251

Biochimica et Biophysica Acta, 541 (1978) 251--262 © Elsevier/North-Holland Biomedical Press

BBA 28565

STUDIES ON THE UTILIZATION OF F E R R I T I N IRON IN THE F E R R O C H E L A T A S E REACTION OF ISOLATED RAT LIVER MITOCHONDRIA

R. ULVIK and I. ROMSLO

Laboratory of Clinical Biochemistry, N-5016 Haukeland sykehus, University of Bergen, Bergen (Norway) (Received October 31st, 1977)

Summary The utilization of ferritin as a source of iron for the ferrochelatase reaction has been studied in isolated rat liver mitochondria. 1. It was found t h a t isolated rat liver mitochondria utilized ferritin as a source of iron for the ferrochelatase reaction in the presence of succinate plus FMN (or FAD). 2. Under optimal experimental conditions, i.e., approx. 50 pmol/1 FMN, 37°C, pH 7.4 and 0.5 mmol/1 Fe(III) (as ferritin iron), the release process, as shown by the formation of deuteroheme, a m o u n t e d to approx. 0.5 nmol iron/min per mg protein. 3. The release process could not be elicited by ultrasonically treated mitochondria, lysosomes, microsomes or cytosol, i.e., the release of iron from ferritin was due to mitochondria and was a function of the in situ orientation of the mitochondrial inner membrane. 4. The release of iron from ferritin by the mitochondria might be of relevance not only for the in situ synthesis of heme in the hepatocyte, but also with respect to the mechanism(s) by means of which iron is mobilized for transport to the erythroid tissue.

Introduction The pathways between the uptake of iron at the cell surface and its subsequent incorporation into protoporphyrin within the mitochondria are as y e t poorly understood. Major areas of concern are whether or n o t transferrin penetrates to the cytosol [1--3], how the iron ions move from the plasma memAbbreviation: HEPES, N-2-hyd~oxyethylpiperazine-Nt-2-ethanesulfonic acid.

252 brane to the mitochondria, and what is the nature of the cytoplasmic iron carrier(s) [1--9]. It has been shown that mitochondria from different tissues and species accumulate iron from iron(III)-sucrose as well as from transferrin by an energydependent process [10--12], and that the iron ions thus accumulated are recovered mainly in the inner membrane and the matrix space [13]. Iron(III)-sucrose is not a physiological donor of iron to the mitochondria [14]. To what extent the same applies to transferrin is a matter of dispute [1--3,5--9,15,16]. Thus, although most investigators favour the assumption that transferrin penetrates to the cytosol, definite p r o o f that transferrin delivers its iron directly to the mitochondria in situ is lacking. As shown by studies on isolated mitochondria, however, the uptake of iron from transferrin reveals some feature which make transferrin a less likely mitochondrial iron donor: the process has a pH optimum at 6.2, and the a m o u n t of iron accumulated is less than 2 pmol/min per mg protein [11,12], i.e., the rate of accumulation is significantly below that reported for the rate of heme synthesis [17]. Furthermore, recent electronmicroscopic studies have shown that transferrin entrapped within the cytosol does n o t adhere to the mitochondria [2]. Barnes et al. [18] studied the ability of ferritin to donate iron to the ferrochelatase, using rat liver mitochondria as the enzyme source and NADH as the electron donor. In this system iron ions were not mobilized from ferritin. More recently Sirivech et al. [19] have shown that ferritin-iron can be rapidly and quantitatively reduced and liberated as Fe(II) by reduced flavins. This observation suggested to us that by draining reducing equivalents from the mitochondria, ferritin may function as an iron source to ferrochelatase in the presence of excess flavins. The present study deals with experiments using ferritin as the source of iron for the ferrochelatase of whole rat liver mitochondria. Evidence is presented that in the presence of FMN (or FAD) plus succinate, rat liver mitochondria mobilize ferritin iron and utilize it for heme synthesis. The release process can n o t be elicited by lysosomes, microsomes or cytosol. A preliminary account of certain aspects of this work has already appeared [20]. Materials and Methods

Preparation of subcellular organelles Rat liver mitochondria were prepared as previously described [21]. Lysosomes, microsomes and cytosol were prepared according to Slinde and Flatmark [22], and taken up in 0.25 tool/1 sucrose, 10 m m o l / 1 H E P E S buffer, pH 7.40 at a concentration of approx. 20 mg protein/ml. The purity of the subcellular fractions was tested by measuring the activity of marker enzymes (Table III). The functional integrity of the mitochondria was tested by measuring the respiratory control ratio with ADP using succinate as the substrate. Only mitochondrial with respiratory control ratios with ADP of greater than 4.0 were used. Submitochondrial particles were prepared by sonicating the mitochondrial

253 suspension for three periods of 30 s in a chamber maintained at +4°C, using an MSE 150 W ultrasonic disintegrator, model MK2, operated with a 9.5-mm end diameter probe and an amplitude reading of 18 pm.

