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Sulfide is an efficient iron releasing agent for mammalian ferritins
Biochimica et Biophysica Acta 1547 (2001) 174^182 www.bba-direct.com
Sul¢de is an e¤cient iron releasing agent for mammalian ferritins Sylvie Cassanelli, Jean-Marc Moulis * CEA, De¨partement de Biologie Mole¨culaire et Structurale, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France Received 11 December 2000; received in revised form 6 March 2001; accepted 8 March 2001
Abstract The most prominent role of mammalian ferritins is to provide an extensive iron-buffering capacity to cells. The large ferritin iron stores can be mobilized in vitro, but the functional relevance of the most efficient iron releasing agents remains elusive. Sulfide is a strongly reducing chemical generated by a series of enzymes. In the presence of limited amounts of sulfide a continuous rate of iron release from ferritin was observed and a majority of the protein iron core was recovered in solution. The rate constants for iron efflux triggered by several reducing or chelating compounds have been measured and compared. Although not as efficient as reduced flavins, sulfide displayed kinetic parameters which suggest a potential physiological role for the chalcogenide in converting the iron storage protein into apoferritin. To further probe the relevance of sulfide in the mobilization of iron, several enzymes, such as NifS, rhodanese, or sulfite reductase generating reduced forms of sulfur by different mechanisms, have been assayed for their ability to catalyze the release of iron from ferritin. The results show that full reduction of sulfur into sulfide is needed to deplete iron from ferritin. These reactions suggest links between sulfur metabolism and intracellular iron homeostasis. ß 2001 Elsevier Science B.V. All rights reserved. Keywords: Sulfur metabolism; Iron homeostasis; Sulfane; Desulfurase; Ferritin
1. Introduction Ferritins are crucial elements of iron homeostasis in most organisms. They have been evidenced in animals, plants and bacteria for instance, but with somewhat di¡erent properties in these various cases. However, ferritins from di¡erent sources share the capacity to store very large amounts of iron (up to about 4500 atoms/molecule) as iron oxides accumu-
Abbreviations: BPS, bathophenantroline; Fz, ferrozine ; PLP, pyridoxal 5P-phosphate * Corresponding author. Fax: +33-438785872; E-mail: [email protected]
lated in the large internal cavity of the protein with varying amounts of phosphate anions. Ferritin subunits are homologous and are assembled in heteropolymers of 24 polypeptide chains [1]. Mammalian ferritins are made up of two kinds (H and L) of subunits and are generally localized in the cytoplasm. The elongated, mainly K-helical, peptides organize into a highly symmetric structure generating the large hollow in which iron is stored. This internal shell can communicate with the exterior of the protein through di¡erent channels corresponding to the main symmetry axes of the molecules. In particular, the 3-fold intersubunit channels appear to interact with the ferrooxidase centers located on H-subunits in a key step of the ferritin iron loading process. Mammalian ferritin biosynthesis is mainly regulated at the translational level by the binding of iron reg-
0167-4838 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 0 1 ) 0 0 1 8 2 - 0
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ulatory proteins to the iron responsive elements lying on the 5P non-coding sequence of the mRNA. Binding and repression of translation occur when iron is scarce or under several other stress conditions [2]. Transcriptional regulation also occurs [3]. In contrast, the plant ferritin genes are organized di¡erently from the mammalian ones and their expression is regulated at the transcriptional level [4,5]. In aerobic organisms ferritins scavenge iron exceeding the normal needs of the cell. Under conditions of oxidative stress, iron catalyzes partial reduction of oxygen and may lead to cell damage by hydroxyl radicals and other products. In metazoans, these deleterious e¡ects are avoided by inactivation of the iron regulatory proteins which detect the increased iron concentration, hence dissociating from the iron responsive element on the 5P non-coding sequence of the mRNA and increasing ferritin translation. However, once stored in ferritin, the fate of the generally large amounts of ferric ions in the protein is not clear [6]. Ferritin may be oxidatively modi¢ed and proteolysed by the proteasome [7], or it may be transported to lysosomes to be degraded, with the metal recycling back to the cytosol [8,9]. Ferritin might also be a dead-end trap for iron, much as metallothioneins seem to be for copper in some instances [10]. Last, iron exit from ferritin, which has been largely demonstrated in vitro, may be used in the synthesis of iron-containing proteins or as an alternative to extracellular uptake for replenishing the intracellular iron pool. The release of iron from ferritin is triggered by reductants and chelators and some chemicals may play both roles [1]. Reductants revert the mineralization process and chelators displace the equilibrium toward ferrihydrite solubilization. These reactants may gain access to the internal cavity through the protein channels, but direct interaction may not be mandatory and long range electron transfer from the protein surface to the inner core has been proposed [11]. Since the signi¢cance of many compounds shown to release iron from ferritin in vitro remains elusive, a survey of metabolites able to carry out the reaction is of interest. We have thus evaluated the ability of reduced species generated by the metabolism of sulfur-containing molecules to expel iron out from mammalian ferritins.
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2. Materials and methods In all experiments reported herein except iron loading of ferritins, strictly anaerobic conditions were implemented by use of argon lines or of an anaerobic chamber (Jacomex, Livry-Gargan, France) ¢lled with puri¢ed argon [12]. 2.1. Proteins Horse spleen ferritins (apo- and holoforms) were obtained from Sigma-Aldrich Co. Recombinant human H-ferritin was kindly provided by Dr. Sonia Levi (DIBIT, Milan, Italy) as frozen ammonium sulfate precipitate with 2 M urea [13] and further puri¢ed by anion-exchange chromatography (DE-52, diethylaminoethyl cellulose, Whatman). The apoferritins were iron-loaded as described [14] and the holoferritins were puri¢ed by gel ¢ltration in 20 mM Tris^HCl pH 7.4. No signi¢cant reactivity di¡erences were observed between native and in vitro ironloaded horse spleen ferritins. The eluted ferritins were analyzed for their iron [15] and protein [16] contents and they were used in iron release experiments without allowing excessive ageing. The lack of signi¢cant iron leakage from these preparations induced by iron chelators alone (see Section 3) indicates that most of the iron added to apoferritins was incorporated into the iron core of the protein. 2.2. Iron release from ferritins Since the reported rates for iron mobilization from ferritin depend somewhat on the metal loading of the protein [17], experiments are reported separately for each ferritin preparation. The reaction volume was 300 Wl in a cuvette of 0.1 cm optical path. Beside the compound used to trigger iron release, the reaction was carried out in 20 mM Tris^HCl pH 7.4 at room temperature and it contained 0.15^1 WM ferritin (0.2^0.5 mM Fe) and excess bathophenantroline (BPS) or ferrozine (Fz) as iron chelators. The formation of ferrous complexes was followed at 535 nm, O = 22 140 M31 cm31 , with BPS, or at 562 nm, O = 27 900 M31 cm31 , with Fz. Hewlett-Packard 8452 or 8453 spectrophotometers were generally used, except for some measurements with nicotin-
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amide-adenine nucleotides which were performed with a Kontron 941 spectrophotometer equipped with a monochromator. Indeed, NADH and NADPH were unstable under the ultraviolet component of the polychromatic incident light of reversed-optics systems and their products had some ability (maybe via the generation of radicals) to release iron out of ferritin as measured by the accumulation of ferrous chelates as a function of the irradiation time (not shown). It was checked that no iron was expelled from ferritin by NADH and NADPH when the reaction was followed with the conventional Kontron 941 spectrophotometer at the visible wavelength corresponding to the absorbance maximum of the ferrous chelates. Alternatively, measurements were carried out with reverseoptics spectrophotometers connected through ¢ber optics not allowing light of wavelengths below 350 nm to pass through. Known concentrations of FMN, FAD, or ribo£avin were fully reduced by a 6-fold excess of NADPH in the presence of Escherichia coli ferredoxin (£avodoxin)-NADP oxidoreductase. The latter was obtained as follows. From the published sequence of the fpr gene [18], the oligonucleotides Fpr5: 5P-CAAAACAGGAGAAACATATGGCTGATTGGG-3P and Fpr3: 5P-ATCACCGTTTGCTGCAGGCATATCGCGGG-3P were used to amplify the gene from E. coli K12 DNA. The fragment was digested with NdeI and PstI, cloned into pT7-7, and the gene was overexpressed in E. coli K38/pGP12 as described [19]. The very fast reactions between reduced £avins and ferritins were followed for extensive periods, but only the linear part of the data over the few ¢rst seconds of the reactions was used to calculate rate constants.
