Studies on the mobilization of iron from ferritin by isolated rat liver mitochondria

256

Biochimica et Biophysica Acta, 588 (1979) 256--271 © Elsevier/North-Holland Biomedical Press

BBA 29105

STUDIES ON THE MOBILIZATION OF IRON F R O M F E R R I T I N BY ISOLATED R A T L I V E R M I T O C H O N D R I A

R. ULVIK and I. ROMSLO

Laboratory of Clinical Biochemistry, University of Bergen, N-5016 Haukeland Sykehus, Bergen (Norway) (Received March 5th, 1979)

Key words: Ferritin; Iron mobilization; FMN; (Rat liver mitochondria)

Summary Rat liver mitochondria and rat liver mitoplasts mobilize iron from ferritin by a mechanism which depends on a respiratory substrate (preferentially succinate), a small molecular weight electron mediator (FMN, phenazine methosulphate or methylene blue) and (near) anaerobic conditions. The release process under optimized conditions (approx. 50 gmol/1 FMN, 1 mmolfl succinate, 0.35 mmol/1 Fe(III} (as ferritin iron}, 37°C and pH 7.40) amounts to 0.9--1.2 nmol iron/mg protein per min. The results suggest that ferritin might function as an intermediate in the cytosolic transport of iron to the mitochondria.

Introduction A main problem concerning the role of ferritin in the intracellular metabolism o f iron relates to whether ferritin functions as an obligatory intermediate in the cytosolic transport of iron [1--3], or whether it exists mainly as a passive storage protein for excess iron [4--6]. The answer to this question is intimately connected to a more thorough understanding of the mechanism(s) involved in the in vivo uptake and removal of iron from ferritin, h o w these processes are regulated, and where in the cell they take place. The demonstration of a mechanism b y means of which iron can be rapidly mobilized from ferritin within the cytosol would suggest that ferritin plays an active role in the intracellular iron metabolism. The operation o f such a mechaAbbreviations: DCIP, 2,6-dichlorophenolindophenol; fer~ozine, 3-(2-pyridyl)-5,6-bis(4-phenylsulphonic acid)-11204-triazine; Hepes, N-2-hydroxyethylpiperazine-Nt-2-ethanesulphonic acid; TMPD, N,N,Nt,N ttetramethyl-p -phenylenediamine.

257 nism has been proposed from experiments with reticulocytes and bone marrow where the time progress curves for the appearance of injected S9Fe into ferritin and hemoglobin were determined [ 1,2], and from experiments where iron was removed from ferritin by a series of small molecular weight reductants, among which reduced FMN was the most effective [7]. The functioning of an FMNdependent oxidoreductase in the release of iron from ferritin in situ, is suggested by the observations that in rats, riboflavin depletion reduces the content of iron in the liver [8], and in iron overload, feeding with riboflavin increases the excretion of iron (Jacobs, A., personal communication). The generation of reduced FMN with mobilization of ferritin iron has been accomplished by NADH [9], by NADH(P)H-yielding reactions (alcohol dehydrogenase, glucose6-phosphate dehydrogenase) [10] and by a NADH-requiring enzyme in the soluble cytosol [11]. In fact, any system with the capacity to reduce added flavin exhibits ferrireductase activity [10]. Also, in the absence of FMN and NADH, microsomes mobilize iron from ferritin under anaerobic conditions [12]. More recently, Zaman and Verwilghen [13] have isolated and partially purified a microsomal NAD(P)H-FMN
Preparation of subcellular organelles. Rat liver mitochondria, submitochondrial particles, lysosomes, microsomes and cytosol were prepared as previously described [16]. Mitoplasts were prepared by incubating the mitochondria with digitonin essentially as described by Chan et al. [18]. The purity of the subcellular fractions as judged from the activities of the marker enzymes, were as previously described [16]. The functional integrity of the mitochondria was tested by measuring the respiratory control ratio with ADP using succinate as the substrate. Only mitochondria with respiratory control ratios with ADP greater than 4.0 were used. Release of iron from ferritin. The mobilization of iron from ferritin was