Preparation of ferritin Horse spleen ferritin (twice crystallized, cadmium free, from Calbiochem (Luzern, Switzerland)) was dialysed against several changes of 1% (w/v) thioglycollate in 0.25 mol/l acetate buffer, pH 4.9 as described by Crichton [23]. After dialysis, residual iron, thioglycollate and acetate buffer were removed by passing the solution through a Sephadex G-50 column (2.6 × 70 cm), equilibrated and eluted with 50 mmol/1 KC1, 10 mmol/l HEPES buffer, pH 7.40. The apoferritin thus prepared contained less than 30 Fe(III) atoms per ferritin molecule. Ferritin ~as prepared from the apoferritin as described by Harrison et al. [24]. Ferrous ammonium sulphate (Fe(NH4)2(SO~):) in the amount required to give an iron c o n t e n t of approx. 1300 Fe(III) atoms per ferritin molecule was added to a mixture of apoferritin (1 mg protein/ml), 20 mmol/1 KIO3, 80 mmol/1 Na2S:O~ and 10 mmol/1 HEPES buffer, pH 7.40. The rate of uptake of iron by apoferritin was followed by measuring the absorption of the ferric o x y hydroxide micelles at 420 nm [24]. Excess iodate and thiosulphate was removed by chromatography on Sephadex G-50 (see above). The ferritin was analyzed by polyacrylamide gel electrophoresis and stained for iron and protein [25].

Determination of deuteroheme formation The mitochondria, approx. 4 mg protein/ml, were preincubated for 5 min at 37°C in a shaking water-bath in a medium containing, in a volume of 1 ml: 0.25 mol/1 sucrose, 10 mmol/l HEPES buffer, pH 7.40, 10 mmol/1 succinate, 50 gmol/1 FMN and 0.5 mmol/1 Fe(III) (equivalent to approx. 0.4 pmol/1 ferritin protein). Further additions or ommissions were as described in the legends to the tables and figures. To minimize photoreduction of FMN [26], the experiments were carried out in light-shielded incubation chambers. At the time indicated, aliquots of 0.5 ml were withdrawn, and transferred to a tube containing 0.5 ml of pyridine, 0.25 ml of 1 mol/l NaOH and 0.5 ml of water. The mixture was divided equally between t w o cuvettes and 1 mg of solid Na:S204 was added to one cuvette and 0.02 ml of 3 mmol/1 K3Fe(CN)6 to the other. The reduced minus the oxidized spectrum was recorded on a Shimadzu MPS 5000 spectrophotometer, and the a m o u n t of deuteroheme synthesized was determined using the extinction coefficient Ae~,o~-1. ~. c~-1 = 15.3 [27].

Enzymic assays and other analytical procedures Rotenone-insensitive NADPH-cytochrome c oxidoreductase was assayed as described by Sottocasa et al. [28], succinate-phenazine methosulphate-oxidoreductase as described by Arrigone and Singer [29], acid phosphatase as described by Walter and Schiitt [30] and lactate dehydrogenase as described by Keiding et al. [31]. The iron c o n t e n t of the ferritins was determined by atomic absorption s p e c t r o p h o t o m e t r y on aliquots appropriately diluted in double quartz-distilled water, using an IL 453 atomic absorption spectrophotometer. Protein was determined by the Folin-Ciocalteau reagent [32].

