Bioinorganic ChemistryâII

4 Iron Transporting System of Mitochondria TORGEIR FLATMARK Department of Biochemistry, University of Bergen, N-5000 Bergen, Norway INGE ROMSLO

Downloaded by UNIV LAVAL on April 9, 2016 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/ba-1977-0162.ch004

Laboratory of Clinical Biochemistry, University of Bergen, N-5016 Haukeland Sykehus, Norway

Mammalian mitochondria have evolved a transport system to accumulate iron from the environment (cytosol) and are able to utilize Fe(III) of certain synthetic low molecular weight complexes. The process represents a unidirectional transport of Fe(II) across the inner membrane and is driven by energy derived from coupled respiration or ATP hydroly sis and by dissipation of an electric potential gradient of K . The uptake requires reducing equivalents, such as those supplied by the respiratory chain. The iron appears to be tightly bound to protein ligands although not detectable by EPR spectroscopy. The iron can be used for biosynthesis of heme and possibly for iron—sulfur centers within the mito chondria. The chemical nature of the cytosolic iron donor and regulatory mechanisms of the mitochondrial accumula tion of iron are discussed. +

Λ11 l i v i n g organisms r e q u i r e i r o n a n d are f a c e d w i t h the p r o b l e m o f a c c u m u l a t i n g a sufficient a m o u n t o f this m e t a l f r o m the e n v i r o n m e n t . I n m a m m a l s i r o n is t a k e n u p p r e d o m i n a n t l y i n the d u o d e n u m a n d j e j u n u m ( 1 ) a n d is t r a n s p o r t e d to b o d y cells i n e x t r a c e l l u l a r fluid b y the specific i r o n - b i n d i n g p r o t e i n t r a n s f e r r i n (2).

T a k e n u p b y i n d i v i d u a l cells, i r o n

is e i t h e r s t o r e d as f e r r i t i n a n d h e m o s i d e r i n (3) or is u s e d for b i o s y n t h e t i c purposes, n o t a b l y synthesis of h e m e p r o t e i n s (4, 5) a n d n o n - h e m e i r o n proteins

(6).

T h u s , i n the o v e r a l l m e t a b o l i s m o f i r o n i n m a m m a l i a n

o r g a n i s m s s e v e r a l p e r m e a b i l i t y b a r r i e r s exist, a n d a d e t a i l e d u n d e r s t a n d i n g o f the t r a n s p o r t m e c h a n i s m o f the m e t a l across b i o l o g i c a l m e m b r a n e s is essential for a n u n d e r s t a n d i n g of its m e t a b o l i s m i n h i g h e r o r g a n i s m s . So far, h o w e v e r , the m e c h a n i s m b y w h i c h i r o n is t a k e n u p a n d t r a n s f e r r e d across b i o l o g i c a l m e m b r a n e s is p o o r l y u n d e r s t o o d . 78

Raymond; Bioinorganic Chemistry—II Advances in Chemistry; American Chemical Society: Washington, DC, 1977.

4.

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Iron Transporting System

W i t h i n t h e p a s t f e w years, there has b e e n c o n s i d e r a b l e progress i n understanding the role played b y the mitochondria i n the cellular homeostasis o f i r o n .

T h u s , e r y t h r o i d cells d e v o i d o f m i t o c h o n d r i a d o n o t

a c c u m u l a t e i r o n ( 7 , 8), a n d i n h i b i t o r s of t h e m i t o c h o n d r i a l r e s p i r a t o r y c h a i n c o m p l e t e l y i n h i b i t i r o n u p t a k e ( 8 ) a n d h e m e biosynthesis ( 9 ) b y r e t i c u l o c y t e s . F u r t h e r m o r e , t h e e n z y m e ferrochelatase ( p r o t o h e m e f e r r o lyase, E C 4.99.1.1) w h i c h catalyzes t h e i n s e r t i o n o f F e ( I I ) p h y r i n s , appears to b e m a i n l y a m i t o c h o n d r i a l e n z y m e ( J O ,

into por-

11,12,13,14)

c o n f i n e d to t h e i n n e r m e m b r a n e (15, 16, 17). F i n a l l y , t h e i m p o r t a n c e of m i t o c h o n d r i a i n t h e i n t r a c e l l u l a r m e t a b o l i s m o f i r o n is also e v i d e n t f r o m


the f a c t t h a t i n disorders w i t h d e r a n g e d h e m e b i o s y n t h e s i s , t h e m i t o c h o n d r i a a r e h e a v i l y l o a d e d w i t h i r o n (see " M i t o c h o n d r i a l I r o n P o o l , " below).

I t w o u l d therefore b e e x p e c t e d that m i t o c h o n d r i a , of a l l m a m -

m a l i a n cells, s h o u l d b e a b l e t o a c c u m u l a t e i r o n f r o m t h e cytosol.

From

t h e p e r m e a b i l i t y characteristics of t h e m i t o c h o n d r i a l i n n e r m e m b r a n e ( 1 8 ) a s p e c i a l i z e d t r a n s p o r t system analogous to t h a t of t h e other m u l t i v a l e n t cations ( f o r r e v i e w , see R e f . 19 ) m a y b e e x p e c t e d .