258 assessed by three different approaches: A. Mitochondria (approx. 3 mg protein/ml) were incubated in a Thunberg tube in 2.0 ml medium (0.25 M sucrose, 10 mM Hepes buffer, pH 7.40, 0.35 mM Fe(III) (equivalent to approx. 0.25 pM ferritin), 1 mM succinate and 175 pM bathophenanthroline). Further additions or omissions are described in the legends to the tables and figures. The tube was repeatedly evacuated and refluxed with nitrogen purified b y passing it over BASF catalyst R3-11 heated to 200°C. The release process was initiated by pouring FMN to a final concentration of 50 pM from the short arm of the Thunberg tube into the anaerobic suspension. After 10 min incubation at 37°C, the tube was opened and 0.5 ml in duplicate was rapidly transferred to cuvettes containing 0.5 ml 2% (w/v) Triton X-100 in distilled water. The absorbance was read at 530 nm against appropriate blanks. The a m o u n t of iron liberated was determined using the extinction coefficient for the iron-bathophenanthroline complex: E 22.15 mM -1 • cm -I [19]. When chelators other than bathophenanthroline were used, the extinction coefficients were 22.14 at 530 nm [19], 8.65 at 530 nm [7] and 27.9 at 562 nm [20] for the iron-bathophenanthroline sulphonate-, 2,2'bipyridyl- and ferrozine complex, respectively. B. Mitochondria (approx. 3 mg protein/ml) were incubated in the closed and temperature-controlled (37°C) chamber of the oxygraph (YSI Model 53 Oxygen monitor) as described in method A in the presence of 50 pM FMN. After having reached anaerobiosis, 0.35 mM Fe(III) (equivalent to 0.25 pM ferritin) was added, and at times intervals aliquots of 0.5 ml were withdrawn and the a m o u n t of iron liberated was determined as described in method A. C. Mitochondria (approx. 3 mg protein/ml) were incubated in an open cuvette (37°C) in the complete medium as described in method B. The formation of the iron-bathophenanthroline complex and the consumption of oxygen were determined b y continuously monitoring the change in absorbance AA = A(A530nm--A560nm) and in the oxygen concentration on an Aminco DW 2 UV/VIS s p e c t r o p h o t o m e t e r equipped with a vibrating platinum electrode. When indicated, a gentle flush of oxygen was applied to the surface of the incubation mixture (Fig. 2b). In some experiments (Fig. 5) the formation of the iron-bathophenanthroline complex was measured in a Thunberg cuvette under complete anaerobic conditions. The reaction was initiated b y pouring FMN to a final concentration of 50 pM from the short arm of the cuvette, and the change in absorbance, AA = A ( A 5 3 0 n m - - A 5 6 o n m ) was recorded as described above. Preparation o f ferritin and other analytical procedures. Horse spleen ferritin (twice crystallized, Cd 2÷ free) was obtained from Calbiochem and was fractionated b y centrifugation on a linear gradient o f sucrose (0.15--0.90 M) in 50 mM borate buffer (pH 8.2) using an SW 27 rotor at 103 000 ×gay for 7 h [21]. T w e n t y 2-ml fractions were removed successively from the top of the gradients to give a series o f ferritin fractions of different iron content. After dialysis against distilled water and subsequently against 10 mM Hepes buffer (pH 7.40}, the fractions were analyzed for protein and iron. Reconstituted ferritin was prepared from horse spleen ferritin (same as above) b y reduction with 0.10 M thiogiycollic acid in 0.25 M acetate buffer, pH 4.9, followed b y reloading with ferrous ammonium sulphate (Fe(NH4):(SO4): • 6H:O) to an average o f 1400 atoms of iron per ferritin molecule and

259 chromatography on a Sephadex G-50 column [16]. The ferritins were analyzed by polyacrylamide gel electrophoresis and stained for iron and protein [22]. The gels were scanned on a Beckman CSD100 Computing Densitometer, slit 0.4 × 5 mm, wavelength 600 nm. The formation of deuteroheme was determined by the pyridine deuterohemochrome method [16] on mitochondria incubated as in the iron mobilization experiments (method B) except that bathophenanthroline was replaced by 37 pM deuteroporphyrin. Rotenone-insensitive NADPH-cytochrome c oxidoreductase [ 23], succinatephenazine methosulphate oxidoreductase [24], acid phosphatase [25], lactate dehydrogenase [26] and adenylate kinase [ 27] were assayed as described in the references. The total iron content of the ferritin was determined by atomic absorption spectrophotometry on aliquots appropriately diluted in double quartz~listilled water, using an IL 453 atomic absorption spectrophotometer. Fe 2+ was determined on aliquots of the ferritin solution diluted in a 0.6 M phosphate buffer, pH 1.5, containing 6 mM 2,2'-bipyridyl. The colour was read at 530 nm after 120 min at room temperature. Protein was determined by the Folin-Ciocalteau reagent [28]. Chemicals. Antimycin A~ bathophenanthroline, bathophenanthroline sulphonate, 2,6
In a previous paper is was shown that ferritin functioned as a source of iron to the ferrochelatase of isolated rat liver mitochondria [16]. These experiments, however, did not permit conclusions with respect to the kinetics of the iron mobilization process, because a series of potentially rate-limiting steps were included: the mobilization of iron from ferritin, the transport of iron

260

4Fig. 1. P o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s ( 7 . 5 % ) o f native h o l o f e r r i t i n in 0 . 4 M g l y c i n e / T r i s b u f f e r ( p H 8 . 5 ) . T h e u p p e r gel w a s stained for i r o n , a n d t h e l o w e r gel w a s s t a i n e d for protein. T h e gel s t a i n e d for i r o n , w a s s c a n n e d o n a B e c k m a n C D S - 1 0 0 C o m p u t i n g D e n s i t o m e t e r (see M a t e r i a l s a n d M e t h o d s ) .