254

Chemicals FAD (grade III), FMN (grade I), NADH (grade III from yeast), a-glycerophosphate, fl-hydroxybutyrate, N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES}, phenazone methosulphate, 2,6-dichlorophenol-indophenol (DCIP} and rotenone were obtained from the Sigma Chemical Co. (St. Louis, Mo., U.S.A.). Ferritin (equine spleen, A grade, twice crystallized, cadmium free) was purchased from Calbiochem (Luzern, Switzerland}, pyridine (Uvasol) was from Merck (Darmstadt, G.F.R.} and deuteroporphyrin IX was obtained from Porphyrin Products (Logan, Utah, U.S.A.). The purity of the deuteroporphyrin IX was examined by thin-layer chromatography after esterification [33], and the chromatograms were scanned on a Shimadzu dual wavelength thin-layer chromatography scanner, model CS-900, equipped with a fluorescence accessory. Only preparations in which more than 90% of the fluorescent material was found as a single spot with an Rf value of approx. 0.74, were used. Other chemicals were of the highest purity commercially available. Double quartz-distilled water was used throughout. Results The mobilization of iron from [SgFe]ferritin by isolated mitochondria coul could n o t be studied in experiments similar to those used for the uptake of iron from transferrin or from iron(III)-sucrose [ 11,19], mainly because the binding of ferritin to the mitochondria masked the release o f minor fractions of iron from the ferritin molecule (Ulvik, R., unpublished observations). Instead, to study the release of iron from ferritin by rat liver mitochondria, we measured the synthesis of deuteroheme, using ferritin and deuteroporphyrin as the substrates. Although this is a highly complex reaction, reflecting several possible rate-limiting steps (i,e., binding of ferritin to the mitochondrial membranes, reductive release of iron from ferritin [19,24,26], transport of iron ions and porphyrins to the M-side of the inner membrane [13,34] and chelation of ferrous iron [18]), the formation of deuteroheme nevertheless should unequivocally prove that iron ions have been mobilized from ferritin. A main prerequisite to the conclusions of the present study concerns the specificity of the binding of iron to the apoferritin molecule. To rule out nonspecific binding of iron, the ferritin solution was taken up in 40 mmol/1 EDTA, pH 7.4 and chromatographed on a Sephadex G-50 column (see Materials and Methods). The ferritin solution after chromatography had the same iron : protein ratio as before chromatography, and it was just as effective in promoting the synthesis of deuteroheme as the ferritin solution before chromatography (Fig. 1). Furthermore, when the ferritin solution was examined by polyacrylamide gel electrophoresis, more than 85% of the ferritin was in the monomeric form, and there was no iron band detectable outside the ferritin bands (Fig. 2). Thus, it seems pertinent to conclude that the ferritin solution did not contain any nonspecifically b o u n d iron, and that there was no iron present which was n o t b o u n d to ferritin.

Effect of respiratory substrates, FMN, FAD and NADH When rat liver mitochondria were incubated with ferritin or FeC13 plus

255

A Fig. 1. R e d u c e d m i n u s o x i d i z e d s p e c t r a of py~idine d e u t e r o h e m o c b x o m e . M i t o c h o n d r i a , a p p r o x . 4 m g p r o t e i n ] m l w e r e i n c u b a t e d as d e s c r i b e d in Materials and M e t h o d s s e c t i o n . T h e r e a c t i o n was i n i t i a t e d b y a d d i n g 37 pmol/1 d e u t e r o p o r p h y r i n . F u r t h e r e x p e r i m e n t a l details w e r e as d e s c r i b e d in T a b l e I. A, spect r u m o b t a i n e d w i t h ferritin p r e p a r e d b y t h e s t a n d a r d p r o c e d u r e (see Materials a n d M e t h o d s s e c t i o n ) ; B, s p e c t r u m o b t a i n e d w i t h ferritln a f t e r t r e a t m e n t w i t h 4 0 m m o l ] l E D T A a n d c h r o m a t o g r a p h y ; C, s p e c t r u m o b t a i n e d in t h e a b s e n c e of ferritin. T h e r a t i o i r o n : f e r r i t i n - p r o t e i n w a s (A), 1 3 5 0 : 1; (B), 1 4 1 0 : 1. T h e c o n c e n t r a t i o n of i r o n (A = B) w a s 0 . 5 4 m m o l / l . Fig. 2. P o l y a c r y l a m i d e gel (5%) disc e l e c t r o p h o r e s i s o f f e r r i t i n in g l y c i n e - T r i s b u f f e r ( p H 8 . 5 ) . T h e gels w e r e s t a i n e d for p r o t e i n ( A ) a n d i r o n (B) (see Materials a n d M e t h o d s ) . T h e a n o d e is at t h e b o t t o m .