T h e relatively

s l o w d e v e l o p m e n t of this field of s t u d y , h o w e v e r , m a i n l y reflects t h e difficulties i n s t u d y i n g t h e c h e m i s t r y of i r o n . Methodological

Problems

I n contrast t o studies o n t h e a c c u m u l a t i o n of C a , S r , M g , L a , 2 +

2 +

2 +

3 +

a n d K ( 19), studies o n the a c c u m u l a t i o n of i r o n b y i s o l a t e d m i t o c h o n d r i a +

h a v e to take i n t o a c c o u n t t h e extreme i n s o l u b i l i t y of F e ( I I I ) at n e u t r a l p H , t h e f o r m a t i o n of i r o n complexes w i t h h i g h s t a b i l i t y constants, a n d t h e r e d o x a c t i v i t y o f i r o n . T h u s , i n o r d e r t o o v e r c o m e these difficulties of w o r k i n g i n t h e p h y s i o l o g i c a l p H r a n g e , i t is necessary t o use a c o m p l e x of i r o n w h i c h is s o l u b l e at n e u t r a l p H . A l t h o u g h there is some e x p e r i m e n t a l e v i d e n c e w h i c h p o i n t s to a b i n d i n g of i r o n ions b y specific c y t o s o l i c p r o t e i n s (see " C y t o s o l i c I r o n Donor," below),

these p r o t e i n s , w i t h t h e e x c e p t i o n of t r a n s f e r r i n , a r e

a v a i l a b l e o n l y i n m i n u t e q u a n t i t i e s , a n d t h e n a t u r e a n d extent of i r o n p r o t e i n i n t e r a c t i o n s are p o o r l y u n d e r s t o o d . T h e r e f o r e , a n u m b e r of n o n p r o t e i n i r o n chelates h a v e b e e n s t u d i e d as p o s s i b l e m o d e l d o n o r

com-

plexes ( T a b l e I ) . B e c a u s e of t h e h i g h s t a b i l i t y constants of, f o r e x a m p l e , the F e ( I I ) / F e ( I I I ) - 8 - h y d r o x y q u i n o l i n e

and F e ( I I I ) - A D P

complexes

(20), these i r o n - c h e l a t e c o m p l e x e s are u n f a v o r a b l e as i r o n donors, a n d i n fact n o e n e r g y - d e p e n d e n t u p t a k e of i r o n has b e e n d e t e c t e d u s i n g these complexes

(21, 23).

W e h a v e f o u n d , h o w e v e r , t h a t t h e f e r r i c c o m p l e x w i t h sucrose is s u i t a b l e f o r this p u r p o s e .

A l t h o u g h t h e c o m p l e x does n o t represent a

h o m o g e n o u s m o l e c u l a r species, i t is stable a n d h i g h l y s o l u b l e i n w a t e r at n e u t r a l p H at a r a t i o of i r o n : s u c r o s e < 1:40 a n d h a s a f a v o r a b l e o v e r -


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BIOINORGANIC

Table I.

CHEMISTRY

II

Iron Complexes Used in the Study of Mitochondrial Iron Accumulation by Heart and Liver Mitochondria Type of Compound

Accumulation

Energydependent

Energyindependent

— — — +

+ -\+ +

References

Nonprotein chelates


Fe(III)-ADP Fe(III)-8-hydroxyquinoline Fe(II)-8-hydroxyquinoline Fe(III)-sucrose°

b

1J> 21 Λ Λ

23,24,27-32

Protein chelates Fe(III)-phosvitin ' Transferrin a

— +

c

d

+ +

e

50 5

3

"Polynuclear Fe(III) complexes. Approximately 7 nmol · 30 sec" · mg of protein" . From avian egg yolk. Partly saturated. See text, h

1

1

c

d e

a l l d i s s o c i a t i o n constant, w h i c h p e r m i t s t h e c o m p l e x

to b e dissociated

b y i n t e r a c t i o n w i t h t h e m i t o c h o n d r i a l m e m b r a n e s ( F i g u r e 1 A ) (23).

A

h i g h c o n c e n t r a t i o n of t h e l i g a n d ( s u c r o s e ) i n t h e i n c u b a t i o n m e d i u m c o n t r i b u t e s t o this s t a b i l i t y . F u r t h e r m o r e , t h e c o m p l e x

is a m p h o t e r i c

(24) a n d s l i g h t l y n e g a t i v e l y c h a r g e d at n e u t r a l p H ; t h e m a i n species has a pZ °c of 5.7. T h e l i g a n d is m e t a b o l i c a l l y i n e r t (25), a n d t h e m i t o c h o n 4

d r i a l i n n e r m e m b r a n e is i m p e r m e a b l e t o t h e c o m p l e x as w e l l as t o t h e l i g a n d (26).

I n spite of s o m e c o m p l i c a t i n g factors r e l a t e d to its u n i q u e

chemistry, the F e ( I I I ) - s u c r o s e

c o m p l e x has b e e n successfully u s e d i n

o u r l a b o r a t o r y t o s t u d y t h e process o f i r o n a c c u m u l a t i o n b y m i t o c h o n d r i a (23,24,

27-32).

