Arrangement of proteins in the mitochondrial inner membrane

Biochimica et Biophysica Acta, 694 (1982) 291-306 Elsevier Biomedical Press

291

BBA 85236

ARRANGEMENT

OF PROTEINS

IN THE MITOCHONDRIAL

INNER MEMBRANE

RODERICK A. CAPALDI Institute of Molecular Biology, University of Oregon, Eugene, OR 97403 (U.S.A.) (Received June 1lth, 1982)

Contents

II.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Lipid composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B, Protein composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

291 291 292

Structural features of the major proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Complex I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Complex II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Complex III . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Cytochrome c oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Cytochrome ¢ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. ATP synthase . . . . . . . . . . . . . . . . . . . . ....... ...............................................

294 294 294 295 296 298 298

III. A model for the arrangement of proteins in the mitochondrial inner membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

299

IV. Arrangement of prosthetic groups in relation to energy coupling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

300

V.

301

Lateral diffusion of proteins and importance for electron transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

VI. Summary and prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

301

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

302

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

302

Introduction 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 has b e e n t h e subject of i n t e n s i v e e x p e r i m e n t a t i o n for m o r e than two decades. Studies have focused on various s t r u c t u r a l f e a t u r e s [1-3], o n the b i o s y n t h e s i s [4,5] as well as o n t h e m e c h a n i s m of e n e r g y t r a n s d u c t i o n of this m e m b r a n e [6-8]. I n this r e v i e w I h a v e c o n c e n t r a t e d o n s t r u c t u r a l features. C o m p o s i t i o n a l data on the inner membrane of bovine heart mitochondria have been tabulated, followed by a 0304-4157/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

d i s c u s s i o n o f the s t r u c t u r e s of t h e m a j o r p r o t e i n c o m p o n e n t s . T h i s i n f o r m a t i o n is t h e n u s e d to derive a three dimensional picture of the mitochondrial inner membrane. Finally, implicat i o n s of r e c e n t s t r u c t u r a l d a t a f o r the m e c h a n i s m o f e n e r g y c o n s e r v a t i o n are discussed. I A . L i p i d composition 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 has b e e n e s t i m a t e d to c o n t a i n b e t w e e n 0 . 3 3 - 0 . 4 4 m g l i p i d / m g p r o t e i n [9-11]. A l m o s t all of this lipid is

292 TABLE I LIPID COMPOSITION OF THE BOVINE MITOCHONDRIAL INNER MEMBRANE

HEART

Phospholipid

% of total phospholipid

Phosphatidylinositol Phosphatidylcholine Phosphatidylethanolamine Cardiolipin Others Refs.

0 47 34 14 15 [9]

0 38- 41 23-26 20-25 8-19 [10]

10 42 34 15 1 [11]

phospholipid (Table I), arranged mainly as a bilayer [12-14], with protein bound to the surface and penetrating through the lipid continuum. A small portion of the lipid may be present in hexagonal or inverted micellar structures [15]. An asymmetry of the lipid bilayer has been clearly demonstrated [ 10-16,17], with phosphatidylethanolamine predominating on the matrix facing (M) side and phosphatidylcholine on the cytoplasmic (C) side. Cardiolipin is also asymmetrically distributed with 75% of this anionic lipid located on the M side

[101. The fatty acid composition of the mitochondrial inner membrane is species, tissue and diet dependent [18,19], but in all cases, there are sufficient amounts of unsaturated fatty acids present to give a highly fluid membrane at physiological temperatures [ 12,13,20].