of Drs. Mahel Zeghouf and Jacques Cove©s (CEA/ Grenoble, France). 3. Results 3.1. Release of ferritin iron by sul¢de The features generally shared by reactants previously shown to release iron from ferritin are their powerful reducing capacity or their metal chelating properties [1]. Although often considered as a toxic component, sul¢de is involved in several metabolic pathways [23^25]. It is a strong reductant, but no reports on the reaction of this relatively common cellular reductant on iron-loaded ferritin seem available. Under acidic (pH 5.4) or alkaline (pH 8.9) conditions, exchange between oxygen and sul¢de in the coordination sphere of the core iron has been described [26,27]. Fig. 1 shows that sul¢de actually expelled the metal from horse spleen ferritin, which mainly contains the L-ferritin subunit, in the presence of iron chelators. Recombinant human H-ferri-
2.3. Enzymes involved in sulfur metabolism Overexpression of Azotobacter vinelandii nifS in E. coli K38/pGP1-2 was carried out using plasmid pDB551 [20] kindly supplied by Prof. Dennis R. Dean (Virginia Tech, Blacksburg, VA, USA). The catalytic conversion of L-cysteine into alanine by 0.16 mg of soluble extracts was quantitated by the formation of methylene blue in the presence of sul¢de [21]. Bovine liver rhodanese was obtained from Sigma. Puri¢ed E. coli sul¢te reductase [22] was a gift
Fig. 1. Iron release from horse spleen ferritin by sodium sul¢de. 185 nM ferritin (415 WM Fe) was treated with increasing concentrations of Na2 S in 20 mM Tris^Cl, pH 7.4 and the formation of BPS ferrous chelate was monitored at 535 nm. Sul¢de concentrations were 0.1 (circles), 0.25 (squares), 0.5 (up-triangles), 1 (down-triangles), 1.5 (hexagons), and 2 mM (diamonds).
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tin homopolymers also reacted with sul¢de under pseudo ¢rst-order kinetics (Fig. 2). Iron release was monitored by the absorbance of ferrous chelates in these experiments: Fz and BPS gave similar results and the rates of iron release due to chelators alone were far smaller, of the order of 1x, than those measured in the presence of sul¢de. About half of the iron core was removed from human H-ferritin in 90 min with a single initial addition of 0.5 mM Na2 S. The reaction of sul¢de with ferritin has thus the ability to trigger the exchange of signi¢cant amounts of iron from the large intracellular store. It was checked that the reaction rate is linear in the 0.15^1 WM ferritin range. 3.2. Comparison of iron release from ferritin with di¡erent reductants Second-order rate constants for iron release from ferritin induced by selected compounds of potential biological signi¢cance were determined (Table 1). The reactions with £avin derivatives were very fast, as previously shown [17,28,29], with rate constants larger by approximately two orders of magnitude than that for sul¢de. However, the latter exceeded those of physiologically relevant thiols, such as Lcysteine or glutathione (Table 1). Other thiols have also been studied for comparison (Table 1). Superoxide is often considered as a mediator of iron release by ferritin, e.g. [30], but the actual rates are di¤cult to measure and remain controversial [31^ 33]. When superoxide was anaerobically generated by dissolving KO2 in our assays, no metal complex with BPS built up from horse spleen ferritin. Similar
Fig. 2. Dependence of the observed rate constants for iron release from ferritin with the sodium sul¢de concentration. The Na2 S data used to derive the values of Table 1 (columns 1 and 3) are plotted for horse spleen ferritin (circles) and human recombinant H-ferritin (squares).
to superoxide, initial reports [34] of iron release from ferritin by nitrogen monoxide have been questioned [35]. 3.3. E¡ect of enzymatically produced sulfane sulfur on holoferritin Sul¢de is very easily oxidized under aerobic conditions. However, the chalcogenide is generated as part of metabolism [23^25] and the possible contribution of several enzymes producing sul¢de has been assessed in the removal of iron from ferritin.
Table 1 Second-order rate constants (M31 min31 ) for iron release from mammalian ferritins HoSFr (2250 Fe/ferritin) FMNH2 Ribo£avin FADH2 Na2 S L-Cysteine BPS NADPH O3 2
HoSFr (1200 Fe/ferritin) 26 000 11 400 3 000 100 10 6 0.2 0 0
Na2 S glutathione thioglycollate
HuH-Fr (600 Fe/ferritin)
680 6 10 6 10
Na2 S glutathione
175 0.3
Each column presents results obtained with the same preparation of ferritin. Rate constants were determined from data as those presented in Fig. 2, for several ferritin concentrations in the 0.1^0.75 WM range. HoSFr is iron-loaded horse spleen ferritin and HuH-Fr is recombinant human H-subunit homopolymer.
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Fig. 3. Iron release from horse spleen ferritin in the presence of NifS. 160 nM ferritin (200 WM Fe) was treated with either 1 mM L-cysteine (open squares) or the E. coli extract containing NifS (open circles). In another experiment with the same protein, L-cysteine was added by successive 0.25 mM increments (arrows) to the mixture of ferritin and NifS (¢lled triangles). The reaction without ferritin produced bound persul¢de that was assayed as sul¢de (open diamonds, right scale).
