- Email: [email protected]
Ferritin iron deposition and mobilisation
267
Journal of Molec&ar CataI,vsis, 7 (1980) 267 - 276 @ Eketier Sequoia S-A., Lausann e -Printed in the Netherlands
FERMTIN
IRON
DEPOSITION
AND MOBILZS_4TiON
ROBER_T R. CRICHTON, FRANC_OESE ERIC PAQUES, ANTOINEZTE PAQUES UnitP de Biochimie. UniuenitL Looualr-la-Neuue (Belgium)
Catirolique
ROMAN, FRANCl-NJZ ROLAND. and ETIENNE VANDAMME de Louuai~.
Pluce L. Pasteur.
I, B-i348
summary We have studied the mechanisms whereby iron is deposited in, and mobilised from, ferritin, the principal protein of iron storage in mammals. Iron deposition involves oxidation of Fe(II), cataIysed by the protein with molecular oxygen as electron acceptor, followed by hydrolysis and deposition of the ferric oxyhydroxide in the interior of the protein shell. We have proposed a model for iron deposition in which Fe(II) binds to catalytic sites situated on adjacent polypeptide chains; dioxygen is then bound between the two iron atoms and is reduced to give a relatively stable Fe(III)-peroxo complex. Incoming Fe(I1) can displace the peroxo complex allowing hydrolysis of the ferric iron and its migration to the interior of the protein. The evidence in support of this model is briefly summarised. We assume that at low pH the rate-limiting step is the deprotonation of a group or groups on the protein with a pK of around 6. Iron mobilisation from ferritin can be readily achieved by reduced flavins in the presence of a suitable iron chelatir_ We have found that iron mobilization from ferri&n by bipyridyl and also by pyridinne-Z-aIdehyde-2pyridyl hydrazone (Paphy) is greatly increased by photoreduced flavin, whereas, for the iron chelators, desfenioxamine B, rhodotorulic acid and 2,3-dihydroxybenzoat.e, iron mobiIis.ation is effectively blocked by the addition of flavin. We suggest that the reduced flavin reacts with the peroxo bridge of the catalytic sites to generate a flavin hydroperoxide and Fe(II).
Introduction The chemistry of iron in aqueous solution is dominated by two reactions -the oxidstion of Fe(H) to Fe(III), and the hydrolysis of the latter to form essentiaIly insoluble ferric oxyhydroxide polymers [I] _ In view of theimportant role of iron in many proteins involved in eleetin transfer, oxygen transport, and oxygen activation (oxidases, hydroxylases, etc_) and detoxification (catalases, peroxidases, c* bacterial superoxide dismutases), the bioavailability of iron in a soluble, non-toxic and yet readily avaiIable form
268
is an essential requirement for aerobic life. Whereas many prokaryotes and lower eukaryotes (such as fun@) have developed low molecular weight siderophcres, which are excreted into the extracellular milieu, complex iron, and then transport it to-the in+&rior of the organism where it is released for internal utilisation [ 21, the higher eukaryotes have developed a system of transport and storage based on high molecular weight protein ligands for the iron. Thus the transport of iron in plasma is accomplished by the or -glotulin, transferrin, which is a glycoprotein of molecular weight around 80 000 and which can transport a maximum of two atoms of ferric iron bound to the protein in intimate association with bicarbonate ion [ 3 J _ Within eukaryotic cells iron is stored in ferritin [4,5], a protein of molecular weight 440 000 composed of 24 polypeptide chains each of molecular weight 18 500 which, in a fashion rather reminiscent of spherical viruses, form a hollow protein shell of esterior diameter around 120 A, in the interior of which a cavity of diameter 65 - 70 A serves to store the iron. The capacity of ferritin to store is considerable; ferritin molecules from horse spleen can accumulate up to 4 500 atoms of iron, essentially as FeO.OH together with some phosphate such that the ‘full ferritin’ contains 0.57 g of iron/g of protein and yet can readily he concentrated to give sol-utions which are 1M m iron in a completely water-soluble form. The purpose of this presentation is to analyse at the molecular level the mechanisms by which iron is deposited in, and mobilised from, ferritin. Before passing to the discussion of these mechanisms, we will first present briefly the structure of the apoferritin molecule (t.hat is, the protein shell of ferritin) and its relation to the micelIar iron core. Apoferritin has been crystallised from a number of sources, but at
present the best character&d apoferritin is that of horse spleen for which an electron den&y map at 2.8 A resolution has been obtained by Professor Pauline Harrison and her collaborators in Sheffield [Cl] _ The protein shell is roughly spherical and access ti the internal cavity in which the iron is depositi is assured by channels 10 - 15,4 in diameter along the four-fold axes. The individual polypeptide chains can best be described as four, long, approximately parallel, a-helices of 38 - 45 A in length oriented at right angles to the four-fold auis, along which lies a shorter helix which lines the channels referred to above. These five cc-helical domains are connected by non-helical regions which represent about 30 - 35% of the structure. The apoferritin molecule is composed of 165 amino acids and a sequence of more than 90% has been establish4 in our laboratory [7] _ There is a considerable amount of acidic ammo acids (22 - 26 according to chemical modification and titration studies, [ 8, 9]), three cysteine residues/subunit, six histidine residues/subunit and a high proportion of non-polar amino acids (45%). The construction of an atomic model of horse spleen apoferritin based on X-ray crystallogaphicand primary structure studies is in progress, and we anticipate that the structure will be solved by atomic resolution in the very near future.
269
The iron core of ferritin contains some degree of internal ordering [IO] and it seems likely that there are intimate contacts between the iron micelle and the apoferritin shell [ 11,12]. It i.s of interest to note that the structure of the ferric oxyhydroxide formed is determined by the protein; thus, whereas polymerisation of ferric osyhyclroxide in several different buffers in the absence of apoferritin gives rise to different forms of polymers, the iron micelle obtained in ferritin in the presence of the protein is independent of the buffer employed ]13]. Finally, it should be pointed out that apoferritin preferentiaIly accumuiates Fe(R), and catalyses the oxidation of Fe(R) to Fe(III) IL4 - X6]_ Thus, we are led to conclude that apoferritin is a ferroxidase and that specific groups in the protein participate in oxidation of iron. In iron release from fe-rritin many studies imply a reductive pathway such that the ferric iron is first reduced to ferrous, most probably by reduced fIavins, before the iron can be released from the protein and participate in the synthesis of ironcontaining enzymes Cl7 - 191.