(and porphyrin) across the mitochondrial inner membrane [30] and the enzymic formation o f heme [31]. To s t u d y more specifically the process of release of iron from ferritin, and the effect of oxygen on this process, mobilized iron was chelated with bathophenanthroline at known concentration of oxygen. Polyacrylamide gel electrophoresis of the reconstituted ferritin used in our previous studies showed that 1--2% of the iron was superimposed on protein near the electrophoretic origin and outside the mono-, di- and multimeric ferritin bands (Fig. 2 of Ref. 16, densitometric trace not shown). Thus, it could be argued that part of the iron mobilized from the reconstituted ferritin origins from iron heterogeneities. Native holoferritin, however, contains only traces of loosely b o u n d iron [7,32,33] presumably as part of a dynamic equilibrium between peroxo complexes on the catalytic sites within the intersubunit channels and the ferrioxyhydroxides in the interior of the protein [34]. In the present study therefore the experiments have been done with native holoferritin (when not otherwise indicated) {see Materials and Methods). The ferritin thus prepared, of iron content approx. 1400 atoms/molecule apoferritin, revealed precise superimposability of iron and protein on polyacrylamide gel electrophoresis, and the partitition of iron between mono-, di- and multimeric ferritins was 87, 12 and 1% with only traces of iron at the electrophoretic origin (Fig. 1). No Fe 2÷ could be detected.

Effect of oxygen, respiration inhibitors and respiratory substrates on the mobilization o f iron from ferritin The time progress curves for deuteroheme synthesis differed depending on whether FeC13 or ferritin was the source of iron [16]. Thus, with FeC13 steadystate rate was reached almost instantaneously. On the other hand, with ferritin there was a delay of approx. 4 rain before the reaction reached steady-state rate. This delay has been explained by the access of the reductant to only a minor fraction of the iron core o f the ferritin molecule [16] or an inherent lesser chemical reactivity of the ferric iron at the outer surface of the ferritin

261 02--

_

20 0211 Nmol

.

/ b

I~'(A530-A560}=001 l 3 mm

~

Fig. 2. E f f e c t of o x y g e n o n t h e release o f i r o n f r o m f e r r i t i n . M i t o c h o n d r i a , a p p r o x . 3 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 m e t h o d C. (a) C o n t r o l ; (b) w i t h a n t i m y c i n A (2 Dg/ml) a d d e d t o t h e i n c u b a t i o n m i x t u r e a t t h e t i m e o f a n a e r o b i o s i s . O x y g e n w a s g e n t l y f l u s h e d a c r o s s t h e sttrface o f t h e i n c u b a t i o n m i x t u r e f r o m t h e t i m e i n d i c a t e d ( a r r o w ) . T h e i n s e r t s h o w s t h e release o f i r o n f r o m f e r r i t i n w h e n m i t o c h o n d r i a a n d s u c c i n a t e w e r e r e p l a c e d b y d i t b i o n i t e (1 m M ) . T h e c o n c e n t r a t i o n s o f f e r r i t i n a n d of F M N w e r e as d e s c r i b e d ( m e t h o d C).

iron cores together with a hindered access of the reductant to the ferritin core by the protein shell [35]. Alternatively, however, the latency may reflect the time necessary to build up appropriate amounts of FMNH2 for ferritin reduction [9]. This latter hypothesis is supported by the experiments reported in Fig. 2 and Tables I and II. The mobilization of "iron from ferritin did n o t start TABLE I E F F E C T O F R E S P I R A T I O N I N H I B I T O R S O N T H E R E L E A S E OF I R O N F R O M F E R R I T I N M i t o c h o n d r i a , a p p r o x . 3 m g p r o t e i n / m l , w e r e i n c u b a t e d u n d e r a n a e r o b i c c o n d i t i o n s in a m e d i u m o f 2 m l : 0 . 2 5 m o l f l sucrose, 10 m m o l f [ H e p e s b u f f e r , p H 7.0, 1 m m o l f l s u c c i n a t e , 0 . 3 5 m m o l f l F e ( I I I ) (as ferritin i r o n ) a n d 1 7 5 ~ m o l f l b a t h o p e n a n t h r o l i n e . 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 F M N ( 5 0 ~ m o l f l ) a n d resp i r a t i o n i n h i b i t o r f r o m t h e s h o r t a r m o f t h e T h u n b e r g t u b e ( m e t h o d A). T h e results are t h e m e a n s a n d t h e r a n g e s (in p a r e n t h e s e s ) f r o m f o u r d i f f e r e n t e x p e r i m e n t s . T e m p e r a t u r e 3 7 ° C , i n c u b a t i o n 10 rain. Addition

Fe ( I I ) ( b a t h o p h e n a n t h z o l i n e ) 3 (nmol/mg protein)

None 10 m M m a l o n a t e 15 ~M r o t e n o n e 2.0 ~ g / m l a n t i m y c i n A 2 mM KCN

10.1 0.4 10.2 10.0 9.9

(7.8--12.1) (0 - - 0 . 9 ) (7.9--11.9) (7.4--11.9) (7.4--10.8)