Energy-Dependent Isolated Rat Liver

Accumulation

of Iron by

Mitochondria

D e t a i l e d k i n e t i c measurements of t h e i n i t i a l rates of i r o n u p t a k e b y rat l i v e r m i t o c h o n d r i a h a v e b e e n c a r r i e d o u t i n this l a b o r a t o r y d u r i n g the last f e w years u s i n g 27-32).

5 9

Fe(III)-sucrose

as t h e d o n o r c o m p l e x (23, 24,

T h e g e n e r a l features g i v e n i n T a b l e I I s u p p o r t t h e c o n c e p t t h a t

i r o n , l i k e other cations, is a c c u m u l a t e d b y a n e n e r g y - d e p e n d e n t as a n e n e r g y - i n d e p e n d e n t

different t i m e , p H , a n d t e m p e r a t u r e T h e energy-dependent

as w e l l

m e c h a n i s m , a n d these t w o processes

have

dependencies.

u p t a k e , w h i c h b y d e f i n i t i o n is sensitive t o

u n c o u p l e r , is s u p p o r t e d b y e n d o g e n o u s r e s p i r a t i o n a n d l o w c o n c e n t r a -


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tions of A T P , a n d u n c o u p l e r - s e n s i t i v e u p t a k e is also i n c r e a s e d b y a n e l e c t r i c p o t e n t i a l g r a d i e n t of K (see T a b l e I i n R e f . 31). T h e e n e r g i z e d +

u p t a k e is a r a p i d process w i t h s a t u r a t i o n k i n e t i c s . I t is s t i m u l a t e d b y K * and M g

2 +

a n d is i n h i b i t e d b y c o m p l e x i n g agents s u c h as p h o s p h a t e ,

c a r b o x y l a t e s , a n d A D P as w e l l as A T P a t h i g h e r concentrations.

I t is

sensitive t o t h e same cations a n d S H - g r o u p reagents w h i c h i n h i b i t C a


Outer m e m b r a n e

Β

Inner m e m b r a n e

2 +

Matrix

Respiratory chain •

Rotenone Antimycin ι Ferro- \ 'chelatase Primarily conserved

-FedlD-r^FeOl)-

ι

1

Fe(Il)-

Ca *(La ) Uncouplers 2

3+

- -Fe(H)-

f w - Fe(lll) /\ " Non-ferritin storage complex(es)"

Figure 1.

Schematic of binding, oxidation-reduction, and transport of iron by

intact rat liver mitochondria. A. Passive binding of Fe(III)-Ln complex and Fe(III) to ligands (acceptor sites) of the outer membrane ana the outer phase of the inner membrane. Fe(III)—Lm represents a soluble chelate complex of Fe(III) with a sufficiently low stability constant, e.g., polynuclear Fe(III)-sucrose complex(es). B. reduction of membrane-hound Fe(III) to Fe(II) by the respiratory chain at the level of cytochrome clcytochrome a and energydependent active transport of Fe(II) across the inner membrane. See text. This model is based on Refs.

23, 24, 27-32.


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BIOINORGANIC

Table II.

CHEMISTRY

II

Properties of the Iron Accumulation by Isolated Rat Liver Mitochondria Using Fe(III)-Sucrose as Iron Donor" 59

Energydependent

Properties Sensitive to uncoupler Supported by endogenous respiration A T P (low cone.) electric potential gradient of K

b

+ +

+
not yet c h a r a c t e r i z e d (see the t r a n s p o r t of F e ( I I )

Figure I B ) .

10 dalton) 6

A s for the m o l e c u l a r basis of

across the m i t o c h o n d r i a l i n n e r m e m b r a n e ,

little

is k n o w n except that i t has m a n y features i n c o m m o n w i t h that of C a (27, 31).

C o n s i d e r a b l e e v i d e n c e suggests that the t r a n s p o r t of C a

m e d i a t e d b y a c a r r i e r (38), still uncertain.

2 +

have been isolated from

w i t h h i g h affinity, a n d a n a c i d i c

p r o t e i n has b e e n f o u n d to increase the C a bilayers

(39).

Cytosolic

Iron Donor

2 +

is

b u t the c h e m i c a l n a t u r e of this c a r r i e r is

S e v e r a l p r o t e i n factors

chondria w h i c h can b i n d C a

2 +

B e c a u s e of the l o w s o l u b i l i t y of F e ( I I )

2 +

mitoglyco-

c o n d u c t a n c e of l e c i t h i n

and F e ( I I I )

in a medium

c o r r e s p o n d i n g to the c y t o p l a s m of l i v i n g cells, it is extremely i m p r o b a b l e that i o n i z e d i r o n exists i n the cytosol.

T h u s , i r o n is present either i n

m e t a b o l i c a l l y active c e l l c o m p o n e n t s or b o u n d to i n t r a c e l l u l a r l i g a n d s , a n d specific i r o n - b i n d i n g m o l e c u l e s

have evolved

to m a i n t a i n i r o n i n

s o l u b l e f o r m useable b y the c e l l . It has b e e n k n o w n for 40 years that m a m m a l i a n cells store i r o n i n the f o r m of t w o c o m p l e x e s k n o w n as f e r r i t i n a n d h e m o s i d e r i n (3).