Results The deposition of iron in ferritin is most readily achieved by incubation of apoferritm with Fe(II j in the presence of an appropriate electron acceptor, of which molecular orrygen is perfectly adequate. It was immediately evident that the study of iron deposition in fen-&in necessitated the use of buffer systems in which (i) the rate of autoxidation of iron was minimal, (ii) the complex&ion of iron by the buffer was not sufficiently important to interfere with the kinetic analysis. In our initial studies [ 151 we used sodium borate-cacodylate buffers of pH 5.5 - 6.0 which satisfied the first, but not the second criteria. The effect of buffer on the kinetics of iron deposition in apoferritin (Fig. 1) are clearly separated into two groups [lS] . Ironcomplexing buffers such as imidazole, bora*cacodylate znd Bicine give saturation kinetics at pH 7.0, whereas non-complexing buffers such as Mes and Mops give rise to S-shaped cumes. These results, together with rapid kinetic studies in boraWcacody1at.e buffers, pH 5.6 - 6.0 [17], suggested that iron deposition in ferritin was at least second order with respect to the iron concentration. A detailed analysis of the inhibition of iron deposition by Cr(III) [IS, 201 showed that an 85 - 90% inhibition was observed upon fixation of one chromium/two subunits. It was subsequently shown that Cr(IIl) blocks fixation of uranyl acetate to apcferritin crystals; the uranyl site was close to the two-fold axis [Is] . The X-ray crystallographic studies [S] showed that the major site of Tb 3c fixation is located close to the twofold axis and only 4.3 # distant from its symmetry-related pair. Inhibition of iron deposition in apofemitin by Tb a has been okserved 1211 zmd was attributed to binding of Tb3+ to carboxyl groups at the Febinding sites. Prior to the publication of these results we had observed that 2,2’-bipyridyl could release iron as Fe(II) from ferritin in the absence of reducing agents
270
Fig.
1. Kinetics
of
iron
deposition
in apoferritin
nt pH
7 ~3 in a series
of
different
buffers,
all at a concentration of lOOmM_ The initial velocity of iron deposition was measured at 310 nm and the iron concentration was varied from 0.028 to 2.05mM (from ref. 16). The curves are, respectively, (0) Imidazole, ( -) Mes, (-) Bonte-c;lcodyIate, (0) Mops, and (A) Bicin _
[ 223 . On the basis of these resuits we were led to prcpose a mechanism for iron deposition in fen-&in involving four steps (Fig. 2): (i) fixation of Fe” by catalytic sites located on two adjacent polypeptide chains; (ii) fixation of a dioxygen molecule between the two i-on atoms; (iii) reduction of the dioxygen to give a peroxo complex in which the oxygen ligand is coordinated to two iron atoms; (iv) hydrolysis of the peroxwomplex accompanied by migration of the ferric oxyhydroxide formed to the interior of the protein. The evidence supporting ihis model will now be discussed. (i)
The
catalytic
site
involves
two
polypeptide
chains
Fe ‘+ by apoferritin using rapid kinetic techniques (stopped flow) in bora*cacodylate buffers, pH 5.8, showed [%I] a second order dependence of the initial velocity as a function of iron concentration, consistent with a mechanism invohtig fixation of two iron atoms. A more detailed kinetic analysis of the effects of buffer and pH was undertaken [IS] and it was found that at pH values between 6-5 and 7.5 the rate of iron deposition in ferritin showed a sigmoidal dependence on the concenStudies
on
the
oxidation
of
271
Fig. 2. Model for ferritin iron oxidation and deposition. fixation of iron(H) and in oxidation are in the channels heferonucleation to which the iron migrates are situated (from ref. 22).
The iron binding sites invo!ved in between the subunits. The sites of in the
iaterior
of
the molecule
of iron in noncomplexing bmuffers(Fig- 1). In ircidazole buffer, sigmoidal dependence was also observed at pH 7.5. We interpreted these results in terms of the fixation of two iron atoms/catalytic site. At lower pH valuq s&uration kinetics were observed_ The very considerable effect of pH on the rate of iron deposition, and the rather similar rates observed in different buffers at pH values between 6.0 and 6.5, led us to conchrde that at low pH the deprotonation of a group or groups on the protein with pK of around 6 was the rate-Iimiting step in ferritin formation, accounting for the saturation kinetics observed [I.61 _ The inhibition of iron deposition upon fEation of one g at. of Cr?/subunit referred to above also supports the view that the catalytic site is composed of two adjacent polypeptide chains 116, 201. Finally, the observation that the major site of Tb3* binding in the apoferritin crystals is locaL& close to the two-fold avis of the apoferritin molecule and only 4.3 A from its symmetry-related pair [S] is consistent wi+h the model observed_ Tb3* ions inhibit iron uptake by apoferritin and are assumed to bind to carboxyl groups at the Fe ‘* binding sited [21] _ Chemical modification studies [S, 9] show that carboxyl groups are certainly involved in the iron oxidation sites. Further, we have recently shown that Zn*, Ni**, Co2* and Tbs+ are all inhibitors of iron deposition in ferritin, confirming previous studies 121,231, and that the inhibition in aJl cases is competitive ]24]_ tration
272
{ii}
The peroxo in &.-mediate The presence of oxygen in an activated form, which we presume to be a peroxo complex with Fe(M), is suggested by the capacity of ferritin to oxidise a number of organic molecules such as 2,2’-bipyridyl and triphenylphosphine. We have been able to detect bipyridyl Naxide in chloroform extracts of the incubation mixture of fen-&in with bipyridyl by thin layer chromatography in the solvent system isopropanol-ethyl aceta&cyclohesane 20/10/70 v/v after repeated chromatography [ 221. Triphenylphosphine oxide formation was detected after incubation of ferritin with triphenylphosphine under argon. Ether extraction allowed us to extract both the phosphine and its oxide; detection and quantification were achieved by 31 P n.m.r. measurements. Using variable amounts of phosphine we con&tent!y observed values of 0.97 mole of triphenylphosphine oxide/subunit formed after incubation with ferritin. Concomitantly 15 - 30 atoms of Fe(II) were released from the protein [ 25]_ These results, together with the iron mobihsation studies using bipyridyl described below, suggest that the peroxo comples is present at the catalytic sites. The considerable enhancement of iron deposition that we have observed [24] in the presence of Cu2+ is also consistent with this intermediate, since Cu2+ is known to destabilise peroxides. Hydrolysis of the perowo intermedicte and internalisafion of ferric oxyhydroxide The iron in ferritin is mostly present in hydrolysed form as ferric oxyhydroxide_ Thus, it is reasonable to assume that the peroxo intermediate hydrolyses to form FeO.OH, which subsequently migrates in’to the interior of the protein shell. Careful analysis of the stoichiometry of oxygen consumption confirms that 4 g at. of Fe(I1) are oxidised per molecule of dioxygen consumed [31,32] _ However, the mechanism proposed in Fig. 2 is unlikely to take place as indicated, and more plausible pathways involving formation of a ~-0x0 complex either following scheme (1) or scheme (2) could precede hydrolysis (3) or (4) _
(1)
0 Fe’
Fe/O\
\
O,Fe
Fe
+
+
H,O
2Fe”
+
4
.O
2Fe /
l?eHo ‘OH
\
+
(2)
Fe
FeRC f
2H’
(3)
273
Fe/O\
Fe + 3H20
+
2FeHo ‘OH
+ SH’.