262

T A B L E II E F F E C T O F R E S P I R A T O R Y S U B S T R A T E S ON T H E R E L E A S E O F I R O N F R O M F E R R I T I N M i t o c h o n d r i a , a p p r o x . 3 m g p r o t e i n / m l , w e r e i n c u b a t e d under a n a e r o b i c c o n d i t i o n s as d e s c r i b e d in T a b l e I. T h e m i t o c h o n d r i a w e r e P r e i n c u b a t e d w i t h the s u b s t r a t e ( w h e n a d d e d ) b e f o r e t h e r e a c t i o n was s t a r t e d w i t h F M N ( 5 0 btM) a d d e d f r o m t h e s h o r t a r m of t h e T h u n b e r g t u b e . 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 (in p a r e n t h e s e s ) f r o m f o u r d i f f e r e n t e x p e r i m e n t s . T e m p e r a t u r e 3 7 ° C , i n c u b a t i o n 10 rain. Addition

F e ( I I ) Coath o p h e n a n t h r o l i n e ) 3 (nmol/mg protein)

None 1.0 m M 1.5 m M 0.1 m M 2.0 m M 2.0 m M

0.9 10.0 1.5 0.8 0.9 0.8

succinate p y r u v a t e + 1.5 m M m a l a t e NADH c~-glycerophosphate a s c o r b a t e + 10 p M T M P D

( 0 . 4 - - 1.9) (6.9--11.2) (0.8-- 2.2) ( 0 . 5 - - 1.2) ( 0 . 4 - - 1.2) ( 0 . 5 - - 1.1)

until there was no oxygen detectable in the incubation medium, and even after the suspension had become apparently anaerobic, there was a delay of approx. 3--4 rain before the release process reached steady-state rate (Fig. 2a). On the other hand if dihydroflavins were generated by dithionite, steady-state rate was reached within a few seconds (Fig. 2, insert) as reported b y Sirivech et al. [7]. When antimycin A was added to the anaerobic incubation mixture to depress the respiratory activity of the mitochondria, oxygen diffused into the cuvette to approx. 4--5 gM and concomitantly the mobilization of iron gradually leveled o f f (Fig. 2b). When these experiments were run in a closed vessel under totally anaerobic conditions (method A), antimycin A and cyanide had no effects on the release process. Malonate, however, reduced the a m o u n t of iron liberated b y approx. 95% (Table I). We interpret these results to mean that FMN drains reducing equivalents from the respiratory chain on the substrate side of the antimycin A-sensitive site(s), and depending on the concentration of oxygen, reduced FMN is reoxidized either b y ferritin or oxygen. The mobilization of endogenous iron from the mitochondria averaged 0.4 nmol/mg protein per 10 min (Fig. 3). When ferritin was added, the amount of iron released to bathophenanthroline increased significantly, particularly in the presence of FMN plus succinate (Table II and Fig. 3). NADH, a-glycerophosphate and ascorbate (with and without TMPD) had no effect on the a m o u n t of iron mobilized from ferritin compared to that obtained in the absence of exogenous substrate. With pyruvate/malate there was only a slight increase in the mobilization o f iron over that in State 1 (Table II). Thus succinate is the preferred ultimate reductant to the mitochondrial ferrireductase. The succinateFMN-mediated release of iron from ferritin surpassed the consumption of iron b y the ferrochelatase measured b y the pyridine d e u t e r o h e m o c h r o m e m e t h o d approx, t w o fold (Fig. 3). The release rate as calculated from the 10-min mobilization experiments (Fig. 3 and Table I), was approx. 1 nmol Fe~÷/s per 8000 nmol Fe 3÷, or 100--120 atoms Fe 2÷ molecule apoferritin per 10 min. This rate corresponds to that obtained with 1.25 ~M reduced FMN [35].

263 c

E

15

c~ 10

z:

u. 5 c

~5

25

T}ME (rain)

Fig. 3. E f f e c t o f s u c c i n a t e and F M N o n t h e release of i r o n f r o m ferritin and o n the f o r m a t i o n o f d e u t e r o h e i n e . M i t o c h o n d r i a , a p p r o x . 3 m g / m l , w e r e i n c u b a t e d as described in m e t h o d B. e , c o n t r o l ; ~, w i t h o u t F M N ; A w i t h o u t ' ferrltin. In parallell e x p e r i m e n t s b a t h o p h e n a n t h r o l i n e w a s r e p l a c e d b y 37 /~M d e u t e r o p o r p h y r i n IX and the f o r m a t i o n o f d e u t e r o h e m e was d e t e r m i n e d (o). For e x p e r i m e n t a l details, see Materials a n d M e t h o d s .