lB. Protein composition The major function of the mitochondrial inner membrane is in energy transduction and this is reflected in the protein composition of the membrane, as shown in Table II. The electron transfer chain has been found to purify as four multicomponent complexes, an NADH-ubiquinone reductase (or complex I), succinate ubiquinone reductase (or complex II), ubiquinol cytochrome c oxidoreductase (complex III or bc 1 complex) and cytochrome c oxidase (complex IV) [21]. The other major electron transfer components are cytochrome c and ubiquinone. Ranges of estimates for the concentrations of the various electron transfer components in the inner membrane of bovine heart mitochondria are provided in Table II. The concentration of complex I has been estimated from the binding of the inhibitor piericidin [22], from EPR using the center 2 signal [23] and from the titer of antibody made against purified complex [24]. The concentrations of complexes II, III, IV and cytochrome c have all been determined by spectral analysis [9,25-27]. In the case of complex III, antibody titer has also been used [30]. The best fit unit stoichiometry, using the range of values listed in Table II, is: 1 complex I: 2 complex II: 3 complex III: 6 cytochrome c: 6 cytochrome c oxidase. The molar ratio of ubiquinone and phospholipid with respect to cytochrome c oxidase have been determined at 6-8 and between 440590, respectively. The concentration of both the

T A B L E II P R O T E I N C O M P O N E N T S OF T H E BOVINE H E A R T 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 Component

Complex I Complex II Complex III Cytochrome c oxidase Cytochrome c A T P synthase A D P - A T P translocase Transhydrogenase Ubiquinone Phospholipid

Conc. range

Monomer

n m o l / m g protein

Ref.

Mr

Ref.

0.06- 0.13 0.19 0.25- 0.53 0.6 - 1.00 0.8 - 1.02 0.52- 0.54 3.4 - 4.6 a 0.05 6 - 8 440 -587

22-24 9 9, 24, 30 9, 25, 27 9, 25, 27 34, 35 37, 38 39 9, 35 9-11

700 000 200000 300 000 160 000 12000 500000 30 0@3 120000

28 29 31 32 33 36 38 39, 40

a Based on 1.7-2.3/Lmol carboxyatractylate bound per g membrane protein [37,38].

293

ATP synthese [34] and the ADP-ATP translocase [37,38] have been obtained from inhibitor-binding studies. The data indicate that there is 1 ATP synthase and between 3 and 5 ADP-ATP translocase molecules for each cytochrome c oxidase in the membrane. The nicotinamide nucleotide transhydrogenase is present in approximately the same amount as complex I based on antibody-binding experiments [39]. Minimum molecular weights of the major components are listed in Table II. These range from 700000 for complex I [28] to 160000 for cytochrome c oxidase [32] and 12000 for cytochrome c [3]. From the data in Table II, it can be calculated that the electron transfer chain, the ATP-ADP translocase and ATP synthase together contribute at least two thirds of all of the protein in the membrane. Other proteins present in the mitochondrial inner membrane in lesser amounts include fl-hydroxybutyrate dehydrogenase [41], fatty acyl carnitine transferase [42], 8-aminolevulinic acid synthetase [431, ferrochelatase [44], L-aglycerophosphate oxidase [45], a phosphate transporter [46] and carriers for dicaboxylic acids, pyruvate, glutamate, other metabolites and ions [47]. In addition, the pyruvate dehydrogenase complex and other citric cycle enzymes remain associated, adding to the protein complement of inner membrane preparations such as sub-

mitochondrial particles and electron transfer particles [481. Most of the polypeptides in the mammalian mitochondrial inner membrane are coded for on nuclear DNA and are made in the cytoplasm [4,5]. A small number are coded for on mitochondrial DNA and are made inside the matrix space of the mitochondrion [5]. These include one polypeptide of complex III (cytochrome b), three subunits of cytochrome c oxidase (I, II and III) and one polypeptide of the ATP synthase (component 5). In all, mtDNA in mammalian mitochondria codes for 13 different polypeptides including a ribosomal protein and several very hydrophobic poly-

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T A B L E III P R O S T H E T I C G R O U P S A N D S U B U N I T N U M B E R OF THE ELECTRON TRANSFER COMPLEXES

CYT. OX.