(4)
We are unable to distinguish for the moment between these alternatives. A closer scrutiny of the kinetics of iron deposition in ferritin at neutral or a&aline pH has shown 1331 a considerable increase in initial velocity at high iron concentrations which would be not inconsistent with a heteronucleation of FeO.OH micelles at internal sites on the protein shell. The hydrolysis of the peroxo intermediate in the case of schemes (2) f (3) would regenerate Fe’+ on the sites and might be accompanied by displacement of the hydrolysed product to the interior. We assume that the hydrolysis is reversible in view of the results of iron mobilisation obtained with bipyridyl (see below)_ Iron
mobilisation
from
ferritin
The most effective way of releasing ferritin iron in vitro is by reduction in the presence of an appropriate chelating agent. Thus, in the preparation of apoferritin from ferritin, the iron is removed by reduction with dithionite in the presence of bipy-ridyl or, alternatively, reduced and complexed by thioglycollate. Of the biological reductants that have been tested, by far the most impressive results have been obtained with reduced riboflavin mono nucleotide (FMNH& in the presence of bipyridyl [5,17,19]. However, to obtain maximum rates of iron release strict anaerobic conditions are necessary. It has been suggested that reduced FMN penetrates to the interior of the protein shell through the channels along the four-fold axes of the molecule and in this way comes in contact with the iron core [5] _ This view receives support from expe_iments in which reduced FMN was immobilised on an inert carrier and it was found that no iron release was observed [19]. However, it is not yet clear whether FMN can penetrate through the channels_ If the role of FMNH2 as biological reductant is not yet clearly stablished, information concerning the chelator or chelators that are employed in the cell is conspicuous by its absence: there is certainly no lack of potential candidates. Our inter-t in ferritin iron mobilisation has been directed to the effects of iron chelators. Since we observed that 2,2’-bipyridyl could release ferritin iron [ 261 we have extended these studies with bipyridyl [22] ; as describ& above an N-oxide of bipyridyl was detected_ Subsequently [27,28], release of iron from horse spIeen ferritin by desferrioxamine B, rhodotoruhc acid, 2,3dihydroxybenzoate, 2,2’-bipyridyl, and pyridine-2-aldehyde-2-pyridyl hydrazone (Paphy) was studied in vitro (Table I). Deferrioxamine B and rhodotomhc acid both release appreciable amounts of ferriti iron. Iron release from ferritin by the other three chelators is less effective: at ImM final concentration the c!aelators can be classed in the order rhodotorulic acid > desferrioxamine B > Paphy > 2,3&hydroxybenzoate > 2,2’bipyridyl. The effects of low molecular weight mediators (FMN, EDTA, ascorbate, citrate, and oxalate) on iron release by the chelators was also
274 TABLE
1
Iron release from ferritin by chelators The amount of iron releed after 2 h and 6 h incubation at 37 “C of 1.14 x 10-6bl horse spleen ferritin in 0 2M Mops (morphohne propane sulphonate) buffer, pH 7 -4, by the different chelators at a final concentration of 1mM alone, and in the presence of a finai concentration of 1mM FMN, was measured at the appropriate xc,, of the chelator. The resu!te are exmed as g at. of iron released/molecule of ferritin and are the ruean of five independent determinations_ All experiments were cerried out in the light, and some of the FMN was thus pho’&reduced to FMNH, (from ref. 28). Chelator
g_ At. of Fe rcleasedjmolecule chelator
Bipyridyl Paphy* Desferrioxamine
Rhodotomlic
B
acid
alone
by chelator
+ 1mM
FMN
2h
6h
2h
6h
59 21-i 62.7
108 50.1 1.182
24 .I3 47.9 118
492 90.5 12.6
22.8
22.7
246
26.5
74-o
151.3
2,3-Dihydroxybwnrante
25.5
37.4
YPyridine-2_aldehyde-2-pyridyl
hydrazone.