Effect of mitoplasts, sonicated mitochondria, lysosomes, microsomes and cytosol on the mobilization of iron from ferritin The mobilization of iron from ferritin and the subsequent incorporation into deuteroporphyrin is a function mainly of intact mitochondria [16]. Thus, whereas ferritin iron was not utilized for synthesis of heme in sonicated mito-

0 2 = 0 ~

30m0 021t

B//

I A(A530-A560)= 0,4-0~~

4A

Fig. 4. E f f e c t of w h o l e m i t o c h o n d r i a , m i t o p l a s t s a n d s o n i c a t e d m i t o c h o n d r i a o n the m o b i l i z a t i o n o f i r o n f r o m ferritin. M l t o c h o n d r i a ( A ) , m i t o p l a s t s ( B ) a n d s o n i c a t e d m i t o e h o n d r i a (C), a p p r o x . 3 m g / m l , w e r e i n c u b a t e d as described in m e t h o d C.

264

A

I ~ (A 530 - A560 ): 0.01

3 rain

+

/

Fig. 5. E f f e c t o f w h o l e m i t o c h o n d r i a , l y s o s o m e s , m i c r o s o m e s and e y t o s o l o n the m o b i l i z a t i o n o f iron f r o m f e r r i t i n . M i t o c h o n d r i a ( A ) , l y s o s o m e s ( B ) , m i c r o s o m e (C) a n d c y t o s o l (D), a p p r o x , 3 m g / m l , w e r e i n c u b a t e d in a T h u n b e r g c u v e t t e as d e s c r i b e d in M e t h o d C.

chondria, FeC13 functioned very effectively as a source of iron to the ferrochelatase reaction. These observations are further supported by the results presented in Fig. 4. Sonicated mitochondria mobilized only negligible amounts of iron from ferritin. Whole mitochondria and mitoplasts, however, mobilized iron from ferritin at essentially the same rates, approx. 1.0 nmol iron/mg protein per min (Fig. 4 and Table I). Note here that adenylate kinase activity released to a 13 000 × g supernatant from the mitochondria at the end o f the iron mobilization experiments amounted to approx. 12% of the total activity, and that the mitoplasts contained less than 16% of the adenylate kinase activity of whole mitochondria. Crichton et al. [12] determined ferrireductase activity in subceUular frac-

T A B L E III ABILITY OF BATHOPHENANTHROLINE AND BATHOPHENANTHROLINE SULPHONATE C H E L A T E I R O N M O B I L I Z E D F R O M F E R R I T I N BY M I T O C H O N D R I A A N D M I C R O S O M E S

TO

M i t o c h o n d r i a o r m i c r o s o m e s , a p p r o x . 3 m g P r o t e i n / m l , w e r e i n c u b a t e d u n d e r a n a e r o b i c c o n d i t i o n s as d e s c r i b e d in Table I, e x c e p t that s u c c i n a t e w a s r e p l a c e d b y 1 5 0 ~uM N A D H in the e x p e r i m e n t s w i t h m i c r o s o m e s . The results are the m e a n s and the ranges (in p a r e n t h e s e s ) f r o m f o u r d i f f e r e n t e x p e r i m e n t s . Temperature 37°C, incubation 10 rain.

Iron mobilized (nmol/mg protein)

175/~M bathophenanthroline 1 7 5 ~tM b a t h o p h e n a n t h r o l i n e

sulphonate

Mitochondria

Microsomes

10.1 (8.1--12.1) 0.5 (0.1-- 0.8)

10.9 (8.8--13.1) 5.1 ( 3 . 2 - - 7 . 0 )

265 T A B L E IV ABILITY OF IRON CHELATORS TO CHELATE IRON MOBILIZED FROM FERRITIN ( A ) M i t o e h o n d r i a , a p p r o x . 3 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 T a b l e I. T h e c o n c e n t r a t i o n o f i r o n c h e l a t o r s w a s l S 0 /~M. (B) E x p e r i m e n t s in ( A ) w e r e r e p e a t e d w i t h d i t h i o n i t e (1 raM) a n d c h e l a t o r (1 r a M ) in t h e a b s e n c e o f s u c c i n a t e a n d m i t o c h o n d r i a . 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 (in p a r e n t h e s e s ) f r o m f o u r d i f f e r e n t e x p e r i m e n t s . T e m p e r a t u r e 3 7 ° C , i n c u b a t i o n 10 rain. n m o l iron mobilized

Bathophenanthroline 2,2'-Bipyridyl Ferrozine Bathophenanthroline sulphonate

A

B

67.5 (61.4--70.0) 17.0 (13.8--19.8) 15.1 (8.8--20.0) 2.4 (1.3-- 3.8)

184.7 240.3 171.2 188.3

(150.1--207.3) (180.2--279.8) (140.3--197.5) (144.0--209.1)

tions f r o m ' r a t liver and they found the highest activity in the microsomes. In these experiments no NADH or FMN was added, and anaerobiosis was obtained b y glucose and glucose oxidase. Using FMN as redox mediator and succinate as the electron donor under anaerobic conditions, neither lysosomes, microsomes nor cytosol did mobilize iron from ferritin (Fig. 5). However, if the microsomes were supplemented with FMN plus N A D H under anaerobic conditions, iron was mobilized from ferritin at a rate comparable to that obtained with mitochondria as judged from the formation of iron bathophenanthroline (Table III).