Complex

No. of different polypeptides

No. of prosthetic groups

]I

b,c~

I II

III

IV

NADH-ubiqui- 26 none reductase Succinate-ubiqui- 5 none reductase Ubiquinol-cytochrome c oxidoreductase Cytochrome c oxidase

10

FMN 6 - 7 FeS centers FAD 2 X 2 FeS 1 X 4 FeS (HiPIP) 2 X b hemes

(b562 -F b566) 7-8

1X c I heine 1 × 2 FeS 2 X a hemes

(a+a 3) 2 X Cu atoms

Fig. 1. Polypeptide composition of the succinic oxidase segment of the bovine heart mitochondrial respiratory chai n. Upper densitometric trace, complex II; middle, complex III. Both samples were run on 12% polyacrylamide gels in 0.1% SDS and 8 M urea as described in references [53,63]. Bottom, cytochrome c oxidase run on a 16% polyacrylamide gel. Numbering systems used for the polypeptides of the different complexes are discussed in the text.

294 C

I2"_

~z

M Fig. 2. Structureof complexIII. The shape and arrangement in the membrane are taken from Ref. 67. The approximate arrangement of subunits is shown with each polypeptideplaced with respect to the bilayer in one or other monomers of the dimer. Each monomer, of course, contains one (or more; see text) copies of each subunit.

peptides of as yet unknown function [49]. Compositional data on the electron transfer complexes of bovine heart mitochondria are given in Table III. The most complicated structurally appears to be complex I with 26 different polypeptides and 6-8 redox centers [50]. Complex II contains only 5 different subunits (Fig. 1, scan a) while complex III and cytochrome e oxidase contain 10 and at least 7-8 respectively (Fig. 2, scans b and c).

II. Structural features of the major proteins The organization of the electron transfer complexes and the ATP synthase are known to varying levels of detail, with complex I the least well understood and cytochrome e oxidase the best characterized.

IIA. Complex 1 The complexity of the NADH-ubiquinone reductase segment is evident from the large number of prosthetic groups and polypeptides present [50]. Table IV lists the components by size and by ease of separation from the complex by chaotropic reagents. Labeling experiments with both water-

soluble and lipophilic protein-modifying reagents have established that the flavoprotein and nonheme iron-containing polypeptides are for the most part extrinsic to the bilayer and mainly on the M side of the inner membrane [50-52]. The so called 'insoluble fraction' represents the bilayer-intercalated part of the complex.

liB. Complex H The molecular weights of the polypeptides in complex II, and the locus of prosthetic groups are given in Table V. Succinate dehydrogenase (a protein composed of two subunits with molecular weights of 73000 and 25000 [55]) is located on the matrix side of the mitochondrial inner membrane as indicated by the accessibility of the flavin to reductants and oxidants in submitochondrial particles but not in intact mitochondria [56], by antibody-binding experiments [57] and through labeling studies with the water-soluble, membrane-impermeant protein-modifying reagents [57]. One of the prosthetic groups, the HiPIP center, is shielded from water and must be located near the bilayer. Removal of succinate dehydrogenase from its membrane environment destroys this center [586O]. The three smallest polypeptides are separated from succinate dehydrogenase by treatment with chaotropic reagents [55,61]. These components retain bound lipid and there is a small amount of b heme present [55,61,62]. The cytochrome b in complex II is spectrally different from the two b cytochromes in complex III [62]. It has a high midpoint potential and is unlikely to function in electron transfer from succinate or N A D H to cytochrome c [611. Amino acid analyses have established that the three smallest molecular weight polypeptides in complex II are very hydrophobic [53,61]. These components are each labeled by arylazidophospholipids, reagents which are converted by ultraviolet light into a highly reactive nitrene that inserts covalently into protein from within the lipid bilayer [63]. The smaller subunit of succinate dehydrogenase is also labeled by arylazidophospholipids, indicating contact of this polypeptide with the fatty acid tails in the bilayer [63]. At least one of the bilayer-intercalated polypeptides (and probably all three) span the mere-

295 TABLE IV POLYPEPTIDES OF BOVINE HEART COMPLEX I Fraction after NaC104 resolution

Polypeptides by M r ( × 103)