A subset of widespread pyridoxal 5P-phosphate (PLP)-dependent enzymes, including cysteine synthases, cystathionine L-lyase and the protein encoded by nifS [36], share the ability to desulfhydrate L-cysteine. The nifS gene was initially identi¢ed in a nitrogen ¢xation operon of A. vinelandii and it is believed to participate in the biogenesis of the nitrogenase enzyme [37]. The enzyme transforms L-cysteine into L-alanine and an enzyme-bound persul¢de: the latter can be released from the enzyme by reduction of the disul¢de bond, with dithiothreitol for example [20]. Fig. 3 shows that addition of NifS to horse spleen ferritin does not sustain iron release at a high rate after an initial burst. This increase in soluble iron can be attributed to endogenous compounds able to expel iron from ferritin. Addition of the NifS substrate, L-cysteine, increases the rate. However, cysteine may have di¡erent roles in the reaction: it is the substrate providing the sulfur atom binding to the active site cysteine, but non-reacted free cysteine can also release this sulfur atom by reaction with enzyme-bound persul¢de. In addition, cysteine alone is able to release iron out of ferritin (Table 1), albeit at a very low rate (Fig. 3). However, the combination of cys-
teine and NifS has always been found more e¤cient than cysteine alone to remove iron out of ferritin. These data suggest that the persul¢de sulfur bound to NifS does not play a direct role in the mobilization of ferritin iron, but, once the enzyme-bound sulfur is displaced, by excess cysteine in the present case, signi¢cant iron amounts are concomitantly liberated from ferritin (Fig. 3). In order to further probe the involvement of reduced sulfur species in this reaction, another enzyme producing persul¢de by a di¡erent mechanism was studied. In mammals, rhodanese is a mitochondrial enzyme not belonging to the family of PLP-dependent transferases. It catalyzes sulfur transfer between thiosulfate and a thiophilic anion. A dithiol can play this role, but cyanide is most often used as the reaction may be of importance in cyanide detoxi¢cation [38]. No cofactors are required. The combination of thiosulfate and bovine liver rhodanese, or thiosulfate alone, only displayed marginal iron release from horse spleen ferritin (Fig. 4). When glutathione is added to the reaction, some iron was mobilized from the protein. The rate of iron release is the same as that with the same amount
Fig. 4. Iron release from horse spleen ferritin in the presence of rhodanese. 160 nM ferritin (200 WM Fe) was treated with 50 mM sodium thiosulfate (squares), 50 mM sodium thiosulfate and 10 Wg bovine liver rhodanese (up-triangles), or 50 mM sodium thiosulfate, 10 Wg bovine liver rhodanese, and 16 mM glutathione (circles). For comparison, the release of iron with 16 mM glutathione is also shown (down-triangles).
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by reacting genuine sul¢de with the protein (Figs. 1 and 2). 3.4. E¡ect of enzymatically produced sul¢de on holoferritin
Fig. 5. Iron release from horse spleen ferritin in the presence of sul¢te reductase. 160 nM ferritin (200 WM Fe) was treated with 1 mM NaHSO3 (squares) and 1.5 mM NADPH (up-triangles). Iron release from ferritin was only observed (circles) when 4.5 Wg of E. coli sul¢te reductase was added. The amount of sul¢de produced by the enzyme (down-triangles) under the same conditions is also shown.
(16 mM) of glutathione alone over the ¢rst few minutes, but, whereas the glutathione e¡ect rapidly levels o¡, the presence of the enzyme ensures a linear release of iron over the next 2 h (Fig. 4). Therefore, in much the same way as NifS, rhodanese can contribute to the depletion of iron from ferritin only when a thiol cleaving the enzyme-bound persul¢de is present. In both cases, it is proposed that the resulting persul¢de carrier disproportionates (step 3 below) into a disul¢de compound (cystine and oxidized glutathione in the present cases) and hydrosul¢de, according to the following sequence of events: EnzSH substrateSH ! EnzSSH
step 1
EnzSSH 2RSH ! EnzSH RSSSR
step 2
RSSSR ! RSSR H2 S
step 3
where EnzSH is the catalytic cysteinyl thiol of the enzyme, the italicized sulfur atom is that provided by the substrate, and RSH is the externally added thiol that may be identical to the substrate of the enzyme. Under the anaerobic conditions prevailing in our assays, the resulting sul¢de would be responsible for removing iron from ferritin, as shown above
The need for a sulfur carrier molecule in the above two examples to observe chelation of iron originating from ferritin has been superseded by the use of yet another enzyme able to generate sul¢de as a direct product of its reaction. Sul¢te reductase is one of the few enzymes catalyzing a six-electron reduction by use of a speci¢c metal-based active site made of a reduced porphyrin, a siroheme, coupled to a [4Fe^ 4S] cluster. The enzyme occurs in plants and several other cells but not in animals. The assimilatory enzyme from E. coli is a polymer of two subunits, in which subunit K contains FMN and FAD and subunit L contains the metal-based sul¢te binding and reducing site. Subunit K conveys electrons from NADPH to subunit L and the reaction catalyzed by the E. coli enzyme directly produces sul¢de, without release of partially reduced sulfur moieties [39]. The substrates of E. coli sul¢te reductase (NADPH and sul¢te) alone or in combination did not expel iron from ferritin (Fig. 5). When the enzyme was added to the reaction a nearly linear production of the BPS ferrous chelate (after an initial short lag) was observed for more than 2 h (Fig. 5). Over this period, a large amount (ca. 15%) of the iron initially entrapped in ferritin was mobilized. Also, omission of sul¢te in the assay did not sustain continuous iron release from ferritin in contrast to the complete system. Sul¢te reductase can indeed act as a £avin reductase through its £avoprotein component (K subunit) [22], but reduction of other compounds, including iron-containing ones [40], by this and other £avoenzymes requires free £avins as mediators. Based on the independently measured activities of the enzyme, 2.3 Wmol NADPH oxidized/min/Wg and 0.8 Wmol S23 produced/min/Wg, it was calculated from Fig. 5 that slightly more than 2 mol sul¢de was needed to release 1 mol of (ferrous) iron from ferritin under these conditions. This ratio can be compared with the value of about 6 S23 /Fe extrapolated from the data of Fig. 3, in agreement with the poor ability of the products of the NifS reaction in the presence of cysteine to deplete iron from ferritin.