studied. FMN effectively blocks the release of ferrite iron by rhodotorulic acid, desferrioxamine B and 2,3dihydroxyknzoate. Iron release by 2,2’bipyridyl and by Paphy is increassed substantially in the presence of FMN; this effect is dependent on photoreducbion of FMN. Ascorbate also increases iron release from ferritin by desEerrioxamine B, rhodotorulic acid, and 2,3dihydroxybenzoate. Of the other mediators, EDTA inhibits iron release by rhodotorulic acid, Paphy and 2,3dihydroxybenzoate; citrate and oxalate have little or no effect. When ferrktin iron is reduced using the system FMN/NADH we observe that desferrioxamke B is unable to complex the fetitin iron; if we subse-
quently add 2,2’-bipyridyl, the iron is immediately released as the 2,2’bipyridyl-Fe(II) complex. The-se studies suggest that the reduced flavii may react with the peroxo-bridge of the catalytic si& (Fig. 3) to generate a fIavin hydroperoxide and Fe(II). If the chelator used is too bulky to have access to the sites where the Fe(H) is bound, on account of steric obstruction by the flavin or if its complexation constant is inferior to that of the sites. the iron will not be removed_ We suspect that this is the case, respectively, for desferrioxamine B and rhodotorulic acid on the one hand, and for 2,3dihydro xybenzoate on the other. In the case of bipyridyl and Paphy, the chelators can not only complex the Fe@) and thus remove it from the siti, thus liberating the sites for ferric oryhydroxide iron from the core to migrate, but may also be oxidised to the corresponding Noxide by the fIavin hydroperoxide, +hus initiating a new catalytic cycle. Ferritin thus seems to be an interesting case of a non-haem iron protein which may he a useful model system for the study of the activation of
275
Fe'<
R = -CH$CHC-HI$H$FUJH~
Fig. 3. Possible mechanism for oxidation of bipyridpl and triphenyl phasphine by ferritin in the prsence of reduced flavin. The active site peroxo (Fig_ 1) reacts with the reduced flab-in to farm a flzvin hydroperoside and Fe(D); the former intenxediate can oxidke
bipyridyl and the phosphine, while in the presence of excess chelator the Fe(E) removed from the sites; allowing re-formation of the Perono-bridge; after reduction the flavin by zn external electron donor the catalytic site can recommence.
;S of
molecular oxygen 129 1, especially in view of its remarkable resistance to denaturation by a large number of orgztnic solvents [30] _ To what extent the peroxo intermediate can be considered an activat.4 oxygen species is not clear. Oxidation of bipyridyl and of triphenylphasphine has been achieved; however, preliminary aampts to osidise olefkic substrates have not so far met with success. No oxide was formed with either ethylene, cyclohexene or tetramethylethylene [SO] _
References P. Aken. CIB4 Symp.. 51 (1977) 1 - 14. J. Nie!a&s. Struct. Boding, 2 (1972) 145 3 P. Aisen and E. B. Brown. Semim Hematol..
1 2
- 170. 14 (1977)
31 - 53.
276 4
R. R. Crichton, SCruc~. Eonding. 17 (1973) 67 - 134. 5 P. M. Harrison, S H. Banyard. FL J_ Hoare. S. M. Russeli and A. Trefy, CfRA Symp.. Sl(l977) 19 - 35. 271 (1978) 6 S. H. Banyard. D. K. Stammers and D. MM.Harrison. Nature (London). 282- 284. and C!_ Wustefeld, in prepsration. 7 R. R. Crichton, M. Heu&erspreute, D. Collet-Cassart 8 K. Wetz and R. R. Crichton, Eur. J. Biochem.. 61(1976) 545 - 550. 6 963 - 6 969_ 9 S. T_ Silk and E. Brcdow, J. Biol. Chem.. 251 (1976) 10 W. H. Massover and J. M. Rowley, Roe. Nat. Accd. Sci. USA. 70 (1973) 3 847 - 3 851. 11 H. B. Stuhrmarm. J. Haas. K. Ibel. M_ EL J- Koch and R. R. Crichton. J. MO!_ E&I.. 100 (1976) 399 - 413. 12 W. H. Massover, J. MoL BioL, 123 (1978) 721 -726. 13 P. M_ Harrison, F. A. Fischbacb, T. G. Hoy and G. H. Haggis, NoCure (London). 216 (1967) 1 188 - 1 190_ 14 I. G. Macara, T. G. Hoy and P. M. Harrison, Biochem. J., 125 (1972) 151 - 162. 15 C. F_ A. Bryce and R. R. Crichton. Biochem. J.. 133 (1973) 301 - 309. 16 E. P. Piques, A. Pgques and R_ R. Crichton. J. Mel- CalaL, 4 (1978) 75 - 82. 17 S. Sirivech, E. Frieden and S. Osaki, Biochem. J.. 143 (1974) 311 - 315_ 18 R. R. Crichton, F. Roman and M. Wauters, Biochem. Sot. Texts., 3 (1975) 946 - 948. 19 T. Jones. R. Spencer and C. Walsh. Biochemistry. 17 (1978) 4 011 - 4 017_ 20 M. Wautcrs. A. M. Michelson and R. R. Crichton, FEBS Lett., 91 (1978) 276 - 279_ 21 A. Treffry, S. H. Banyard. R- J. Hoare and P_ M. Harrison. in E. B. Brown. P. Aisen, J. Fielding and R. R. Crichton (eds.), Pr0feLz.s of Iron Metabolism. Grune and Stratton, New York, San Francisco and London, 1977, pp. 3 - Il. 22 R. R. Crichton and F. Roman, J. Mol. Catal.. 4 (1978) 75 - 82. 23 I. G_ h¶acara. T_ G. Hoy and P. M. Harrison, Biochem. J.. 135 (1973) 785 - 789_ 24 E. Vandamme and R. R. Crichton, in prepamtion. 25 A. Crutzen, K. Bonemann and R. R. Crichton, in preparation. 26 J. Dognin and R. R. Crichton, FEBS Left.. 54 (1975) 234 - 236. 27 F. Roman. F. Roland and R_ R_ Cricbton. Arch. Int. Pl;ystiL Biochem.. 87 (1979) in press. 28 F. Roman, F. Roland and R. R_ tiichton. in preparation_ 29 R R. Crichton. CUBA Symp., 63 (1979) 67 - 72_ 30 A. CruLzen, Ph. D. Thesis, Univenite Catholiquc de Louvain, 1979. 31 M. Wauters, Ph. _D_ thesis. Universiti Catholique de Louvain, 1978. 32 G. Melino. S. Stefanini, E. Chiancone, E. Antonini and A. Finazzi-Agro, FEBS ktt., 86 (1978) 136 -, 138. 33 E. P. Piques, Ph. D. thesrS. Universite Catholique de Louvain, 1979.