Whereas

f e r r i t i n is a w a t e r - s o l u b l e p r o t e i n c o n t a i n i n g a b o u t 2 0 % i r o n b y w e i g h t , h e m o s i d e r i n represents i n s o l u b l e granules i n w h i c h t y p i c a l f e r r i t i n m o l e cules m a y be w h o l l y or p a r t l y r e p l a c e d b y a m o r p h o u s i r o n deposits (40).

electron-dense

U n t i l recently ferritin a n d hemosiderin were consid-

e r e d solely as storage c o m p o u n d s of i r o n w h i c h c o u l d be d r a w n o n w h e n required.

Recent

findings

i n d i c a t e t h a t f e r r i t i n also p a r t i c i p a t e s i n t h e

t r a n s p o r t of i r o n f r o m d e g r a d e d r e d cells i n the r e t i c u l o e n d o t h e l i a l system to the hepatocytes

(41).

O u r k n o w l e d g e of other proteins a n d m o l e c u l e s

i n the c y t o s o l w h i c h b i n d s i r o n as w e l l as the m e c h a n i s m ( s ) the c y t o s o l i c

involved in

transport of i r o n i n m a m m a l i a n cells is as yet l i m i t e d .

V i r t u a l l y n o t h i n g is k n o w n a b o u t the f o r m ( s ) i n w h i c h i r o n is d e l i v e r e d


4.

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to the m i t o c h o n d r i a i n v i v o . A l t h o u g h a N A D H : ( f e r r i t i n - F e ( I I I ) ) o x i n

d o r e d u c t a s e system has b e e n d e m o n s t r a t e d i n l i v e r h o m o g e n a t e s

(42),

its p h y s i o l o g i c a l significance i n the r a p i d c y t o s o l i c t r a n s p o r t of i r o n is s t i l l u n c e r t a i n (43, 44, 45, 46).

I n the m u c o s a l cells, f e r r i t i n m a y f u n c t i o n

as p a r t of a stable i r o n p o o l i n e q u i l i b r i u m w i t h a m o r e l a b i l e p o o l ( 1 ). O t h e r possible donors h a v e therefore b e e n s e a r c h e d for. It has b e e n p r o p o s e d that a l o w m o l e c u l a r w e i g h t of p h o s v i t i n n a t u r e (47)

phosphoprotein

w h i c h b i n d s i r o n w i t h h i g h affinity m a y

i n v o l v e d i n the c y t o s o l i c t r a n s p o r t of i r o n i n l i v e r cells (48, 49),

be

but no

e n e r g i z e d u p t a k e of i r o n has b e e n d e m o n s t r a t e d s u c h as i n i s o l a t e d r a t liver mitochondria using a F e ( I I I ) - p h o s v i t i n from avian egg yolk Downloaded by UNIV LAVAL on April 9, 2016 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/ba-1977-0162.ch004

w t 40,000) as the substrate ( 5 0 ) .

(mol

T h e b i o c h e m i c a l significance of

the

c y t o s o l i c i r o n b i n d i n g p h o s p h o p r o t e i n is therefore s t i l l u n c e r t a i n . I n m a m m a l s , i r o n is t r a n s p o r t e d to cells of the b o d y v i a the e x t r a c e l l u l a r f l u i d t i g h t l y b o u n d to t r a n s f e r r i n as a m o n o - or d i f e r r i c c o m p l e x , and

the m e c h a n i s m of i r o n b i n d i n g a n d release has b e e n s t u d i e d ex-

t e n s i v e l y i n recent years ( f o r r e v i e w , see R e f . 2 ) .

Iron can be released

f r o m t r a n s f e r r i n b y cells s u c h as e r y t h r o i d cells ( b o n e m a r r o w cells a n d r e t i c u l o c y t e s ), b u t the m e c h a n i s m b y w h i c h these cells use i r o n f r o m this c a r r i e r is s t i l l p o o r l y u n d e r s t o o d .

O n e of the hypotheses p r o p o s e d is t h a t

the process of i r o n exchange b e t w e e n t r a n s f e r r i n a n d e r y t h r o p o i e t i c cells i n v o l v e s at least f o u r stages i n c l u d i n g a progressive u p t a k e of t r a n s f e r r i n m o l e c u l e s i n t o the c y t o s o l b y surface endocytosis

(for r e v i e w , see

51 ), a n d at this stage i r o n b e c o m e s a v a i l a b l e to the c e l l .

Ref.

It has

been

p r o p o s e d that m i t o c h o n d r i a take a n active p a r t i n the d i s s o c i a t i o n of t h e iron-transferrin complex

(8, 52),

b u t at least i n v i t r o p a r t l y s a t u r a t e d

t r a n s f e r r i n f u n c t i o n s as a p o o r i r o n d o n o r chondria i n a conventional

for i s o l a t e d rat l i v e r m i t o -

i n c u b a t i o n m e d i u m at n e u t r a l p H

an e n e r g i z e d u p t a k e of o n l y 2 - 3 p m o l · 15 m i n " · m g of p r o t e i n " 1

(53); was

1

m e a s u r e d as c o m p a r e d w i t h 3 0 - 4 0 p m o l · 15 m i n " · m g of p r o t e i n " 1

pH

6.2.