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4. Discussion The comparative data of Table 1 indicate that, whereas sul¢de is far less e¤cient than reduced £avins to mobilize iron from mammalian ferritins, it reacts more rapidly and with higher yields than physiologically abundant reductants such as glutathione, NADPH, or cysteine. Despite the wealth of structural and kinetic data available, the detailed mechanism of iron release from ferritin remains somewhat obscure. The most frequently evoked features of e¤cient iron releasing agents are a low reduction potential and easy access to the iron core [1]. The large reducing power and relatively small size of sul¢de meet these criteria. Previous evidence showed that sul¢de reaches the iron oxide surface of the ferritin core at low or high pH [26,27]; from our experiments at pH 7.4, sul¢de interacts also at neutral pH with the ferritin core, where it may reduce ferric ions and help solubilize ferrous salts. The reduction potential of the S³/HS3 couple can be estimated at 3280 mV at pH 7 and sul¢de may thus also provide electrons to a variety of accepting sites at the ferritin surface to trigger long range electron transfer through the protein shell [11,41]. Therefore, no mechanistic reasons appear to contradict the possible involvement of sul¢de in the release of ferritin iron. However, no egress of iron from ferritin was observed with sul¢de in previous studies [26,27], probably because no e¤cient external iron acceptors, such as the chelators used here, were provided. Iron loading of the ferritins was carried out in the absence of phosphate in the present study and the release of iron from mixed hydroxide-phosphate cores may quantitatively di¡er from the reported results [42,43]. Nevertheless, no signi¢cant qualitative di¡erences are expected. Many parameters seem to a¡ect the rates of iron egress from ferritin [17], but, in contrast to iron uptake, no clear-cut di¡erences between the reactivity of each subunit exist since homogeneous recombinant human H-ferritin and horse spleen, mainly L-, ferritin display rates of the same order of magnitude with sul¢de (Table 1). These observations are in line with the proposed mechanism of ferritin iron exit implementing localized unfolding of the protein at the junction of three undi¡erentiated subunits [44]; in the present experiments, such
unfolding may be facilitated by the chelators added to the ferritin solutions. Although the involvement of intact ferritin as iron supplier in animal cells remains to be clari¢ed [6], there might be circumstances under which intracellular leakage of the metal occurs. Such conditions, with emphasis on oxidative, mainly superoxide, stress, have been implicated in a variety of cellular damages, but the signi¢cance of iron release under oxidative conditions is still far from clear and contradicts the documented cytoprotective role of ferritin [31^33,45]. In contrast to the above scenario, a role for iron release from ferritin triggered by sul¢de, i.e. under reducing conditions, may be considered. The physiological sources of sul¢de are several. Among the enzymes used in this work with mammalian ferritins, only sul¢te reductase is not present in animal cells. Rhodanese occurs in mitochondria [38]. Eukaryotic homologues of nifS have been found and their products appear to be directed to several subcellular compartments, including the cytosol and mitochondria [46]. The subcellular localization of these enzymes may not impede their contribution to the release of iron from cytoplasmic ferritin: the data in Figs. 3 and 4 show that an intermediate (per)sulfur carrier allowing a further reducing step is required, and such a sulfur transport agent may be able to easily cross membranes. In addition, other enzymes distinct from, albeit homologous to, NifS that generate persul¢de have been described (e.g. [47,48]). Furthermore, enzymes of sulfur-amino acids metabolism, such as O-acetylserine sulfhydrylases, catalyzing the last step of cysteine biosynthesis, L-cystathionase, or cystathionine Q-lyase, involved in the synthesis of cystathionine, share the ability to desulfhydrate cysteine [49,50]. All these enzymes should therefore be able to trigger iron release from ferritin, as long as the sulfur moiety they generate can be mobilized, i.e. reduced to sul¢de, by accessory components. Intracellular glutathione is abundant, with estimates around 5 mM, under normal conditions, but a steady state concentration of sul¢de 500-fold smaller (10 WM) than that of the tripeptide is as e¤cient to release iron from human H-ferritin (Table 1). With horse spleen ferritin and assuming an intracellular ribo£avin concentration of about 0.1 WM, 10 WM sul¢de would produce as much iron from ferritin as
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the fully reduced vitamin. From these rough calculations, it can be concluded that sul¢de may be a valid alternative to previous candidates to quantitatively mobilize intracellular iron from ferritin (Fig. 1). Mammalian tissues [50,51] and other eukaryotic cells [52,53] do produce sul¢de, up to 160 WM in the brain [54]. From the data reported herein, such concentrations can clearly interfere with the iron £uxes in and out of ferritin [55]. Moreover a synergic action of sul¢de with nitrogen monoxide in smooth muscle relaxation has been proposed [54], and nitrogen monoxide may modulate iron homeostasis [2]. Therefore, the metabolic or signaling schemes relating several intracellular redox mediators [53,56] may now be considered with the possible involvement of increased iron availability triggered by sul¢de as demonstrated here. Acknowledgements We thank Dr. Sonia Levi (DIBIT, Milan, Italy), Prof. Dennis R. Dean (Virginia Tech, Blacksburg, VA, USA), Drs. Mahel Zeghouf and Jacques Cove©s (CEA/Grenoble, France) for providing us with some of the biological materials used in this work. References [1] P.M. Harrison, P. Arosio, The ferritins: molecular properties, iron storage function and cellular regulation, Biochim. Biophys. Acta 1275 (1996) 161^203. [2] M.W. Hentze, L.C. Ku«hn, Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide and oxidative stress, Proc. Natl. Acad. Sci. USA 93 (1996) 8175^8182. [3] Y. Tsuji, H. Ayaki, S.P. Whitman, C.S. Morrow, S.V. Torti, F.M. Torti, Coordinate transcriptional and translational regulation of ferritin in response to oxidative stress, Mol. Cell. Biol. 20 (2000) 5818^5827. [4] J.-F. Briat, S. Lobre¨aux, N. Grignon, G. Vansuyt, Regulation of plant ferritin synthesis: how and why, Cell. Mol. Life Sci. 56 (1999) 155^166. [5] J. Wei, E.C. Theil, Identi¢cation and characterization of the iron regulatory element in the ferritin gene of a plant (Soybean), J. Biol. Chem. 275 (2000) 17488^17493. [6] P. Ponka, C. Beaumont, D.R. Richardson, Function and regulation of transferrin and ferritin, Semin. Hematol. 35 (1998) 35^54.