Journal of Molec&ar CataI,vsis, 7 (1980) 267 - 276 @ Eketier Sequoia S-A., Lausann e -Printed in the Netherlands
FERMTIN
IRON
DEPOSITION
AND MOBILZS_4TiON
ROBER_T R. CRICHTON, FRANC_OESE ERIC PAQUES, ANTOINEZTE PAQUES UnitP de Biochimie. UniuenitL Looualr-la-Neuue (Belgium)
Catirolique
ROMAN, FRANCl-NJZ ROLAND. and ETIENNE VANDAMME de Louuai~.
Pluce L. Pasteur.
I, B-i348
summary We have studied the mechanisms whereby iron is deposited in, and mobilised from, ferritin, the principal protein of iron storage in mammals. Iron deposition involves oxidation of Fe(II), cataIysed by the protein with molecular oxygen as electron acceptor, followed by hydrolysis and deposition of the ferric oxyhydroxide in the interior of the protein shell. We have proposed a model for iron deposition in which Fe(II) binds to catalytic sites situated on adjacent polypeptide chains; dioxygen is then bound between the two iron atoms and is reduced to give a relatively stable Fe(III)-peroxo complex. Incoming Fe(I1) can displace the peroxo complex allowing hydrolysis of the ferric iron and its migration to the interior of the protein. The evidence in support of this model is briefly summarised. We assume that at low pH the rate-limiting step is the deprotonation of a group or groups on the protein with a pK of around 6. Iron mobilisation from ferritin can be readily achieved by reduced flavins in the presence of a suitable iron chelatir_ We have found that iron mobilization from ferri&n by bipyridyl and also by pyridinne-Z-aIdehyde-2pyridyl hydrazone (Paphy) is greatly increased by photoreduced flavin, whereas, for the iron chelators, desfenioxamine B, rhodotorulic acid and 2,3-dihydroxybenzoat.e, iron mobiIis.ation is effectively blocked by the addition of flavin. We suggest that the reduced flavin reacts with the peroxo bridge of the catalytic sites to generate a flavin hydroperoxide and Fe(II).
Introduction The chemistry of iron in aqueous solution is dominated by two reactions -the oxidstion of Fe(H) to Fe(III), and the hydrolysis of the latter to form essentiaIly insoluble ferric oxyhydroxide polymers [I] _ In view of theimportant role of iron in many proteins involved in eleetin transfer, oxygen transport, and oxygen activation (oxidases, hydroxylases, etc_) and detoxification (catalases, peroxidases, c* bacterial superoxide dismutases), the bioavailability of iron in a soluble, non-toxic and yet readily avaiIable form
268
is an essential requirement for aerobic life. Whereas many prokaryotes and lower eukaryotes (such as fun@) have developed low molecular weight siderophcres, which are excreted into the extracellular milieu, complex iron, and then transport it to-the in+&rior of the organism where it is released for internal utilisation [ 21, the higher eukaryotes have developed a system of transport and storage based on high molecular weight protein ligands for the iron. Thus the transport of iron in plasma is accomplished by the or -glotulin, transferrin, which is a glycoprotein of molecular weight around 80 000 and which can transport a maximum of two atoms of ferric iron bound to the protein in intimate association with bicarbonate ion [ 3 J _ Within eukaryotic cells iron is stored in ferritin [4,5], a protein of molecular weight 440 000 composed of 24 polypeptide chains each of molecular weight 18 500 which, in a fashion rather reminiscent of spherical viruses, form a hollow protein shell of esterior diameter around 120 A, in the interior of which a cavity of diameter 65 - 70 A serves to store the iron. The capacity of ferritin to store is considerable; ferritin molecules from horse spleen can accumulate up to 4 500 atoms of iron, essentially as FeO.OH together with some phosphate such that the ‘full ferritin’ contains 0.57 g of iron/g of protein and yet can readily he concentrated to give sol-utions which are 1M m iron in a completely water-soluble form. The purpose of this presentation is to analyse at the molecular level the mechanisms by which iron is deposited in, and mobilised from, ferritin. Before passing to the discussion of these mechanisms, we will first present briefly the structure of the apoferritin molecule (t.hat is, the protein shell of ferritin) and its relation to the micelIar iron core. Apoferritin has been crystallised from a number of sources, but at
present the best character&d apoferritin is that of horse spleen for which an electron den&y map at 2.8 A resolution has been obtained by Professor Pauline Harrison and her collaborators in Sheffield [Cl] _ The protein shell is roughly spherical and access ti the internal cavity in which the iron is depositi is assured by channels 10 - 15,4 in diameter along the four-fold axes. The individual polypeptide chains can best be described as four, long, approximately parallel, a-helices of 38 - 45 A in length oriented at right angles to the four-fold auis, along which lies a shorter helix which lines the channels referred to above. These five cc-helical domains are connected by non-helical regions which represent about 30 - 35% of the structure. The apoferritin molecule is composed of 165 amino acids and a sequence of more than 90% has been establish4 in our laboratory [7] _ There is a considerable amount of acidic ammo acids (22 - 26 according to chemical modification and titration studies, [ 8, 9]), three cysteine residues/subunit, six histidine residues/subunit and a high proportion of non-polar amino acids (45%). The construction of an atomic model of horse spleen apoferritin based on X-ray crystallogaphicand primary structure studies is in progress, and we anticipate that the structure will be solved by atomic resolution in the very near future.
269
The iron core of ferritin contains some degree of internal ordering [IO] and it seems likely that there are intimate contacts between the iron micelle and the apoferritin shell [ 11,12]. It i.s of interest to note that the structure of the ferric oxyhydroxide formed is determined by the protein; thus, whereas polymerisation of ferric osyhyclroxide in several different buffers in the absence of apoferritin gives rise to different forms of polymers, the iron micelle obtained in ferritin in the presence of the protein is independent of the buffer employed ]13]. Finally, it should be pointed out that apoferritin preferentiaIly accumuiates Fe(R), and catalyses the oxidation of Fe(R) to Fe(III) IL4 - X6]_ Thus, we are led to conclude that apoferritin is a ferroxidase and that specific groups in the protein participate in oxidation of iron. In iron release from fe-rritin many studies imply a reductive pathway such that the ferric iron is first reduced to ferrous, most probably by reduced fIavins, before the iron can be released from the protein and participate in the synthesis of ironcontaining enzymes Cl7 - 191.