T h e l o w d o n o r a c t i v i t y of t r a n s f e r r i n a r o u n d p H 7.0 as

p a r e d w i t h its p o t e n c y at l o w e r p H p r i m a r i l y reflects the of the i r o n b i n d i n g a b i l i t y of t r a n s f e r r i n (51).

1

at

com-

pH-dependence

T h u s , there is n o c o n c l u -

sive e v i d e n c e to s u p p o r t the recent p r o p o s a l ( 5 2 ) t h a t t r a n s f e r r i n is t h e i m m e d i a t e d o n o r of the m i t o c h o n d r i a l i r o n i n v i v o . A l o w m o l e c u l a r w e i g h t p r o t e i n , different f r o m m e t a l l o t h i o n i n e w h i c h r e v e r s i b l y b i n d s i r o n w i t h h i g h affinity has b e e n i s o l a t e d f r o m r a b b i t r e t i c u l o c y t e c y t o s o l (54, 55, 56).

A l t h o u g h v e r y l i t t l e is yet k n o w n a b o u t

its p h y s i o l o g i c a l p r o p e r t i e s , the m o l e c u l a r w e i g h t is a r o u n d 6000,

and

i r o n appears to b e r e v e r s i b l y b o u n d u n d e r p h y s i o l o g i c a l c o n d i t i o n s . T h i s p r o t e i n m a y be a b l e to m o b i l i z e i r o n f r o m the p l a s m a m e m b r a n e

and

d o n a t e i t for h e m e a n d f e r r i t i n biosynthesis ( 5 6 ) , b u t n o d e f i n i t i v e p h y s i o l o g i c a l role for " s i d e r o c h e l i n " has b e e n e s t a b l i s h e d .


88

BIOINORGANIC C H E M I S T R Y

Cell Differentiation

and Mitochondrial

Iron

II

Metabolism

T h e p a r e n c h y m a t o u s l i v e r cells ( hepatocytes ) h o l d a k e y p o s i t i o n i n the o v e r a l l m e t a b o l i s m of i r o n (57, 58, 59, 60), a n d since f u n c t i o n a l l y i n t a c t l i v e r m i t o c h o n d r i a c a n b e c o n v e n i e n t l y p r e p a r e d at h i g h y i e l d , these m i t o c h o n d r i a h a v e b e e n most extensively s t u d i e d so far. T h e i r o n t r a n s p o r t i n g system d i s c u s s e d a b o v e for l i v e r m i t o c h o n d r i a is present also i n m i t o c h o n d r i a f r o m other tissues a n d a n i m a l species ( T a b l e I I I ) . Q u a n t i t a t i v e l y , e r y t h r o i d cells of the b o n e m a r r o w p l a y the most i m p o r tant role i n the o v e r a l l m e t a b o l i s m of i r o n (61),

a n d i t w a s therefore not

u n e x p e c t e d to find that the e n e r g i z e d u p t a k e of i r o n b y i s o l a t e d r e t i c u l o Downloaded by UNIV LAVAL on April 9, 2016 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/ba-1977-0162.ch004

c y t e m i t o c h o n d r i a exceeds that of m i t o c h o n d r i a i s o l a t e d f r o m , for e x a m p l e , l i v e r , k i d n e y , a n d heart ( T a b l e I V ). T h u s , a r e l a t i o n s h i p appears to exist b e t w e e n the rate a n d extent of h e m e p r o t e i n t u r n o v e r i n m i t o c h o n d r i a i s o l a t e d f r o m different tissues a n d t h e i r e n e r g i z e d i r o n a c c u m u l a t i o n (30).

T h u s , it is e v i d e n t that c e l l u l a r d i f f e r e n t i a t i o n is expressed at the

m i t o c h o n d r i a l l e v e l b y m o d u l a t i o n of the a c t i v i t y of essential f u n c t i o n s r e l a t e d to i r o n transport a n d h e m e biosynthesis. Biochemical Transporting

Significance System of

T h e r o l e of

of the Iron Mitochondria

m i t o c h o n d r i a i n the r e g u l a t i o n of

cytoplasmic

Ca

2 +

a c t i v i t y is w e l l d o c u m e n t e d a n d n o w g e n e r a l l y a c c e p t e d (62, 63, 64, 65, R e c e n t studies h a v e e s t a b l i s h e d i m p o r t a n t f u n c t i o n s also i n i r o n

66).

metabolism.

In mammals, approximately 7 0 %

of

the

body

iron

is

i n v o l v e d i n the transport a n d storage of o x y g e n , a n d p r o t o h e m e I X , the prosthetic group

of

hemoglobin

a n d m y o g l o b i n , represents the

abundant organic iron-containing compound. protoheme

I X no d o u b t

most

T h u s , the biosynthesis of

q u a n t i t a t i v e l y represents the most i m p o r t a n t

p a t h w a y of the i n t r a c e l l u l a r m e t a b o l i s m of i r o n (61).