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[23] D.M. Greenberg, Metabolism of Sulfur Compounds, 3rd edn., Academic Press, New York, 1975. [24] H. Beinert, A tribute to sulfur, Eur. J. Biochem. 267 (2000) 5657^5664. [25] J. Imsande, Iron, sulfur, and chlorophyll de¢ciencies: a need for an integrative approach in plant physiology, Physiol. Plant 103 (1998) 139^144. [26] T. Douglas, D.P.E. Dickson, S. Betteridge, J. Charnok, C.D. Garner, S. Mann, Synthesis and structure of an iron(III) sul¢de-ferritin bioinorganic nanocomposite, Science 269 (1995) 54^57. [27] T.G. St.Pierre, W. Chua-anusorn, P. Sipos, I. Kron, J. Webb, Reaction of hydrogen sul¢de with native horse spleen ferritin, Inorg. Chem. 32 (1993) 4480^4482. [28] S. Sirivech, E. Frieden, S. Osaki, The release of iron from horse spleen ferritin by reduced £avins, Biochem. J. 143 (1974) 311^315. [29] F. Funk, J.-P. Lenders, R.R. Crichton, W. Schneider, Reductive mobilisation of ferritin iron, Eur. J. Biochem. 152 (1985) 167^172. [30] J.M. McCord, Iron, free radicals, and oxidative injury, Semin. Hematol. 35 (1998) 5^12. [31] B.J. Bolann, R.J. Ulvik, On the limited ability of superoxide to release iron from ferritin, Eur. J. Biochem. 193 (1990) 899^904. [32] L.R. Harris, M.H. Cake, D.J. Macey, Iron release from ferritin and its sensitivity to superoxide ions di¡ers among vertebrates, Biochem. J. 301 (1994) 385^389. [33] T. Paul, E¡ect of prolonged superoxide £ux on transferrin and ferritin, Arch. Biochem. Biophys. 382 (2000) 253^261. [34] D.W. Reif, R.D. Simmons, Nitric oxide mediates iron release from ferritin, Arch. Biochem. Biophys. 283 (1990) 537^541. [35] J.-P. Laulhe©re, M. Fontecave, Nitric oxide does not promote iron release from ferritin, BioMetals 9 (1996) 10^14. [36] F.W. Alexander, E. Sandmeier, P.K. Mehta, P. Christen, Evolutionary relationships among pyridoxal-5P-phosphate dependent enzymes, Eur. J. Biochem. 219 (1994) 953^960. [37] M.R. Jacobson, V.L. Cash, M.C. Weiss, N.F. Laird, W.E. Newton, D.R. Dean, Biochemical and genetic analysis of the nifUSVWZM cluster from Azotobacter vinelandii, Mol. Gen. Genet. 219 (1989) 49^57. [38] J. Westley, Thiosulfate: cyanide sulfurtransferase (Rhodanese), Methods Enzymol. 77 (1981) 285^291. [39] B.R. Crane, E.D. Getzo¡, The relationship between structure and function for the sul¢te reductases, Curr. Opin. Struct. Biol. 6 (1996) 744^756. [40] J. Cove©s, M. Fontecave, Reduction and mobilization of iron by a NAD(P)H:£avin oxidoreductase from Escherichia coli, Eur. J. Biochem. 211 (1993) 635^641. [41] J.L. Johnson, D.C. Norcross, P. Arosio, R.B. Frankel, G.D. Watt, Redox reactivity of animal apoferritins and apohetero-
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Sul¢de is an e¤cient iron releasing agent for mammalian ferritins Sylvie Cassanelli, Jean-Marc Moulis * CEA, De¨partement de Biologie Mole¨culaire et Structurale, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France Received 11 December 2000; received in revised form 6 March 2001; accepted 8 March 2001
Abstract The most prominent role of mammalian ferritins is to provide an extensive iron-buffering capacity to cells. The large ferritin iron stores can be mobilized in vitro, but the functional relevance of the most efficient iron releasing agents remains elusive. Sulfide is a strongly reducing chemical generated by a series of enzymes. In the presence of limited amounts of sulfide a continuous rate of iron release from ferritin was observed and a majority of the protein iron core was recovered in solution. The rate constants for iron efflux triggered by several reducing or chelating compounds have been measured and compared. Although not as efficient as reduced flavins, sulfide displayed kinetic parameters which suggest a potential physiological role for the chalcogenide in converting the iron storage protein into apoferritin. To further probe the relevance of sulfide in the mobilization of iron, several enzymes, such as NifS, rhodanese, or sulfite reductase generating reduced forms of sulfur by different mechanisms, have been assayed for their ability to catalyze the release of iron from ferritin. The results show that full reduction of sulfur into sulfide is needed to deplete iron from ferritin. These reactions suggest links between sulfur metabolism and intracellular iron homeostasis. ß 2001 Elsevier Science B.V. All rights reserved. Keywords: Sulfur metabolism; Iron homeostasis; Sulfane; Desulfurase; Ferritin
1. Introduction Ferritins are crucial elements of iron homeostasis in most organisms. They have been evidenced in animals, plants and bacteria for instance, but with somewhat di¡erent properties in these various cases. However, ferritins from di¡erent sources share the capacity to store very large amounts of iron (up to about 4500 atoms/molecule) as iron oxides accumu-
Abbreviations: BPS, bathophenantroline; Fz, ferrozine ; PLP, pyridoxal 5P-phosphate * Corresponding author. Fax: +33-438785872; E-mail: [email protected]
lated in the large internal cavity of the protein with varying amounts of phosphate anions. Ferritin subunits are homologous and are assembled in heteropolymers of 24 polypeptide chains [1]. Mammalian ferritins are made up of two kinds (H and L) of subunits and are generally localized in the cytoplasm. The elongated, mainly K-helical, peptides organize into a highly symmetric structure generating the large hollow in which iron is stored. This internal shell can communicate with the exterior of the protein through di¡erent channels corresponding to the main symmetry axes of the molecules. In particular, the 3-fold intersubunit channels appear to interact with the ferrooxidase centers located on H-subunits in a key step of the ferritin iron loading process. Mammalian ferritin biosynthesis is mainly regulated at the translational level by the binding of iron reg-
0167-4838 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 0 1 ) 0 0 1 8 2 - 0
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ulatory proteins to the iron responsive elements lying on the 5P non-coding sequence of the mRNA. Binding and repression of translation occur when iron is scarce or under several other stress conditions [2]. Transcriptional regulation also occurs [3]. In contrast, the plant ferritin genes are organized di¡erently from the mammalian ones and their expression is regulated at the transcriptional level [4,5]. In aerobic organisms ferritins scavenge iron exceeding the normal needs of the cell. Under conditions of oxidative stress, iron catalyzes partial reduction of oxygen and may lead to cell damage by hydroxyl radicals and other products. In metazoans, these deleterious e¡ects are avoided by inactivation of the iron regulatory proteins which detect the increased iron concentration, hence dissociating from the iron responsive element on the 5P non-coding sequence of the mRNA and increasing ferritin translation. However, once stored in ferritin, the fate of the generally large amounts of ferric ions in the protein is not clear [6]. Ferritin may be oxidatively modi¢ed and proteolysed by the proteasome [7], or it may be transported to lysosomes to be degraded, with the metal recycling back to the cytosol [8,9]. Ferritin might also be a dead-end trap for iron, much as metallothioneins seem to be for copper in some instances [10]. Last, iron exit from ferritin, which has been largely demonstrated in vitro, may be used in the synthesis of iron-containing proteins or as an alternative to extracellular uptake for replenishing the intracellular iron pool. The release of iron from ferritin is triggered by reductants and chelators and some chemicals may play both roles [1]. Reductants revert the mineralization process and chelators displace the equilibrium toward ferrihydrite solubilization. These reactants may gain access to the internal cavity through the protein channels, but direct interaction may not be mandatory and long range electron transfer from the protein surface to the inner core has been proposed [11]. Since the signi¢cance of many compounds shown to release iron from ferritin in vitro remains elusive, a survey of metabolites able to carry out the reaction is of interest. We have thus evaluated the ability of reduced species generated by the metabolism of sulfur-containing molecules to expel iron out from mammalian ferritins.