Results The deposition of iron in ferritin is most readily achieved by incubation of apoferritm with Fe(II j in the presence of an appropriate electron acceptor, of which molecular orrygen is perfectly adequate. It was immediately evident that the study of iron deposition in fen-&in necessitated the use of buffer systems in which (i) the rate of autoxidation of iron was minimal, (ii) the complex&ion of iron by the buffer was not sufficiently important to interfere with the kinetic analysis. In our initial studies [ 151 we used sodium borate-cacodylate buffers of pH 5.5 - 6.0 which satisfied the first, but not the second criteria. The effect of buffer on the kinetics of iron deposition in apoferritin (Fig. 1) are clearly separated into two groups [lS] . Ironcomplexing buffers such as imidazole, bora*cacodylate znd Bicine give saturation kinetics at pH 7.0, whereas non-complexing buffers such as Mes and Mops give rise to S-shaped cumes. These results, together with rapid kinetic studies in boraWcacody1at.e buffers, pH 5.6 - 6.0 [17], suggested that iron deposition in ferritin was at least second order with respect to the iron concentration. A detailed analysis of the inhibition of iron deposition by Cr(III) [IS, 201 showed that an 85 - 90% inhibition was observed upon fixation of one chromium/two subunits. It was subsequently shown that Cr(IIl) blocks fixation of uranyl acetate to apcferritin crystals; the uranyl site was close to the two-fold axis [Is] . The X-ray crystallographic studies [S] showed that the major site of Tb 3c fixation is located close to the twofold axis and only 4.3 # distant from its symmetry-related pair. Inhibition of iron deposition in apofemitin by Tb a has been okserved 1211 zmd was attributed to binding of Tb3+ to carboxyl groups at the Febinding sites. Prior to the publication of these results we had observed that 2,2’-bipyridyl could release iron as Fe(II) from ferritin in the absence of reducing agents
270
Fig.
1. Kinetics
of
iron
deposition
in apoferritin
nt pH
7 ~3 in a series
of
different
buffers,
all at a concentration of lOOmM_ The initial velocity of iron deposition was measured at 310 nm and the iron concentration was varied from 0.028 to 2.05mM (from ref. 16). The curves are, respectively, (0) Imidazole, ( -) Mes, (-) Bonte-c;lcodyIate, (0) Mops, and (A) Bicin _
[ 223 . On the basis of these resuits we were led to prcpose a mechanism for iron deposition in fen-&in involving four steps (Fig. 2): (i) fixation of Fe” by catalytic sites located on two adjacent polypeptide chains; (ii) fixation of a dioxygen molecule between the two i-on atoms; (iii) reduction of the dioxygen to give a peroxo complex in which the oxygen ligand is coordinated to two iron atoms; (iv) hydrolysis of the peroxwomplex accompanied by migration of the ferric oxyhydroxide formed to the interior of the protein. The evidence supporting ihis model will now be discussed. (i)
The
catalytic
site
involves
two
polypeptide
chains
Fe ‘+ by apoferritin using rapid kinetic techniques (stopped flow) in bora*cacodylate buffers, pH 5.8, showed [%I] a second order dependence of the initial velocity as a function of iron concentration, consistent with a mechanism invohtig fixation of two iron atoms. A more detailed kinetic analysis of the effects of buffer and pH was undertaken [IS] and it was found that at pH values between 6-5 and 7.5 the rate of iron deposition in ferritin showed a sigmoidal dependence on the concenStudies
on
the
oxidation
of
271
Fig. 2. Model for ferritin iron oxidation and deposition. fixation of iron(H) and in oxidation are in the channels heferonucleation to which the iron migrates are situated (from ref. 22).
The iron binding sites invo!ved in between the subunits. The sites of in the
iaterior
of
the molecule
of iron in noncomplexing bmuffers(Fig- 1). In ircidazole buffer, sigmoidal dependence was also observed at pH 7.5. We interpreted these results in terms of the fixation of two iron atoms/catalytic site. At lower pH valuq s&uration kinetics were observed_ The very considerable effect of pH on the rate of iron deposition, and the rather similar rates observed in different buffers at pH values between 6.0 and 6.5, led us to conchrde that at low pH the deprotonation of a group or groups on the protein with pK of around 6 was the rate-Iimiting step in ferritin formation, accounting for the saturation kinetics observed [I.61 _ The inhibition of iron deposition upon fEation of one g at. of Cr?/subunit referred to above also supports the view that the catalytic site is composed of two adjacent polypeptide chains 116, 201. Finally, the observation that the major site of Tb3* binding in the apoferritin crystals is locaL& close to the two-fold avis of the apoferritin molecule and only 4.3 A from its symmetry-related pair [S] is consistent wi+h the model observed_ Tb3* ions inhibit iron uptake by apoferritin and are assumed to bind to carboxyl groups at the Fe ‘* binding sited [21] _ Chemical modification studies [S, 9] show that carboxyl groups are certainly involved in the iron oxidation sites. Further, we have recently shown that Zn*, Ni**, Co2* and Tbs+ are all inhibitors of iron deposition in ferritin, confirming previous studies 121,231, and that the inhibition in aJl cases is competitive ]24]_ tration
272
{ii}
The peroxo in &.-mediate The presence of oxygen in an activated form, which we presume to be a peroxo complex with Fe(M), is suggested by the capacity of ferritin to oxidise a number of organic molecules such as 2,2’-bipyridyl and triphenylphosphine. We have been able to detect bipyridyl Naxide in chloroform extracts of the incubation mixture of fen-&in with bipyridyl by thin layer chromatography in the solvent system isopropanol-ethyl aceta&cyclohesane 20/10/70 v/v after repeated chromatography [ 221. Triphenylphosphine oxide formation was detected after incubation of ferritin with triphenylphosphine under argon. Ether extraction allowed us to extract both the phosphine and its oxide; detection and quantification were achieved by 31 P n.m.r. measurements. Using variable amounts of phosphine we con&tent!y observed values of 0.97 mole of triphenylphosphine oxide/subunit formed after incubation with ferritin. Concomitantly 15 - 30 atoms of Fe(II) were released from the protein [ 25]_ These results, together with the iron mobihsation studies using bipyridyl described below, suggest that the peroxo comples is present at the catalytic sites. The considerable enhancement of iron deposition that we have observed [24] in the presence of Cu2+ is also consistent with this intermediate, since Cu2+ is known to destabilise peroxides. Hydrolysis of the perowo intermedicte and internalisafion of ferric oxyhydroxide The iron in ferritin is mostly present in hydrolysed form as ferric oxyhydroxide_ Thus, it is reasonable to assume that the peroxo intermediate hydrolyses to form FeO.OH, which subsequently migrates in’to the interior of the protein shell. Careful analysis of the stoichiometry of oxygen consumption confirms that 4 g at. of Fe(I1) are oxidised per molecule of dioxygen consumed [31,32] _ However, the mechanism proposed in Fig. 2 is unlikely to take place as indicated, and more plausible pathways involving formation of a ~-0x0 complex either following scheme (1) or scheme (2) could precede hydrolysis (3) or (4) _
(1)
0 Fe’
Fe/O\
\
O,Fe
Fe
+
+
H,O
2Fe”
+
4
.O
2Fe /
l?eHo ‘OH
\
+
(2)
Fe
FeRC f
2H’
(3)
273
Fe/O\
Fe + 3H20
+
2FeHo ‘OH
+ SH’.