deuteroporphyrin, we could not detect formation of deuteroheme during 10min incubation (Table I). On the other hand, when the medium was supplemented with succinate, formation of deuteroheme from FeC13 was found of approx. 0.6 nmol/min per mg protein. With ferritin as the iron source, addition of succinate did n o t result in any formation of deuteroheme. However, by supplementing the medium with FMN plus succinate, ferritin was found to release its iron at a rate comparable to that of FeC13. A possible interpretation of these results may be thai whereas FMN is required for the mobilization of iron from ferritin [19], succinate is necessary to accelerate the overall ferrochelatase reaction [35]. If so, it should be possible to replace succinate with pyruvate/malate and have the same overall rate of deuteroheme synthesis [35]. As seen from Table I, however, neither pyruvate/malate nor NADH, alone or in combination, could replace succinate in the present assay system. Note here that NADH depressed the formation of deuteroheme from FeCI~ as well as from ferritin, presumably by competitively trapping the iron ions liberated [36]. ~-Glycerophosphate and ~-hydroxybutyrate behaved essentially as pyruvate/malate (data n o t shown). The rate of formation of deuteroheme increased with increasing concentra-

256 TABLE I E F F E C T OF R E S P I R A T O R Y S U B S T R A T E S , FMN AND N A D H ON T H E SYNTHESIS OF D E U T E R O HEME

M i t o c h o n d r i a , a p p r o x , 4 mg p r o t e i n / m l , were p r e i n c u b a t e d for 5 m i n (see Materials and M e t h o d s ) in the presence o f ferritin (equivalent to 1 4 0 n m o l F e ( n I ) / m g m i t o c h o n d r i a l protein) or FeCI 3 (equivalent to 40 n m o l F e ( I I I ) / m g m i t o c h o n d r i a l protein). Further a d d i t i o n s were 50 p m o l f l F M N , 50 p m o l / l N A D H , 10 m m o l f l succinate, 5 m m o l / l p y r u v a t e and 5 m m o l / l malate as indicated. T h e reaction w a s initiated by adding 3 7 D m o l ] l d e u t e r o p o r p h y r i n . T h e a m o u n t o f d e u t a r o h e m e s y n t h e s i z e d during the f o l l o w i n g 10 min w a s d e t e r m i n e d by the p y r i d i n e d e u t e r o h e m o c h r o m e m e t h o d . T h e results are the m e a n s and the ranges (in parentheses) f r o m five separate e x p e r i m e n t s . Additions

None FMN NADH NADH + FMN Succinate Succinate + FMN Succinate + F M N + N A D H Pyruvate/malate Pyruvate/malate + FMN Pyruvate/malate + NADH Pyruvate/maiate + NADH + FMN

A m o u n t o f d e u t a r o h e m e s y n t h e s i z e d ( n m o l / m g protein) Ferritin

FeCI 3

n.d. * n.d. n.d. n.d. n.d. 5.4 ( 3 . 2 - - 8 . 0 ) 1.7 ( 1 . 1 - - 2 . 6 ) n.d. n.d. n.d, n.d.

n.d. n.d. n.d. n.d. 6.7 ( 5 . 4 - - 8 . 2 ) 6.4 ( 5 . 4 - - 8 . 3 ) 2.4 ( 1 . 2 - - 3 . 7 ) 3.8 ( 1 . 2 - - 6 . 1 ) 2.2 ( 1 . 7 - - 4 . 2 ) 2.1 ( 1 . 4 - - 2 . 6 ) 2.3 ( 0 . 6 - - 4 . 1 )

* n.d., n o t detectable.

tions of FMN, reaching a m a x i m u m level at approx. 50 pmol/1 (Fig. 3). N o t only FMN, but FAD as well was effective in mediating the release of iron from ferritin (Fig. 3). At all levels, however, FMN was more effective than FAD in promoting the synthesis of deuteroheme and, whereas the efficiency of FMN remained fairly constant in the range 5 0 - - 1 0 0 pmol/1, at concentrations of FAD beyond 50 pmol/1 the formation of deuteroheme was slightly decreased.

