oxidation and hydrolysiseactions from occurring within the cytosol of theell. In this way other proteins and nucleic acids arerotected from the toxic effects of ‘‘free’’ iron.

This work was supported in part by Grant R37 GM20194 fromhe National Institute of General Medical Sciences (N.D.C.) andy the Wellcome Trust (P.M.H.). We are indebted to Dr. T. J.tillman for Figs. 1a–1c and 4–6, Dr. T. Douglas for Fig. 1d, androfessor S. Mann for Fig. 2.

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