175
2. Materials and methods In all experiments reported herein except iron loading of ferritins, strictly anaerobic conditions were implemented by use of argon lines or of an anaerobic chamber (Jacomex, Livry-Gargan, France) ¢lled with puri¢ed argon [12]. 2.1. Proteins Horse spleen ferritins (apo- and holoforms) were obtained from Sigma-Aldrich Co. Recombinant human H-ferritin was kindly provided by Dr. Sonia Levi (DIBIT, Milan, Italy) as frozen ammonium sulfate precipitate with 2 M urea [13] and further puri¢ed by anion-exchange chromatography (DE-52, diethylaminoethyl cellulose, Whatman). The apoferritins were iron-loaded as described [14] and the holoferritins were puri¢ed by gel ¢ltration in 20 mM Tris^HCl pH 7.4. No signi¢cant reactivity di¡erences were observed between native and in vitro ironloaded horse spleen ferritins. The eluted ferritins were analyzed for their iron [15] and protein [16] contents and they were used in iron release experiments without allowing excessive ageing. The lack of signi¢cant iron leakage from these preparations induced by iron chelators alone (see Section 3) indicates that most of the iron added to apoferritins was incorporated into the iron core of the protein. 2.2. Iron release from ferritins Since the reported rates for iron mobilization from ferritin depend somewhat on the metal loading of the protein [17], experiments are reported separately for each ferritin preparation. The reaction volume was 300 Wl in a cuvette of 0.1 cm optical path. Beside the compound used to trigger iron release, the reaction was carried out in 20 mM Tris^HCl pH 7.4 at room temperature and it contained 0.15^1 WM ferritin (0.2^0.5 mM Fe) and excess bathophenantroline (BPS) or ferrozine (Fz) as iron chelators. The formation of ferrous complexes was followed at 535 nm, O = 22 140 M31 cm31 , with BPS, or at 562 nm, O = 27 900 M31 cm31 , with Fz. Hewlett-Packard 8452 or 8453 spectrophotometers were generally used, except for some measurements with nicotin-
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amide-adenine nucleotides which were performed with a Kontron 941 spectrophotometer equipped with a monochromator. Indeed, NADH and NADPH were unstable under the ultraviolet component of the polychromatic incident light of reversed-optics systems and their products had some ability (maybe via the generation of radicals) to release iron out of ferritin as measured by the accumulation of ferrous chelates as a function of the irradiation time (not shown). It was checked that no iron was expelled from ferritin by NADH and NADPH when the reaction was followed with the conventional Kontron 941 spectrophotometer at the visible wavelength corresponding to the absorbance maximum of the ferrous chelates. Alternatively, measurements were carried out with reverseoptics spectrophotometers connected through ¢ber optics not allowing light of wavelengths below 350 nm to pass through. Known concentrations of FMN, FAD, or ribo£avin were fully reduced by a 6-fold excess of NADPH in the presence of Escherichia coli ferredoxin (£avodoxin)-NADP oxidoreductase. The latter was obtained as follows. From the published sequence of the fpr gene [18], the oligonucleotides Fpr5: 5P-CAAAACAGGAGAAACATATGGCTGATTGGG-3P and Fpr3: 5P-ATCACCGTTTGCTGCAGGCATATCGCGGG-3P were used to amplify the gene from E. coli K12 DNA. The fragment was digested with NdeI and PstI, cloned into pT7-7, and the gene was overexpressed in E. coli K38/pGP12 as described [19]. The very fast reactions between reduced £avins and ferritins were followed for extensive periods, but only the linear part of the data over the few ¢rst seconds of the reactions was used to calculate rate constants.
of Drs. Mahel Zeghouf and Jacques Cove©s (CEA/ Grenoble, France). 3. Results 3.1. Release of ferritin iron by sul¢de The features generally shared by reactants previously shown to release iron from ferritin are their powerful reducing capacity or their metal chelating properties [1]. Although often considered as a toxic component, sul¢de is involved in several metabolic pathways [23^25]. It is a strong reductant, but no reports on the reaction of this relatively common cellular reductant on iron-loaded ferritin seem available. Under acidic (pH 5.4) or alkaline (pH 8.9) conditions, exchange between oxygen and sul¢de in the coordination sphere of the core iron has been described [26,27]. Fig. 1 shows that sul¢de actually expelled the metal from horse spleen ferritin, which mainly contains the L-ferritin subunit, in the presence of iron chelators. Recombinant human H-ferri-
2.3. Enzymes involved in sulfur metabolism Overexpression of Azotobacter vinelandii nifS in E. coli K38/pGP1-2 was carried out using plasmid pDB551 [20] kindly supplied by Prof. Dennis R. Dean (Virginia Tech, Blacksburg, VA, USA). The catalytic conversion of L-cysteine into alanine by 0.16 mg of soluble extracts was quantitated by the formation of methylene blue in the presence of sul¢de [21]. Bovine liver rhodanese was obtained from Sigma. Puri¢ed E. coli sul¢te reductase [22] was a gift
Fig. 1. Iron release from horse spleen ferritin by sodium sul¢de. 185 nM ferritin (415 WM Fe) was treated with increasing concentrations of Na2 S in 20 mM Tris^Cl, pH 7.4 and the formation of BPS ferrous chelate was monitored at 535 nm. Sul¢de concentrations were 0.1 (circles), 0.25 (squares), 0.5 (up-triangles), 1 (down-triangles), 1.5 (hexagons), and 2 mM (diamonds).
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tin homopolymers also reacted with sul¢de under pseudo ¢rst-order kinetics (Fig. 2). Iron release was monitored by the absorbance of ferrous chelates in these experiments: Fz and BPS gave similar results and the rates of iron release due to chelators alone were far smaller, of the order of 1x, than those measured in the presence of sul¢de. About half of the iron core was removed from human H-ferritin in 90 min with a single initial addition of 0.5 mM Na2 S. The reaction of sul¢de with ferritin has thus the ability to trigger the exchange of signi¢cant amounts of iron from the large intracellular store. It was checked that the reaction rate is linear in the 0.15^1 WM ferritin range. 3.2. Comparison of iron release from ferritin with di¡erent reductants Second-order rate constants for iron release from ferritin induced by selected compounds of potential biological signi¢cance were determined (Table 1). The reactions with £avin derivatives were very fast, as previously shown [17,28,29], with rate constants larger by approximately two orders of magnitude than that for sul¢de. However, the latter exceeded those of physiologically relevant thiols, such as Lcysteine or glutathione (Table 1). Other thiols have also been studied for comparison (Table 1). Superoxide is often considered as a mediator of iron release by ferritin, e.g. [30], but the actual rates are di¤cult to measure and remain controversial [31^ 33]. When superoxide was anaerobically generated by dissolving KO2 in our assays, no metal complex with BPS built up from horse spleen ferritin. Similar
Fig. 2. Dependence of the observed rate constants for iron release from ferritin with the sodium sul¢de concentration. The Na2 S data used to derive the values of Table 1 (columns 1 and 3) are plotted for horse spleen ferritin (circles) and human recombinant H-ferritin (squares).
to superoxide, initial reports [34] of iron release from ferritin by nitrogen monoxide have been questioned [35]. 3.3. E¡ect of enzymatically produced sulfane sulfur on holoferritin Sul¢de is very easily oxidized under aerobic conditions. However, the chalcogenide is generated as part of metabolism [23^25] and the possible contribution of several enzymes producing sul¢de has been assessed in the removal of iron from ferritin.