Effect of ferritin concentration When the formation of deuteroheme was measured at increasing concentrations of ferritin, a saturation level was reached at approx. 0.5 m m o l Fe(III)/1 (Fig. 4). With FeC13 a saturation level was reached at approx. 0.1 mmol/1. The apparent Km obtained for ferritin with respect to iron was 0.07 mmol/l (Fig. 4), and for FeC13 0.01 mmol/1 (Fig. 4 and ref. 35). It has been shown that the rate of release of iron from ferritin depends to a large extent on the size of the ferric o x y hydroxide micelle within the ferritin core [24]. Our results agree with this observation. Thus, when the ratio iron : ferritin protein was varied between 700 and 1 8 0 0 the maximal release of iron was found at a ratio of approx. 1300.

Time course of deuteroheme formation At 37°C the time course of deuteroheme formation, using FeC13 as the iron source, proceeded at a steady-state rate almost instantaneously from the time of mixing of the t w o substrates and the mitochondria (Fig. 5). With ferritin as the iron source, the maximal rate and the linear slope of the progress curve

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was reached after a lag phase of 3--5 min (Fig. 5). Note that the maximal rate of deuteroheme synthesis in the presence of ferritin was close to that obtained in the presence of FeC13, approx. 0.5 nmol/min per mg protein. An important point here concerns the extent to which the mitochondrial preparations contain any heme oxygenase [37]. As shown in Fig. 6, when EDTA was added to the incubation medium the formation of deuteroheme stopped instantaneously, but the amount of deuteroheme synthesized remained constant for at least 45 min. Furthermore, in the absence of EDTA, the

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Fig. 5. T i m e c o u r s e o f t h e d e u t e r o h e m e f o r m a t i o n . M i t o c h o n d r i a 0 a p p r o x . 4 m g p r o t e i n / m l , w e r e i n c u b a t e d in Materials a n d M e t h o d s e x c e p t t h a t t h e r e a c t i o n w a s i n i t i a t e d w i t h 0 . 1 m m o l / l FeCI 3 ( ~ ) o r 0 . 5 m m o l / 1 ferritin i r o n ( o ) . F u r t h e r e x p e r i m e n t a l d e t a i l s w e r e as d e s c r i b e d i n T a b l e I. Fig. 6. S t a b i l i t y o f d e u t e r o h e m e s y n t h e s i z e d . M i t o c h o n d r t a , a p p r o x . 4 m g p r o t e i n / m l 0 w e r e i n c u b a t e d in d u p l i c a t e ( e 0 o ) as d e s c r i b e d in Fig. 5. T h e c o n c e n t r a t i o n o f f e n d t i n i r o n w a s 0 . 5 m m o l / L A t t h e p o i n t i n d i c a t e d , E D T A , final c o n c e n t r a t i o n 5 m m o l f t , w a s a d d e d t o o n e o f t h e i n c u b a t i o n m e d i a ( e ) . T h e a m o u n t o f d e u t e r o h e m e s y n t h e s i z e d w a s d e t e r m i n e d as d e s c r i b e d i n T a b l e L

258 amount of deuteroheme synthesized leveled off at a value equivalent to the amount of deuteroporphyrin added. Thus, the break-down of deuteroheme in the present system is negligible.

Effect of lysosomes, microsomes and cytosol on the formation of deu teroheme by sonicated mitochondria It is known that sonicated mitochondria utilize FeCI~ as the iron source to the ferrochelatase reaction with great avidity [18,35]. On the other hand, sonicated mitochondria did not utilize ferritin as the iron source to the synthesis of deuteroheme (Table II). This could not be ascribed to a detrimental effect on the ferrochelatase of the ultrasonic treatment (see Table II and ref. 34). Possibly, therefore, one or more of the initial step{s) in the release of iron from ferritin depends on the in situ orientation of the mitochondrial inner membrane. Osaki and Sirivech [38] reported on an oxygen-sensitive, allupurinol-insensitive enzyme in beef liver which reductively released iron from ferritin in the presence of NADH and FMN. Ultracentrifugal analysis indicated that the enzyme activity was localized mainly in the soluble cytosol. In rat liver Crichton et al. [26] found the highest ferriductase activity in the microsomal fraction, but with considerable activity also in the lysosomes and the mitochondria. In the present system, in which all the experiments were done in open vessels under continuous stirring, neither lysosomes, microsomes nor cytosol (Tables II and III) were able to mobilize iron from ferritin in a form utilizable for deuteroheme biosynthesis in sonicated mitochondria. It may be argued from the experiments with FeC13 (see Table II) that iron