(4)
We are unable to distinguish for the moment between these alternatives. A closer scrutiny of the kinetics of iron deposition in ferritin at neutral or a&aline pH has shown 1331 a considerable increase in initial velocity at high iron concentrations which would be not inconsistent with a heteronucleation of FeO.OH micelles at internal sites on the protein shell. The hydrolysis of the peroxo intermediate in the case of schemes (2) f (3) would regenerate Fe’+ on the sites and might be accompanied by displacement of the hydrolysed product to the interior. We assume that the hydrolysis is reversible in view of the results of iron mobilisation obtained with bipyridyl (see below)_ Iron
mobilisation
from
ferritin
The most effective way of releasing ferritin iron in vitro is by reduction in the presence of an appropriate chelating agent. Thus, in the preparation of apoferritin from ferritin, the iron is removed by reduction with dithionite in the presence of bipy-ridyl or, alternatively, reduced and complexed by thioglycollate. Of the biological reductants that have been tested, by far the most impressive results have been obtained with reduced riboflavin mono nucleotide (FMNH& in the presence of bipyridyl [5,17,19]. However, to obtain maximum rates of iron release strict anaerobic conditions are necessary. It has been suggested that reduced FMN penetrates to the interior of the protein shell through the channels along the four-fold axes of the molecule and in this way comes in contact with the iron core [5] _ This view receives support from expe_iments in which reduced FMN was immobilised on an inert carrier and it was found that no iron release was observed [19]. However, it is not yet clear whether FMN can penetrate through the channels_ If the role of FMNH2 as biological reductant is not yet clearly stablished, information concerning the chelator or chelators that are employed in the cell is conspicuous by its absence: there is certainly no lack of potential candidates. Our inter-t in ferritin iron mobilisation has been directed to the effects of iron chelators. Since we observed that 2,2’-bipyridyl could release ferritin iron [ 261 we have extended these studies with bipyridyl [22] ; as describ& above an N-oxide of bipyridyl was detected_ Subsequently [27,28], release of iron from horse spIeen ferritin by desferrioxamine B, rhodotoruhc acid, 2,3dihydroxybenzoate, 2,2’-bipyridyl, and pyridine-2-aldehyde-2-pyridyl hydrazone (Paphy) was studied in vitro (Table I). Deferrioxamine B and rhodotomhc acid both release appreciable amounts of ferriti iron. Iron release from ferritin by the other three chelators is less effective: at ImM final concentration the c!aelators can be classed in the order rhodotorulic acid > desferrioxamine B > Paphy > 2,3&hydroxybenzoate > 2,2’bipyridyl. The effects of low molecular weight mediators (FMN, EDTA, ascorbate, citrate, and oxalate) on iron release by the chelators was also
274 TABLE
1
Iron release from ferritin by chelators The amount of iron releed after 2 h and 6 h incubation at 37 “C of 1.14 x 10-6bl horse spleen ferritin in 0 2M Mops (morphohne propane sulphonate) buffer, pH 7 -4, by the different chelators at a final concentration of 1mM alone, and in the presence of a finai concentration of 1mM FMN, was measured at the appropriate xc,, of the chelator. The resu!te are exmed as g at. of iron released/molecule of ferritin and are the ruean of five independent determinations_ All experiments were cerried out in the light, and some of the FMN was thus pho’&reduced to FMNH, (from ref. 28). Chelator
g_ At. of Fe rcleasedjmolecule chelator
Bipyridyl Paphy* Desferrioxamine
Rhodotomlic
B
acid
alone
by chelator
+ 1mM
FMN
2h
6h
2h
6h
59 21-i 62.7
108 50.1 1.182
24 .I3 47.9 118
492 90.5 12.6
22.8
22.7
246
26.5
74-o
151.3
2,3-Dihydroxybwnrante
25.5
37.4
YPyridine-2_aldehyde-2-pyridyl
hydrazone.