T h e role p l a y e d

b y m i t o c h o n d r i a is therefore v e r y i m p o r t a n t since the final step i n t h e Table III. The Iron Content and Most Common Forms of Iron in Isolated Rat Liver Mitochondria" nmol · mg of protein'

1

T o t a l iron H e m e proteins ( c y t o c h r o m e s ) I r o n - s u l f u r centers Other non-heme i r o n c

b

3.9 ± 0.9 (n = 12) 1.38 - 1 . 4 0 not d e t e r m i n e d 1.47-1.53

F r o m R e f . 67. D e t e r m i n e d f r o m the r e d u c e d m i n u s o x i d i z e d difference spectra. I r o n w h i c h u n d e r m i l d r e d u c i n g c o n d i t i o n s (ascorbate + A ^ N , i V ' , i V ' - t e t r a m e t h y l p - p h e n y l e n e d i a m i n e ) reacts w i t h b a t h o p h e n a n t h r o l i n e sulfonate i n the presence of the F e ( I I ) i o n o p h o r e X - 5 3 7 A ( H o f f m a n - L a R o c h e Inc., N u t l e y , N . J . ) . a

b

c


4.

F L A T M A R K

Table IV.

AND

ROMSLO


89

Energy-Dependent Accumulation of Iron and Calcium by Mitochondria Isolated from Reticulocytes and Various Organs of R a b b i t a

Mitochondria Reticulocyte Kidney Liver Heart Spleen Downloaded by UNIV LAVAL on April 9, 2016 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/ba-1977-0162.ch004

α

b

c

Ion Accumulated (nmol/mg protein)

Respiratory Control Values

Iron

Calcium

Ratio Fe:Ca

18.3 10.5 8.0 3.6 2.2

20.1 105.9 50.5 174.9 34.6

0.910 0.099 0.158 0.021 0.042

a,b

1.6 3.9 2.7 2.9 1.6

0

F r o m R e f . 29. Endogenous respiration. State 4 r e s p i r a t i o n .

biosynthesis of h e m e , the i n s e r t i o n of F e ( I I )

into protoporphyrin I X ,

appears to be m a i n l y a m i t o c h o n d r i a l e n z y m e (10, 11, 12, 13, 14)

prob

a b l y confined to the i n n e r face of the i n n e r m e m b r a n e (15, 16, 17).

The

conclusions b a s e d o n our i n v i t r o studies are also s u p p o r t e d b y several observations i n v i v o . T h u s , i n reticulocytes the u p t a k e of i r o n b y m i t o c h o n d r i a has b e e n s h o w n to d e p e n d o n m e t a b o l i c energy

(8),

and i n

disorders w i t h d e r a n g e d h e m e biosynthesis, the m i t o c h o n d r i a are h e a v i l y l o a d e d w i t h i r o n (see Mitochondrial

below).

Iron Pool and Its

Regulation

W h e n m a m m a l s ingest m o r e i r o n t h a n t h e y n e e d for

biosynthetic

purposes, this excess is stored i n most tissues, b u t m a i n l y i n l i v e r , s p l e e n , and bone marrow.

A t p h y s i o l o g i c a l levels of i r o n i n the tissues, the

storage f o r m is p r e d o m i n a n t l y f e r r i t i n i n the cytosol, b u t at h i g h e r c o n centrations of i r o n , h e m o s i d e r i n is also f o r m e d same c e l l c o m p a r t m e n t

(19).

the m i t o c h o n d r i a c o n t a i n some n o n - h e m e

i n the

i r o n different f r o m the i r o n -

sulfur proteins of the r e s p i r a t o r y c h a i n (37, 67). is detectable b y E P R spectroscopy (37)

and deposited

I n a d d i t i o n to the cytosolic i r o n deposit, A f r a c t i o n of this i r o n

b y its resonance at a r o u n d g =

4.3

c h a r a c t e r i s t i c of F e ( I I I ) i n the h i g h - s p i n state a n d is accessible to

r e d u c t i o n b y substrates (37)

a n d to r e a c t i o n w i t h i r o n chelators

(67).

T a b l e I I I gives the d i s t r i b u t i o n of i r o n i n l i v e r m i t o c h o n d r i a i n rat. A c c o r d i n g to i r o n a n d h e m e analyses there is m o r e n o n - h e m e t h a n h e m e i r o n , a n d the n o n - h e m e i r o n , w h i c h is not i n v o l v e d i n the e l e c t r o n t r a n s p o r t c h a i n , accounts

for a significant p o o l of the m i t o c h o n d r i a l i r o n .

R e c e n t studies b y H a n s t e i n et a l . (68)

seem to i n d i c a t e t h a t the m a g n i

t u d e of this i r o n p o o l m a y v a r y w i t h the t o t a l i r o n b o d y content. I r o n l o a d i n g of m i t o c h o n d r i a is o b s e r v e d i n s i d e r o b l a s t i c a n e m i a i n b o t h a n i m a l s a n d m a n (for r e v i e w , see R e f . 69).