T A B L E II EFFECT OF LYSOSOMES, MICROSOMES HEME BY SONICATED MITOCHONDRIA

AND CYTOSOL ON THE FORMATION

OF DEUTERO-

Ultrasonically-treated mitochondria or whole mitochondria, approx. 4 mg protein/ml were preincubated for 5 rain (see Materials and Methods) i n t h e p r e s e n c e o f f e r r i t i n o r F e C I 3 , F M N a n d s u c c i n a t e as d e s c r i b e d i n T a b l e I. W h e n i n d i c a t e d , w h o l e m i t o c h o n d r i a lysosomes, m i c r o s o m e s or cytosol were added at concentrations o f a p p r o x . 3 m g p r o t e i n / m l . T h e r e a c t i o n w a s i n i t i a t e d b y a d d i n g 37 ~ m o l / l d e u t e r o p o r p h y r i n . T h e r e s u l t s are t h e m e a n s a n d t h e r a n g e s ( i n p a r e n t h e s e s ) f r o m t h r e e s e p a x a t e e x p e r i m e n t s .

A m o u n t of deuteroheme synthesized (~mol/l) Ferritin + sonicated mitochondria Ferritin + whole mitochond~ia FeCl 3 + sonicated mitochondria Ferritin + sonicated mitochondria + lysosomes Ferritin + sonicated mitochondria + microsomes Ferritin + sonicated mitochond~da + cytosol FeCl 3 + sonicated mitochondria + lysosomes FeCI3 + sonicated mitochondria + microsomes FeC13 + s o n i c a t e d m i t o c h o n d r i a + cytosol Ferritin + sonicated mitochondria + whole mitochondria Ferritin + sonicated mitochondria + whole mitochondria Feritin + sonicated mitochondria + whole mitochondrla Ferritin + sonicated mitochondria + whole mitochondria * n.d., n o t d e t e c t a b l e .

1.8 (1.0--3.6) 13.6 (12.3--15.4) 26.8 (22.3--30.1) n.d. *

0.1

+ lysosomes + microsomes + cytosol

0.9 9.4 6.0 16.1 23.9 17.5 17.4 16.6

(0--0.3)

(0--1.4) (6.0--14.3) (4.1--9.2) (10.3--20.1) (22.1--26.6) (13.5--21.4) (14.8--20,1) (15.2--17.3)

259

T A B L E III RELATIVE SPECIFIC ACTIVITY MICROSOMES AND CYTOSOL

OF

MARKER

ENZYMES

IN M I T O C H O N D R I A ,

LYSOSOMES,

T h e subcellular f r a c t i o n s w e r e o b t a i n e d by d i f f e r e n t i a l c e n t r i f u g a t i o n ( s e e Materials and M e t h o d s ) . T h e relative s p e c i f i c a c t i v i t y o f t h e m ~ r k e r e n z y m e s ( o n a p r o t e i n basis) o f the various f r a c t i o n s w a s c a l c u l a t e d w i t h r e f e r e n c e to t h e s p e c i f i c a c t i v i t y o f t h e m a ~ k e r e n z y m e s o f t h e c~ude h o m o g e n a t e ( s e e Materials and M e t h o d s ) as 1.

Mitochondria Lysosomes Microsomes Cytosol

R o t e n o n e insensitive NADPH-cytochrome c oxidoreductase

Succinate-phenazine methosulfate oxidoreductase

Lactate dehydrogenase

Acid phosphatase

0.15 1.55 3.91 0.45

3.60 0.43 0.16 0.10

0.06 0.26 0.39 1.04

0.30 2.91 0.56 0.57

ions released from ferritin by subcellular fractions other than mitochondria, bind to these fractions and thereby become unavailable for deuteroheme synthesis. As seen from Table II, however, the synthesis of deuteroheme from ferritin in the presence of sonicated mitochondria plus whole mitochondria surpassed that in the presence of whole or sonicated mitochondria alone and, in these experiments, further addition of lysosomes, microsomes or cytosol had only a slight effect on the rate of deuteroheme synthesis. Thus, it seems justifiable to conclude that the release of iron from ferritin is a function mainly of the mitochondria. Discussion