Table 1 Second-order rate constants (M31 min31 ) for iron release from mammalian ferritins HoSFr (2250 Fe/ferritin) FMNH2 Ribo£avin FADH2 Na2 S L-Cysteine BPS NADPH O3 2
HoSFr (1200 Fe/ferritin) 26 000 11 400 3 000 100 10 6 0.2 0 0
Na2 S glutathione thioglycollate
HuH-Fr (600 Fe/ferritin)
680 6 10 6 10
Na2 S glutathione
175 0.3
Each column presents results obtained with the same preparation of ferritin. Rate constants were determined from data as those presented in Fig. 2, for several ferritin concentrations in the 0.1^0.75 WM range. HoSFr is iron-loaded horse spleen ferritin and HuH-Fr is recombinant human H-subunit homopolymer.
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Fig. 3. Iron release from horse spleen ferritin in the presence of NifS. 160 nM ferritin (200 WM Fe) was treated with either 1 mM L-cysteine (open squares) or the E. coli extract containing NifS (open circles). In another experiment with the same protein, L-cysteine was added by successive 0.25 mM increments (arrows) to the mixture of ferritin and NifS (¢lled triangles). The reaction without ferritin produced bound persul¢de that was assayed as sul¢de (open diamonds, right scale).
A subset of widespread pyridoxal 5P-phosphate (PLP)-dependent enzymes, including cysteine synthases, cystathionine L-lyase and the protein encoded by nifS [36], share the ability to desulfhydrate L-cysteine. The nifS gene was initially identi¢ed in a nitrogen ¢xation operon of A. vinelandii and it is believed to participate in the biogenesis of the nitrogenase enzyme [37]. The enzyme transforms L-cysteine into L-alanine and an enzyme-bound persul¢de: the latter can be released from the enzyme by reduction of the disul¢de bond, with dithiothreitol for example [20]. Fig. 3 shows that addition of NifS to horse spleen ferritin does not sustain iron release at a high rate after an initial burst. This increase in soluble iron can be attributed to endogenous compounds able to expel iron from ferritin. Addition of the NifS substrate, L-cysteine, increases the rate. However, cysteine may have di¡erent roles in the reaction: it is the substrate providing the sulfur atom binding to the active site cysteine, but non-reacted free cysteine can also release this sulfur atom by reaction with enzyme-bound persul¢de. In addition, cysteine alone is able to release iron out of ferritin (Table 1), albeit at a very low rate (Fig. 3). However, the combination of cys-
teine and NifS has always been found more e¤cient than cysteine alone to remove iron out of ferritin. These data suggest that the persul¢de sulfur bound to NifS does not play a direct role in the mobilization of ferritin iron, but, once the enzyme-bound sulfur is displaced, by excess cysteine in the present case, signi¢cant iron amounts are concomitantly liberated from ferritin (Fig. 3). In order to further probe the involvement of reduced sulfur species in this reaction, another enzyme producing persul¢de by a di¡erent mechanism was studied. In mammals, rhodanese is a mitochondrial enzyme not belonging to the family of PLP-dependent transferases. It catalyzes sulfur transfer between thiosulfate and a thiophilic anion. A dithiol can play this role, but cyanide is most often used as the reaction may be of importance in cyanide detoxi¢cation [38]. No cofactors are required. The combination of thiosulfate and bovine liver rhodanese, or thiosulfate alone, only displayed marginal iron release from horse spleen ferritin (Fig. 4). When glutathione is added to the reaction, some iron was mobilized from the protein. The rate of iron release is the same as that with the same amount
Fig. 4. Iron release from horse spleen ferritin in the presence of rhodanese. 160 nM ferritin (200 WM Fe) was treated with 50 mM sodium thiosulfate (squares), 50 mM sodium thiosulfate and 10 Wg bovine liver rhodanese (up-triangles), or 50 mM sodium thiosulfate, 10 Wg bovine liver rhodanese, and 16 mM glutathione (circles). For comparison, the release of iron with 16 mM glutathione is also shown (down-triangles).
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by reacting genuine sul¢de with the protein (Figs. 1 and 2). 3.4. E¡ect of enzymatically produced sul¢de on holoferritin
Fig. 5. Iron release from horse spleen ferritin in the presence of sul¢te reductase. 160 nM ferritin (200 WM Fe) was treated with 1 mM NaHSO3 (squares) and 1.5 mM NADPH (up-triangles). Iron release from ferritin was only observed (circles) when 4.5 Wg of E. coli sul¢te reductase was added. The amount of sul¢de produced by the enzyme (down-triangles) under the same conditions is also shown.
(16 mM) of glutathione alone over the ¢rst few minutes, but, whereas the glutathione e¡ect rapidly levels o¡, the presence of the enzyme ensures a linear release of iron over the next 2 h (Fig. 4). Therefore, in much the same way as NifS, rhodanese can contribute to the depletion of iron from ferritin only when a thiol cleaving the enzyme-bound persul¢de is present. In both cases, it is proposed that the resulting persul¢de carrier disproportionates (step 3 below) into a disul¢de compound (cystine and oxidized glutathione in the present cases) and hydrosul¢de, according to the following sequence of events: EnzSH substrateSH ! EnzSSH
step 1
EnzSSH 2RSH ! EnzSH RSSSR
step 2
RSSSR ! RSSR H2 S
step 3
where EnzSH is the catalytic cysteinyl thiol of the enzyme, the italicized sulfur atom is that provided by the substrate, and RSH is the externally added thiol that may be identical to the substrate of the enzyme. Under the anaerobic conditions prevailing in our assays, the resulting sul¢de would be responsible for removing iron from ferritin, as shown above
The need for a sulfur carrier molecule in the above two examples to observe chelation of iron originating from ferritin has been superseded by the use of yet another enzyme able to generate sul¢de as a direct product of its reaction. Sul¢te reductase is one of the few enzymes catalyzing a six-electron reduction by use of a speci¢c metal-based active site made of a reduced porphyrin, a siroheme, coupled to a [4Fe^ 4S] cluster. The enzyme occurs in plants and several other cells but not in animals. The assimilatory enzyme from E. coli is a polymer of two subunits, in which subunit K contains FMN and FAD and subunit L contains the metal-based sul¢te binding and reducing site. Subunit K conveys electrons from NADPH to subunit L and the reaction catalyzed by the E. coli enzyme directly produces sul¢de, without release of partially reduced sulfur moieties [39]. The substrates of E. coli sul¢te reductase (NADPH and sul¢te) alone or in combination did not expel iron from ferritin (Fig. 5). When the enzyme was added to the reaction a nearly linear production of the BPS ferrous chelate (after an initial short lag) was observed for more than 2 h (Fig. 5). Over this period, a large amount (ca. 15%) of the iron initially entrapped in ferritin was mobilized. Also, omission of sul¢te in the assay did not sustain continuous iron release from ferritin in contrast to the complete system. Sul¢te reductase can indeed act as a £avin reductase through its £avoprotein component (K subunit) [22], but reduction of other compounds, including iron-containing ones [40], by this and other £avoenzymes requires free £avins as mediators. Based on the independently measured activities of the enzyme, 2.3 Wmol NADPH oxidized/min/Wg and 0.8 Wmol S23 produced/min/Wg, it was calculated from Fig. 5 that slightly more than 2 mol sul¢de was needed to release 1 mol of (ferrous) iron from ferritin under these conditions. This ratio can be compared with the value of about 6 S23 /Fe extrapolated from the data of Fig. 3, in agreement with the poor ability of the products of the NifS reaction in the presence of cysteine to deplete iron from ferritin.