Effect of hydrophobic and hydrophilic iron chelators So far, experiments have been done with bathophenanthroline as the iron chelator. Table IV shows the results obtained with other iron chelators. With dithionite as reductant, the iron chelators were equally effective in binding the iron mobilized from ferritin. With mitochondria, however, the efficiency of the iron chelators differed widely. This could n o t be ascribed to inhibition of the mitochondrial respiration by the chelators [36]. Thus, the ability of the chelators to bind iron apparently parallelled their hydrophobicity. In contrast, the iron mobilized by the NADH-FMN-dependent microsomal ferrireductase, could be chelated b y bathophenanthroline as well as b y bathophenanthroline sulphonate {Table III).

Effect of artificial redox dyes Most studies on release of iron from ferritin in vitro have been made with FMN as redox mediator [7,9--13]. As shown in Table V artificial redox dyes p r o m o t e the mobilization of iron as well. Thus, phenazine methosulphate mobilized iron from ferritin at a rate essentially similar to that of FMN at equimolar concentrations. However, neither DCIP nor TMPD did release iron from ferritin. Indeed, DCIP reduced the a m o u n t of iron mobilized b y phenazine methosulphate. Essentially similar results were obtained with dithionite as the reductant, i.e. neither DCIP nor TMPD did increase the amount of iron liberated b y dithionite (data n o t shown). Taken together with the results of Table I we interpret the results of

266 TABLE V E F F E C T OF R E D O X DYES ON T H E M O B I L I Z A T I O N OF I R O N FROM F E R R I T I N M i t o c h o n d r i a , a p p r o x . 3 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 T a b l e I. 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 t h e r e d o x d y e (all a t final c o n c e n t r a t i o n 50 DM). T h e results are t h e m e a n s a n d the r a n g e s (in p a x e n t h e s e s ) f r o m f o u r d i f f e r e n t e x p e r i m e n t s . T e m p e r a t u r e 3 7 ° C , i n c u b a t i o n 10 rain. Addition

Fe (II) C o a t b o p h e n a n t h r o l i n e ) 3 (nmol/mg protein)

FMN Methylene blue Phenazine methosulphate

10.2 3.8 9.7 1.7 0 7.2

N,N,N',N,-Tetramethyl-p-phenylenediamine 2,6-Dichlorophenolindophenol Phenazine methosulphate + 2,6-dichlorophenolindiphenol

(8.2--11.1) (3.4-- 4.0) (7,3--10.3) ( 1 . 2 - - 2.0) (8.9-- 7.7)

Table V to mean that ferritin gains its reducing equivalents from the respiratory chain at a redox potential on the substrate side of DCIP and on the oxygen side of phenazine methosulphate.

Effect o f p H and temperature on the release of iron from ferritin The mobilization of iron from ferritin has a pH optimum at 7.40 (Fig. 6). The effect of temperature is shown in Fig. 7 (Arrhenius plot). A temperature optimum is found at 37°C, and the Arrhenius plot reveals a discontinuity at approx. 27°C. The energies of activation in the temperature intervals 20--27 and 27--37°C were 100 and 14 kJ/mol, respectively. Mobilization o f iron from native holoferritin and from reconstituted ferritin So far, the experiments have all been done with native holoferritin. As I

I

I

o~ ,0.1 10

o ~

-0.1

5

-0.3

E c 1

65

I

7.5 pH

I

i

l

I

8.5

32

33

34

ix

103

Fig. 6. E f f e c t o f p H o n t h e m o b i l i z a t i o n o f i r o n f r o m ferritin. M i t o c h o n d r i a , a p p r o x . 3 m g p r o t e l 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 m e t h o d B. T h e i n c u b a t i o n p e r i o d w a s 1 0 m i n . Fig. 7. A r r h e n i u s p l o t f o r t h e m o b i l i z a t i o n o f i r o n f r o m ferritin. M i t o c h o n d r i a , a p p r o x . 3 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 m e t h o d B. T h e r e a c t i o n r a t e ( n m o l i r o n / r a i n p e r m g p r o t e i n ) was calcul a t e d f r o m t h e l i n e a r p a r t o f t h e t i m e p r o g r e s s c u r v e ( i n t e r v a l 5 - - 1 0 rain).