T h e p r o t o t y p e of this


90

BIOINORGANIC

CHEMISTRY

II

g r o u p of anemias is p y r i d o x i n e deficiency, w h e r e t h e n u m b e r o f sideroblasts i n the b o n e m a r r o w increases

( 7 0 ) , a n d their mitochondria are

heavily loaded w i t h iron. Electron-dense iron particles have been found i n t h e space b e t w e e n the m i t o c h o n d r i a l cristae, a n d t h e m i t o c h o n d r i a a r e morphologically

distorted

a n d swollen.

Neither electron microscopic

(71) n o r i m m u n o l o g i c a l studies (72) s u p p o r t t h e c o n c l u s i o n that these p a r t i c l e s represent f e r r i t i n as o r i g i n a l l y p r o p o s e d (73).

T h e r e is e x p e r i

m e n t a l e v i d e n c e that t h e a c c u m u l a t i o n o f m i t o c h o n d r i a l i r o n results f r o m a n i m p a i r e d h e m e b i o s y n t h e t i c p a t h w a y of, f o r e x a m p l e , the i r o n use ( f o r r e v i e w , see R e f . 6 9 ) , as w e l l as a n i n c r e a s e d i r o n u p t a k e b y e r y t h r o i d cells f r o m i n h i b i t e d h e m e synthesis (74).

Thus, the cytosolic hemin pool


appears to b e a n a t u r a l n e g a t i v e f e e d b a c k r e g u l a t o r of i r o n u p t a k e

(75)

b y i n h i b i t i n g the d i s s o c i a t i o n of i r o n - t r a n s f e r r i n c o m p l e x i n e r y t h r o i d cells (76) as w e l l as the e n e r g y - d e p e n d e n t

iron accumulation

(77).

H o w e v e r , d u r i n g the most a c t i v e phase o f h e m e synthesis i n m a t u r i n g e r y t h r o i d cells, there is a m a r k e d shift i n the l o c a l i z a t i o n o f c e l l u l a r i r o n f r o m a m e m b r a n e b o u n d p o o l to t h e c y t o s o l (78).

M o r e o v e r , as

s h o w n b y Y o d a a n d Israel ( 7 9 ) , m i t o c h o n d r i a i n c u b a t e d i n c e l l sap o r sucrose synthesize e q u i v a l e n t amounts release o n l y a s m a l l a m o u n t

o f h e m e , b u t those i n sucrose

of h e m e to t h e s u r r o u n d i n g m e d i u m .

In

other w o r d s , t h e release o f h e m e f r o m the m i t o c h o n d r i a seems to d e p e n d o n p r o t e i n ( s ) i n the s u s p e n d i n g m e d i u m . T h u s , t h e c y t o s o l appears t o f a c i l i t a t e t h e u p t a k e of i r o n b y the m i t o c h o n d r i a as w e l l as t h e release o f h e m e f r o m the m i t o c h o n d r i a .

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4.