studied. FMN effectively blocks the release of ferrite iron by rhodotorulic acid, desferrioxamine B and 2,3dihydroxyknzoate. Iron release by 2,2’bipyridyl and by Paphy is increassed substantially in the presence of FMN; this effect is dependent on photoreducbion of FMN. Ascorbate also increases iron release from ferritin by desEerrioxamine B, rhodotorulic acid, and 2,3dihydroxybenzoate. Of the other mediators, EDTA inhibits iron release by rhodotorulic acid, Paphy and 2,3dihydroxybenzoate; citrate and oxalate have little or no effect. When ferrktin iron is reduced using the system FMN/NADH we observe that desferrioxamke B is unable to complex the fetitin iron; if we subse-
quently add 2,2’-bipyridyl, the iron is immediately released as the 2,2’bipyridyl-Fe(II) complex. The-se studies suggest that the reduced flavii may react with the peroxo-bridge of the catalytic si& (Fig. 3) to generate a fIavin hydroperoxide and Fe(II). If the chelator used is too bulky to have access to the sites where the Fe(H) is bound, on account of steric obstruction by the flavin or if its complexation constant is inferior to that of the sites. the iron will not be removed_ We suspect that this is the case, respectively, for desferrioxamine B and rhodotorulic acid on the one hand, and for 2,3dihydro xybenzoate on the other. In the case of bipyridyl and Paphy, the chelators can not only complex the Fe@) and thus remove it from the siti, thus liberating the sites for ferric oryhydroxide iron from the core to migrate, but may also be oxidised to the corresponding Noxide by the fIavin hydroperoxide, +hus initiating a new catalytic cycle. Ferritin thus seems to be an interesting case of a non-haem iron protein which may he a useful model system for the study of the activation of
275
Fe'<
R = -CH$CHC-HI$H$FUJH~
Fig. 3. Possible mechanism for oxidation of bipyridpl and triphenyl phasphine by ferritin in the prsence of reduced flavin. The active site peroxo (Fig_ 1) reacts with the reduced flab-in to farm a flzvin hydroperoside and Fe(D); the former intenxediate can oxidke
bipyridyl and the phosphine, while in the presence of excess chelator the Fe(E) removed from the sites; allowing re-formation of the Perono-bridge; after reduction the flavin by zn external electron donor the catalytic site can recommence.
;S of
molecular oxygen 129 1, especially in view of its remarkable resistance to denaturation by a large number of orgztnic solvents [30] _ To what extent the peroxo intermediate can be considered an activat.4 oxygen species is not clear. Oxidation of bipyridyl and of triphenylphasphine has been achieved; however, preliminary aampts to osidise olefkic substrates have not so far met with success. No oxide was formed with either ethylene, cyclohexene or tetramethylethylene [SO] _
References P. Aken. CIB4 Symp.. 51 (1977) 1 - 14. J. Nie!a&s. Struct. Boding, 2 (1972) 145 3 P. Aisen and E. B. Brown. Semim Hematol..
1 2
- 170. 14 (1977)
31 - 53.
276 4
R. R. Crichton, SCruc~. Eonding. 17 (1973) 67 - 134. 5 P. M. Harrison, S H. Banyard. FL J_ Hoare. S. M. Russeli and A. Trefy, CfRA Symp.. Sl(l977) 19 - 35. 271 (1978) 6 S. H. Banyard. D. K. Stammers and D. MM.Harrison. Nature (London). 282- 284. and C!_ Wustefeld, in prepsration. 7 R. R. Crichton, M. Heu&erspreute, D. Collet-Cassart 8 K. Wetz and R. R. Crichton, Eur. J. Biochem.. 61(1976) 545 - 550. 6 963 - 6 969_ 9 S. T_ Silk and E. Brcdow, J. Biol. Chem.. 251 (1976) 10 W. H. Massover and J. M. Rowley, Roe. Nat. Accd. Sci. USA. 70 (1973) 3 847 - 3 851. 11 H. B. Stuhrmarm. J. Haas. K. Ibel. M_ EL J- Koch and R. R. Crichton. J. MO!_ E&I.. 100 (1976) 399 - 413. 12 W. H. Massover, J. MoL BioL, 123 (1978) 721 -726. 13 P. M_ Harrison, F. A. Fischbacb, T. G. Hoy and G. H. Haggis, NoCure (London). 216 (1967) 1 188 - 1 190_ 14 I. G. Macara, T. G. Hoy and P. M. Harrison, Biochem. J., 125 (1972) 151 - 162. 15 C. F_ A. Bryce and R. R. Crichton. Biochem. J.. 133 (1973) 301 - 309. 16 E. P. Piques, A. Pgques and R_ R. Crichton. J. Mel- CalaL, 4 (1978) 75 - 82. 17 S. Sirivech, E. Frieden and S. Osaki, Biochem. J.. 143 (1974) 311 - 315_ 18 R. R. Crichton, F. Roman and M. Wauters, Biochem. Sot. Texts., 3 (1975) 946 - 948. 19 T. Jones. R. Spencer and C. Walsh. Biochemistry. 17 (1978) 4 011 - 4 017_ 20 M. Wautcrs. A. M. Michelson and R. R. Crichton, FEBS Lett., 91 (1978) 276 - 279_ 21 A. Treffry, S. H. Banyard. R- J. Hoare and P_ M. Harrison. in E. B. Brown. P. Aisen, J. Fielding and R. R. Crichton (eds.), Pr0feLz.s of Iron Metabolism. Grune and Stratton, New York, San Francisco and London, 1977, pp. 3 - Il. 22 R. R. Crichton and F. Roman, J. Mol. Catal.. 4 (1978) 75 - 82. 23 I. G_ h¶acara. T_ G. Hoy and P. M. Harrison, Biochem. J.. 135 (1973) 785 - 789_ 24 E. Vandamme and R. R. Crichton, in prepamtion. 25 A. Crutzen, K. Bonemann and R. R. Crichton, in preparation. 26 J. Dognin and R. R. Crichton, FEBS Left.. 54 (1975) 234 - 236. 27 F. Roman. F. Roland and R_ R_ Cricbton. Arch. Int. Pl;ystiL Biochem.. 87 (1979) in press. 28 F. Roman, F. Roland and R. R_ tiichton. in preparation_ 29 R R. Crichton. CUBA Symp., 63 (1979) 67 - 72_ 30 A. CruLzen, Ph. D. Thesis, Univenite Catholiquc de Louvain, 1979. 31 M. Wauters, Ph. _D_ thesis. Universiti Catholique de Louvain, 1978. 32 G. Melino. S. Stefanini, E. Chiancone, E. Antonini and A. Finazzi-Agro, FEBS ktt., 86 (1978) 136 -, 138. 33 E. P. Piques, Ph. D. thesrS. Universite Catholique de Louvain, 1979.