267 TABLE VI MOBILIZATION OF IRON FROM NATIVE HOLOFERRITIN AND FROM RECONSTITUTED FERRITIN M i t o c h o n d r i a , a p p r o x . 3 m g / m l , w e r e i n c u b a t e d u n d e r a n a e r o b i c c o n d i t i o n s (as d e s c r i b e d in T a b l e I) in t h e p r e s e n c e o f 1 7 5 / ~ M b a t h o p h e n a n t h r o l i n e a n d n a t i v e h o l o f e ~ i t i n or r e c o n s t i t u t e d f e r r i t i n at a c o n c e n t r a t i o n o f 0 . 3 5 m M F e ( I I I ) . R e d o x m e d i a t o r s (at a c o n c e n t r a t i o n o f 50 DM) a n d r e s p i r a t o r y s u b s t r a t e s (at a c o n c e n t r a t i o n o f 1 r a M ) w e r e a d d e d as i n d i c a t e d . T h e r e a c t i o n w a s s t a r t e d b y a d d i n g e i t h e r r e d o x m e d i a t o r , o r f e r r i t i n (in t h o s e e x p e r i m e n t s w h e r e a r e d o x m e d i a t o r w a s n o t P r e s e n t ) . T h e results are t h e m e a n s a n d t h e r a n g e s (in p a r e n t h e s e s ) f r o m f o u r d i f f e r e n t e x p e r i m e n t s . T e m p e r a t u r e 3 7 ° C , i n c u b a t i o n t i m e 10 rain. Addition

Fe(II)(bathophenanthroline)3 (nmol/mg protein)

FMN FMN + succinate Phenazine methosulphate + suecinate DCIP + succinate Suecinate Ascorbate

N a t i v e h o l o ferritin

R e c o n s t i t u t e d ferritin

1.9 10.0 9.5 0.1 1.0 1.2

1.8 ( 0 . 6 - - 2.1) 10.6 ( 8 . 3 - - 1 4 . 7 ) 15.5 ( 1 3 . 7 - - 1 7 . 4 ) 0.5 ( 0 . 2 - - 0 . 6 ) 1.1 ( 0 . 7 - - 1.7) 1.6 ( 1 . 1 - - 2 . 3 )

( 0 . 6 - - 2.2) (6.3--12.4) (8.0--10.3) (0 - - 0 . 2 ) (0.8-- 1.2) (0.8-- 2.2)

shown in Table VI reconstituted ferritin behaves essentially as native holoferritin, with respect to the effect of FMN, succinate, ascorbate and DCIP. With phenazine methosulphate, however, iron is more easily mobilized from reconstituted ferritin (Table VI). This may be ascribed to the mobilization of excess iron loosely bound within the intersubunit channels [34], or the more rapid mobilization of iron from slight iron heterogeneities of the reconstituted ferritin [33] (see Materials and Methods). Note, however, that under the experimental conditions described in our previous paper [ 1 6 ] , both the rate and the amount of iron mobilized from reconstituted ferritin were as that from native holoferritin (Table VI and Fig. 8).

c o L o.

15

10 ~z o

5 -6 E

5

15

30

TIME ( rnin )

Fig. 8. E f f i c i e n c y o f n a t i v e h o l o f e r r i t i n a n d r e c o n s t i t u t e d f e r r i t l n as a s o u r c e o f i r o n t o t h e f e r r o c h e l a t a s c r e a c t i o n o f w h o l e m i t o c h o n d r i a . M i t o c h o n d r l a , a p p r o x . 3 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 m e t h o d B, e x c e p t t h a t b a t h o p h e n a n t h r o l i n e w a s r e p l a c e d b y 37 szM d e u t e r o p o r p h y r l n I X . F o r m a t i o n o f d e u t e r o h e m e w a s m e a s u r e d w i t h n a t i v e h o l o f e r r i t i n (o) a n d r e c o n s t i t u t e d f e r r l t i n ( e ) ( a t e q u i m o l a r c o n c e n t r a t i o n s w i t h r e s p e c t t o i r o n ) . F o r f u r t h e r e x p e r i m e n t a l details, see Materials a n d M e t h o d s .