The release of iron from ferritin depends on the relatively free access to the centre of the ferritin molecule for a reductant [24]. Of the many small molecular weight reductants tested, reduced riboflavin, FMNH~, FADH:, dithionite, cysteine, glutathione and ascorbate were the most effective in that order of efficiency [19]. From the high rate at which FMNH: mobilized iron from ferritin, i.e. approx. 4.0 nmol/min at 200 pmol FMNH:/1 [19], it has been suggested [26] that FMNH2 is the most likely reducing agent in tissues to be significant physiologically. Osaki and Sirivech [38] reported on an enzyme for ferritin reduction in liver homogenates of various species of vertebrates. The enzyme which mobilized iron from ferritin only at oxygen concentration ~ 2 ~mol/1, required NADH plus FMN and was localized mainly in the cytosol. Crichton et al. [26] described a ferriductase of rat liver which mobilized iron from ferritin under anaerobic conditions in the absence of NADH and FMN. The maximal activity was found in the microsomal fraction. The results of these workers deviated from those reported in the present study. Thus, in our hands the mobilization of iron could be elicited only by mitochondria (Table II), and it proceeded under conditions n o t strictly anaerobic (see Materials and Methods). These discrepancies, however, are more apparent than real. That is, they could be explained by different approaches used for generation of reductants [26,38], as well as differences in the purity of the subcellular fractions tested (refs. 26, 38 and Table III}.

260 In agreement with the results of Barnes et al. [18] we found that whereas sonicated mitochondria utilized FeCl3 as the source of iron to the ferrochelatase reaction with great avidity (0.73 and 0.68 nmol deuteroheme synthesized per min per mg protein in their system and in our system, respectively) ferritin iron was n o t mobilized. Together with the very marked increase in the rate of deuteroheme synthesis when ferritin was incubated with a mixture of sonicated and whole mitochondria, these results (Table II) suggest that the in situ orientation of the mitochondrial inner membrane is necessary to have iron mobilized from ferritin. It is unlikely that the macromolecular and partly immobilized lipoprotein • flavin complexes inherent to the mitochondrial inner membrane [39] should penetrate to the ferric oxy hydroxide micelle within the ferritin core. Therefore we understand the mobilization of iron to be due to a pool of noncovaJ lently b o u n d FMN. With respect to the mechanism(s} by which the mitochondria supply reducing equivalent to FMN, we have no detailed knowledge. Apparently succinate is the ultimate electron donor. The pathway(s) from succinate to FMN, however, is as y e t n o t known. The progress curves for FMN and FAD are in keeping with the results reported b y Sirivech et al. [19], and with those of Harrison et al. [24] on the ease with which FMNH2 penetrates the inter-subunit channels compared to FADH2. At steady-state reaction rate, the rate of deuteroheme synthesis was essentially the same whether ferric iron was added as FeC13 or as ferritin, at appropriate concentrations (Fig. 5). There was, however, a great difference in the K~-values of the two iron donors (Fig. 4). This is most easily explained by differences in the availability of iron from the two iron donors. Thus, when FeC13 is added, part of the iron is immediately b o u n d to ligands of the mitochondria in a form which makes iron available for deuteroheme synthesis [18, 35]. On the other hand, with ferritin only a minor fraction of the iron is accessible to the reducing substances that have to penetrate the protein shell [40]. This model may explain the characteristic lag period obtained with ferritin (Figs. 5 and 6). Iron delivered to erythroid cells [4], bone marrow cells [4] or liver cells [41] is recovered in part as intracellular ferritin. The controversial subject is, however, whether or n o t ferritin is an essential intermediate in the transfer of iron from the stroma to the mitochondria [1,4,5,7,9]. The results reported here point to an interaction between ferritin and liver mitochondria not previously described. Compared to the uptake of iron from transferrin by isolated rat liver mitochondria [11,12], the characteristics of the ferritin iron release process are more compatible with those which were to be expected of a physiological iron donor c o m p o u n d , quantitatively as well as qualitatively [14,17, 42]. The release of iron from ferritin may be relevant not only to the in situ synthesis of heme in the hepatocytes b u t also to the mechanism(s) by which iron of liver ferritin is mobilized for transport. Trump et al. [43] found that ferritin was taken up by lysosomes and autophagic vacuoles, after which iron was returned to the cell sap for further ferritin synthesis or was extruded into

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