FLATMARK AND ROMSLO


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18. Klingenberg, M., Essays Biochem. (1970) 6, 119. 19. Azzone, G. F., Massari, S., Biochim. Biophys. Acta (1973) 301, 195. 20. Martell, A. E., "Stability Constants of Metal-Ion Complexes," p. 390, The Chemical Society, London, 1964. 21. Cederbaum, A. I., Wainio, W. W., J. Biol. Chem. (1972) 247, 4593. 22. Stickland, Ε. H., Davis, B. C., Biochim. Biophys. Acta (1965) 104, 596. 23. Flatmark, T., Romslo, I.,J.Biol. Chem. (1975) 250, 6433. 24. Romslo, I., Flatmark, T., Biochim. Biophys. Acta (1973) 305, 29. 25. Pappius, H. M., Elliott, K. A. C., Can. J. Biochem. (1956) 34, 1007. 26. Malamed, S., Recknagel, R. O.,J.Biol. Chem. (1959) 234, 3027. 27. Romslo, I., Flatmark, T., Biochim. Biophys. Acta (1973) 325, 38. 28. Ibid. (1974) 347, 160. 29. Romslo, I., Biochim. Biophys. Acta (1974) 357, 34. 30. Romslo, I., FEBS Lett. (1974) 43, 144. 31. Romslo, I., Biochim. Biophys. Acta (1975) 387, 69. 32. Romslo, I., Flatmark, T., Biochim. Biophys. Acta (1975) 387, 80. 33. Neilands, J. B., ADV. CHEM. SER. (1977) 162, 3. 34. Flatmark,T.,J.Biol. Chem. (1967) 242, 2454. 35. Racker, E., Essays Biochem. (1970) 6, 1. 36. Koller, M.-E., Romslo, I., Flatmark, T., Biochim. Biophys. Acta (1976) 449, 480. 37. Flatmark, T., Beinert, H., unpublished data. 38. Carafoli, E., Sottocasa, G., Dyn. Energy-transducing Membr. I.U.B. Symp. 1973 (1974) 455. 39. Carafoli, E., Prestipino, G. F., Ceccarelli, O., Conti, F., "Membrane Pro teins in Transport and Phosphorylation," p. 85, North-Holland, Amster dam, 1974. 40. Jacobs, Α., Worwood, M., "Progress in Hematology," Vol. 10, p. 1, Grune and Stratton, New York, 1975. 41. Siimes, Μ. Α., Dallman, P. R., Br. J. Haematol. (1974) 28, 7. 42. Sirivech, S., Frieden, E., Osaki, S., Biochem. J. (1974) 143, 311. 43. Hoy, T. G., Harrison, P. M., Shabbir, M., Macara, I. G., Biochem. J. (1974) 137, 67. 44. Hoy, T. G., Harrison, P. M., Shabbir, M., Biochem. J. (1974) 139, 603. 45. Primosigh, J. V., Thomas, E. D., J. Clin. Invest. (1968) 47, 1473. 46. Zail, S. S., Charlton, R. W., Torrance, J. O., Bothwell, T. H.,J.Clin. Invest. (1964) 43, 670. 47. Pinna, L. Α., Clari, G., Moret, V., Biochim. Biophys. Acta (1971) 236, 270. 48. Clari, G., Pinna, L. Α., Moret, V., Siliprandi, N., FEBS Lett. (1971) 17, 300. 49. Donella,A.,Pinna, L. Α., Moret, V., FEBS Lett. (1972) 26, 249. 50. Ulvik, R., Romslo, I., unpublished data. 51. Morgan, Ε. H., "Iron in Biochemistry and Medicine," p. 29, Academic, London, 1974. 52. Neuwirt, J., Borova, J. Poňka, P., "Proteins of Iron Storage and Transport in Biochemistry and Medicine," p. 161, North-Holland, Amsterdam, 1975. 53. Ulvik, R., Koller, M. -E., Prante, P. H., Romslo, I., Scand. J. Clin. Lab. Invest. (1976) 36, 539. 54. Bates, G. W., Workman, E. F., Fed. Proc. (1975) 34, 643. 55. Workman, E. F., Bates, G. W., Biochem. Biophys. Res. Commun. (1974) 58, 787. 56. Workman, E. F., Bates, G. W., "Proteins of Iron Storage and Transport in Biochemistry and Medicine," p. 155, North-Holland, Amsterdam, 1975. 57. Levitt, M., Schacter, Β. Α., Zipursky, Α., Israels, L. G., J. Clin. Invest. (1968) 47, 1281. 58. Pollycove, M., Mortimer, R., J. Clin. Invest. (1961) 40, 753. 59. Snyder, A. L., Schmid, R.,J.Lab. Clin. Med. (1965) 65, 817.



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60. Theorell, H., Beznak, M., Bonnichsen, R., Paul, K. -G., Akeson,Å.,Acta Chem. Scand. (1951) 5, 445. 61. Noyes, W. D., Hosain, F., Finch, C. Α.,J.Lab. Clin. Med. (1964) 64, 574. 62. Borle, A. B., Fed. Proc. (1973) 32, 1944. 63. Carafoli, E., Biochimie (1973) 55, 755. 64. Carafoli, E., Tiozzo, R., Lugli, G., Crovetti, F., Kratzing, G., J. Mol. Cell. Cardiol. (1974) 6, 361. 65. Lehninger, A. L., Bioçhem. J. (1970) 119, 129. 66. Spencer, T., Bygrave, F. L., Bioenergetics (1973) 4, 347. 67. Tangerås, Α., Flatmark, T., unpublished data. 68. Hanstein, W. G. ,Sacks, P. V., Muller-Eberhard, U., Biochem. Biophys. Res. Commun. (1975) 67, 1175. 69. Cartwright, G. E., Deiss, Α., Ν. Engl. J. Med. (1975) 292, 185. 70. Deiss, Α., Kurth, D., Cartwright, G. E.,J.Clin. Invest. (1966) 45, 353. 71. Arstila, A. U., Bradford, W. D., Kinney, T. O., Trump, B. F., Am. J. Pathol. (1970) 58, 419. 72. Romslo, I., Flatmark, T., unpublished data. 73. Bessis, M., Breton-Gorius, J., C. R. Acad. Sci. (Paris) (1957) 244, 2846. 74. Poňka, P., Neuwirt, J., N. Engl. J. Med. (1975) 293, 406. 75. Poňka, P., Neuwirt, J., Br. J. Haematol. (1974) 28, 1. 76. Poňka, P., Neuwirt, J., Borová, J., Enzyme (1974) 17, 91. 77. Koller, M.-E., Prante, P. H., Ulvik, R., Romslo, I., Biochem. Biophys. Res. Commun. (1976) 71, 339. 78. Denton, M. J., Delves, H. T., Arnstein, H. R. V., Biochem. Biophys. Res. Commun. (1974) 61, 8. 79. Yoda, B., Israels, L. G., Can. J. Biochem. (1972) 50, 633. RECEIVED July 26, 1976. This work was supported by the Norwegian Research Council for Science and the Humanities (grant No. C.11.14-3) and the Nor wegian Cancer Society.


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