268

Discussion The results here reported both confirm and extend our previous observation that ferritin functions as a source of iron to the ferrochelatase of liver mitochondria [16]. The release process which can be elicited only by whole mitochondria or mitoplasts depends on a small molecular weight redox mediator, a respiratory substrate and (near) anaerobic conditions, and it mobilizes iron to the inner membrane at a rate which surpasses the activity of the ferrochelatase approx, two-fold [16]. As to mobilization of nonspecifically bound iron from reconstituted ferritin, neither native holoferritin nor reconstituted ferritin contained any ferrous iron as judged from their reaction with bipyridyl, and the a m o u n t of iron mobilized from native holoferritin and from reconstituted ferritin was essentially the same (i.e. 8--10% of the total amount of ferritin iron, or more than five times the amount of iron not b o u n d to mono-, di- or multimers of the reconstituted and native holoferritin (see Table VI). Furthermore native holoferritin and reconstituted ferritin behaved essentially the same with respect to FMN, respiratory substrates, reducing agents and as a source of iron to the ferrochelatase (see Table VI and Fig. 8). Therefore, the results previously reported [16] cannot be ascribed to mobilization of nonspecifically b o u n d iron, or if so, this iron behaves indistinguishably from iron within the protein. The release process has an absolute requirement for a small molecular weight redox mediator (Fig. 3 and Table V). The efficiency of the reductant depends on its molecular size [37] and ability to penetrate through [38] or within the intersubunit channels of the ferritin molecule [39]. Furthermore, the efficiency of the reductant depends on its ability to undergo cyclic redox reactions [6,12] and to transfer reducing equivalents from the respiratory chain to ferritin (Table V). According to Jones et al. [35] the acidic pI of ferritin (aspartate and glutamate residues in or near the intersubunit channels), may set up an electrostatic hindrance to polyanionic reductants (like FMNH:), which therefore largely become rate determining in the release process. With ferritin as the iron donor, the ferrochelatase activity reaches a maxim u m level at approx. 50 gM FMN [16], or three times the total concentration of FMN found in the rat liver [40]. These figures, however, cannot be directly compared because, firstly the concentration of freely diffusable FMN in the liver cell is not known [35] and secondly, only a minor fraction of FMN added to the mitochondria is reduced (see Results). The much higher efficiency of succinate compared to that of the other respiratory substrates (Table II) indicates a relative substrate specificity with drainage of reducing equivalents at the level of the succinate dehydrogenase. Several findings support this concept: (1) antimycin A and rotenone have no effect on the release process in anaerobic mitochondria (Table I); (2) malonate inhibits the mobilization of iron in totally anaerobic mitochondria (Table I); (3) phenazine methosulphate functions as a redox mediator, DCIP and TMPD do not (Table V), and (4) ascorbate at concentrations similar to those of succinate does not effect significant release of iron (Tables II and VI). However, the slight but still significant effect of NADH-dependent substrates (Table II) does not fit the assumption of a specific succinate-FMN-dependent ferrireductase. Therefore, we believe that the apparent substrate specificity relates more

269 to the capacity of the respiratory chain to generate a high thermodynamic force in that segment of the respiratory chain from where reducing equivalents are drained to ferritin. The effect of antimycin A and of the various redox dyes suggests that the interaction between the mitochondria and ferritin takes place on t h e substrate side of the c y t o c h r o m e b segment of the respiratory chain. This conclusion rests on the assumption that the reductants all penetrate through or within the intersubunit channels of the ferritin molecule [37--39]. According to the molecular weights and chemical conformation this is to be expected (Harrison, P., personal communication). According to Osaki and Sirivech [11] the ferrireductase of soluble cytosol is inhibited by oxygen concentration greater than 2 ~M. In an aqueous solution and at equimolar concentrations of oxygen, the reaction rate for the oxidation of reduced FMN greatly exceeds that of Fe 2÷ [41,42]. This difference becomes even more evident when an Fe 2. chelator is present [43]. Hence the inhibitory effect of oxygen on the release process is most likely primarily due to oxidation of reduced FMN. The critical concentration of oxygen as calculated from Fig. 2 is approx. 4--5 pM. This should be considered a minimum value, measured at the b o t t o m of an open cuvette (see Materials and Methods). From this figure the relative rate of oxidation of reduced FMN b y oxygen compared to that of 0.35 mM ferritin iron (see Materials and Methods) can be estimated 70:1. In aqueous solutions, however, the relative rates are 2200:1 [44]. Apparently therefore, the mitochondrial ferrireductase favours the reduction of ferritin relative to oxygen, be it due either to differences in the mode of interaction between reduced FMN and ferritin iron in aqueous solutions and within the lipophilic mitochondrial membranes [45], or to the less readily reoxidation of reduced FMN in the mitochondrial system. Thus, provided the mitochondria are supplemented with a respiratory substrate, the mitochondrial ferrireductase does n o t depend on strict anaerobiosis. Indeed, the limiting oxygen concentration is close to that found in liver cells in situ [46]. Iron is mobilized from ferritin only by whole mitochondria and mitoplasts (Fig. 4). Ultrasonically treated mitochondria with a high respiratory rate do not mobilize iron from ferritin when FMN is the mediator. This is in agreement with the observation that ferritin does n o t function as an iron source to the ferrochelatase of sonicated mitochondria [16,47]. So far, we have no explanation to this finding. The temperature sensitivity of the release process further substantiates the importance of the lipophilic inner membrane (Fig. 7). Thus, the drop in the activation energy for the iron release process coincides with the well-known phase changes in the phospholipids of the mitochondrial inner membrane [48]. With FMN as the redox mediator under totally anaeronic conditions neither microsomes, lysosomes nor cytosol did mobilize iron from ferritin in the time period studied {Fig. 5). When supplemented with NADH plus FMN, however, microsomes mobilized iron at a rate comparable to that obtained with the mitochondria when succinate was used as the electron d o n o r (Table III). Another difference between the microsomal and the mitochondrial ferrireductase relates to the effect of the iron chelators. Thus, the iron mobilized b y the microsomal ferrireductase is chelated b y h y d r o p h o b i c as well as b y hydrophilic iron chelators. On the other hand, in the mitochondrial system, iron released

270

from ferritin is chelated only by bathophenathroline, the only one of the chelators tested known to be able to penetrate within the inner membrane [36] (Tables III and IV). Future studies aim at more detailed investigations of the iron
11 12

13

14

15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

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