Iron-protein fraction Flavoprotein fraction Insoluble(membrane) fraction

75, 49, 30, 22, 18, 15.5 a, 15.5 a, 15.5 a 53, 27, 15.5 42, 39, 33, 26 a, 26 ~, 25, 23.5, 22 a, 22 a, 21, 20, 18, 16.5, 8, 5

Resolved by two-dimensional gel electrophoresis with isoelectric focusing in first dimension and SDS-polyacrylamide gel electrophoresis in the second dimension; from [50].

brane. Component CII-3 can be labeled by [35S]diazobenzenesulfonate from both the M and C sides of the mitochondrial inner membrane [57]. Cleavage of this polypeptide with chymotrypsin leads to loss of succinate ubiquinone reductase activity but not succinate-dichloroindophenol reductase activity [64]. HC. Complex I I I

The molecular weights of the ten different polypeptides and the locus of the prosthetic groups in complex III are given in Table VI. These data are for the bovine heart enzyme [65,66]; the composition of complex III from other eukaryotes appears similar [67,68]. The stoichiometry of the prosthetic groups is two b hemes, onecl-type heine and one non-heme center. The stoichiometry of subunits is not clear. It has variously been reported that the two largest polypeptides are present in 2 copies each per complex [69], in 1 copy each [66] and in the ratio of one copy of core I and two copies of II

[70]. Cytochrome b may be a single polypeptide containing two distinct hemes (b566 and b562) [71] or there could be two identical polypeptides (in different conformations to explain spectral differences) with one heme bound to each [66]. Other components appear to be present in one copy each per monomer [66]. Our understanding of the structure of complex III (and cytochrome c oxidase: see later) has been greatly aided by the preparation of two dimensional crystals of the protein and by analysis of these crystals by electron microscopy and image reconstruction [67,72,73]. A low resolution (25 A) structure of complex III from Neurospora crassa reveals an elongated molecule, 150 ~, in the largest dimension and 70 A across. The protein spans the lipid bilayer extending 70 A from one side (thought

TABLE VI

TABLE V

P O L Y P E P T I D E C O M P O S I T I O N O F BEEF H E A R T UBIQUINOL CYTOCHROME c OXIDOREDUCTASE (COMPLEX III)

POLYPEPTIDE COMPOSITION OF BOVINE HEART SUCCINATE-UBIQUINONE REDUCTASE (COMPLEX II)

Polypeptide (No. from Fig. 1)

Mr

Function and prosthetic groups

I II

45 500 44000 42 000 31000 24 600 15 000 9000 7 800 4 800 3 000

core protein I core protein II cytochrome b cytochrome c~ non-heine iron protein

CII.4 (nomenclature from Ref. 53) has been resolved into two

polypeptides under highly resolving gel conditions, Fig. 1 and Ref. 54.

III

Polypeptide

Mr

Prosthetic group

SD l

73 000

SD 2 CII.3 C lI-4a+ C ii_4b+

25 500 13500 8 000 6 500

Flavin Non-heme iron centers 1 and 2 HiPIP center (Q-binding protein?)

IV V VI VII VIII IX X

a Calculated from sequence data.

antimycin-binding peptide

296 to be equivalent to the M side in mitochondria) and 30 A from the other surface (C side) [67,73]. The complex is a dimer in the membrane [67,73]. Bovine heart and yeast complex III, being compositionally similar to the Neurospora enzyme, are likely to have the same gross shape and disposition in the membrane. A model of the arrangement of polypeptides in the complex from these species, based on chemical-labeling, cross-linking and fractionation studies [67,74-78], is shown in Fig. 2. Polypeptides I and II provide the major portion of the M domain while cytochrome q and the nonheme iron protein contribute most of the smaller C domain. Cytochrome b, in turn, contributes most of the bilayer intercalated part of the complex. The sequence of cytochrome b from several different sources of mitochondria has been obtained by sequencing of m t D N A [49,79-81]. The apoprotein is from 380-385 amino acids long in different species and there is considerable sequence homology. This polypeptide is characterized by the presence of seven long ( > 20 residues) stretches of predominantly hydrophobic amino acids. Each stretch has a favorable free energy for being in the lipid bilayer when folded into a helix (calculated by summing the free energy for transfer from water to a hydrocarbon medium of all of the residues in the stretch of sequence: see refs. 82,83). Cytochrome b thus probably spans the lipid bilayer seven (or more) times as shown in Fig. 3. Both of the core proteins, as well as polypeptides VI, VII and VIII, are also labeled by arylazidophospholipid [78], although to a less extent than cytochrome b, and may contribute to the buried domain. Localization of the apoproteins for cytochrome c~ and the non-heme iron protein to the C side places two of the prosthetic groups on this side of the membrane. The locus of the b heroes is more ambiguous. According to Case and Leigh [84] both b hemes are on the C side. This conclusion is based on the effect of nickel on the EPR signal of these redox centers when added from different sides of the membrane. In contrast, Papa [85] places one b heme on the C side (6562) and the other (b566) on the M side. This localization was based on spectral perturbation studies using p H changes to alter the environment of the hemes in mitochondria and submitochondrial particles.