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4. Discussion The comparative data of Table 1 indicate that, whereas sul¢de is far less e¤cient than reduced £avins to mobilize iron from mammalian ferritins, it reacts more rapidly and with higher yields than physiologically abundant reductants such as glutathione, NADPH, or cysteine. Despite the wealth of structural and kinetic data available, the detailed mechanism of iron release from ferritin remains somewhat obscure. The most frequently evoked features of e¤cient iron releasing agents are a low reduction potential and easy access to the iron core [1]. The large reducing power and relatively small size of sul¢de meet these criteria. Previous evidence showed that sul¢de reaches the iron oxide surface of the ferritin core at low or high pH [26,27]; from our experiments at pH 7.4, sul¢de interacts also at neutral pH with the ferritin core, where it may reduce ferric ions and help solubilize ferrous salts. The reduction potential of the S³/HS3 couple can be estimated at 3280 mV at pH 7 and sul¢de may thus also provide electrons to a variety of accepting sites at the ferritin surface to trigger long range electron transfer through the protein shell [11,41]. Therefore, no mechanistic reasons appear to contradict the possible involvement of sul¢de in the release of ferritin iron. However, no egress of iron from ferritin was observed with sul¢de in previous studies [26,27], probably because no e¤cient external iron acceptors, such as the chelators used here, were provided. Iron loading of the ferritins was carried out in the absence of phosphate in the present study and the release of iron from mixed hydroxide-phosphate cores may quantitatively di¡er from the reported results [42,43]. Nevertheless, no signi¢cant qualitative di¡erences are expected. Many parameters seem to a¡ect the rates of iron egress from ferritin [17], but, in contrast to iron uptake, no clear-cut di¡erences between the reactivity of each subunit exist since homogeneous recombinant human H-ferritin and horse spleen, mainly L-, ferritin display rates of the same order of magnitude with sul¢de (Table 1). These observations are in line with the proposed mechanism of ferritin iron exit implementing localized unfolding of the protein at the junction of three undi¡erentiated subunits [44]; in the present experiments, such
unfolding may be facilitated by the chelators added to the ferritin solutions. Although the involvement of intact ferritin as iron supplier in animal cells remains to be clari¢ed [6], there might be circumstances under which intracellular leakage of the metal occurs. Such conditions, with emphasis on oxidative, mainly superoxide, stress, have been implicated in a variety of cellular damages, but the signi¢cance of iron release under oxidative conditions is still far from clear and contradicts the documented cytoprotective role of ferritin [31^33,45]. In contrast to the above scenario, a role for iron release from ferritin triggered by sul¢de, i.e. under reducing conditions, may be considered. The physiological sources of sul¢de are several. Among the enzymes used in this work with mammalian ferritins, only sul¢te reductase is not present in animal cells. Rhodanese occurs in mitochondria [38]. Eukaryotic homologues of nifS have been found and their products appear to be directed to several subcellular compartments, including the cytosol and mitochondria [46]. The subcellular localization of these enzymes may not impede their contribution to the release of iron from cytoplasmic ferritin: the data in Figs. 3 and 4 show that an intermediate (per)sulfur carrier allowing a further reducing step is required, and such a sulfur transport agent may be able to easily cross membranes. In addition, other enzymes distinct from, albeit homologous to, NifS that generate persul¢de have been described (e.g. [47,48]). Furthermore, enzymes of sulfur-amino acids metabolism, such as O-acetylserine sulfhydrylases, catalyzing the last step of cysteine biosynthesis, L-cystathionase, or cystathionine Q-lyase, involved in the synthesis of cystathionine, share the ability to desulfhydrate cysteine [49,50]. All these enzymes should therefore be able to trigger iron release from ferritin, as long as the sulfur moiety they generate can be mobilized, i.e. reduced to sul¢de, by accessory components. Intracellular glutathione is abundant, with estimates around 5 mM, under normal conditions, but a steady state concentration of sul¢de 500-fold smaller (10 WM) than that of the tripeptide is as e¤cient to release iron from human H-ferritin (Table 1). With horse spleen ferritin and assuming an intracellular ribo£avin concentration of about 0.1 WM, 10 WM sul¢de would produce as much iron from ferritin as
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the fully reduced vitamin. From these rough calculations, it can be concluded that sul¢de may be a valid alternative to previous candidates to quantitatively mobilize intracellular iron from ferritin (Fig. 1). Mammalian tissues [50,51] and other eukaryotic cells [52,53] do produce sul¢de, up to 160 WM in the brain [54]. From the data reported herein, such concentrations can clearly interfere with the iron £uxes in and out of ferritin [55]. Moreover a synergic action of sul¢de with nitrogen monoxide in smooth muscle relaxation has been proposed [54], and nitrogen monoxide may modulate iron homeostasis [2]. Therefore, the metabolic or signaling schemes relating several intracellular redox mediators [53,56] may now be considered with the possible involvement of increased iron availability triggered by sul¢de as demonstrated here. Acknowledgements We thank Dr. Sonia Levi (DIBIT, Milan, Italy), Prof. Dennis R. Dean (Virginia Tech, Blacksburg, VA, USA), Drs. Mahel Zeghouf and Jacques Cove©s (CEA/Grenoble, France) for providing us with some of the biological materials used in this work. References [1] P.M. Harrison, P. Arosio, The ferritins: molecular properties, iron storage function and cellular regulation, Biochim. Biophys. Acta 1275 (1996) 161^203. [2] M.W. Hentze, L.C. Ku«hn, Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide and oxidative stress, Proc. Natl. Acad. Sci. USA 93 (1996) 8175^8182. [3] Y. Tsuji, H. Ayaki, S.P. Whitman, C.S. Morrow, S.V. Torti, F.M. Torti, Coordinate transcriptional and translational regulation of ferritin in response to oxidative stress, Mol. Cell. Biol. 20 (2000) 5818^5827. [4] J.-F. Briat, S. Lobre¨aux, N. Grignon, G. Vansuyt, Regulation of plant ferritin synthesis: how and why, Cell. Mol. Life Sci. 56 (1999) 155^166. [5] J. Wei, E.C. Theil, Identi¢cation and characterization of the iron regulatory element in the ferritin gene of a plant (Soybean), J. Biol. Chem. 275 (2000) 17488^17493. [6] P. Ponka, C. Beaumont, D.R. Richardson, Function and regulation of transferrin and ferritin, Semin. Hematol. 35 (1998) 35^54.
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