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Fig. 3. Predicted folding of cytochrome b. The predicted folding of cytochrome b based on sequence data is shown. Amino acids conservedin yeast, AspergiUus, bovine, human, and mouse (see text) are given by the one letter code. The following other symbols are used: O for positively-chargedamino acid residue, for negatively-chargedresidue, [] for glutamine or asparagine. In several positions along the chain a positively-charged amino acid can be substituted for a negatively-chargedresidue in different sequences, e.g., ~ ) or Q with the outer symbol showing the more common situation. The symbol • represents a position in the chain with varient but always uncharged residues. • shows the locus of insertions in the sequence e.g. in yeast and/or neurospora as compared to the sequence of bovine or human cytochrome b.

liD. Cytochrome c oxidase Cytochrome c oxidase from a variety of eukaryotic sources contains the eight polypeptides listed in Table VII [86-88]. The mammalian enzyme also contains variable amounts of four or five other polypeptides [86-89,90], which are readily removed without loss of either electron transfer or proton pumping function [91]. The four prosthetic groups are localized to subunits I and II. The evidence for this comes from subunit dissociation experiments [92] and from comparisons with cytochrome c oxidase from Paracoceus denitrificans [93]. The enzyme from this prokaryote is spectrally similar to the enzyme from eukaryotic sources with two hemes (a and as) and two copper atoms but only two subunits [93].

297 TABLE VII POLYPEPTIDE COMPOSITION OF BOVINE HEART CYT O C H R O M E c OXIDASE I II III IV V VI VII Ser VII Ile

56065 a 26021 a 29 918 a 17 153 12 436 10026 5541 4 962

F-Met-Phe-Ile-Asn F-Met-Ala-Tyr-Pro Met-Thr-His-Gln Ala-Ser-Gly-Gly Ser-His-Gly-Ser Acely-Ala-Glu-Asp Ile Ser His Tyr Glu Ile Thr-Ala-Lys

a b c

14000 11000 8 480

Ala-Ser-Gly-Gly Ala-Ser-Ala-Ala Ser-Thr-Ala-Leu

a Molecular weights calculated from m t D N A sequence data.

These subunits have antibody cross-reactivity [93] and sequence homology (Buse, G., personal communication) with the two largest subunits (I and II) of the yeast and bovine enzyme. One of the copper atoms appears to be liganded to subunit II through one or both cysteine residues (Cys 196, Cys 200) near the C terminus of this subunit [94,95]. Two different two dimensional crystals of bovine heart cytochrome c oxidase have been obtained and studied by electron microscopy and imaging methods [96,97]. The enzyme is integrated into a bilayer as a dimer in one crystal form [96] and is present as a monomer in detergent-rich sheets in the second form [97]. The two crystals have been studied independently. Both show a Y shaped protein oriented with the stalk of the Y or C domain extending 50-55 ,~ from the C side of the membrane (Fig. 4). The two arms of the Y or M 1 and M 2 domains split on the C side of the membrane and each M domain crosses the lipid bilayer to extend at most 10-15 ,~ from the M side of the inner membrane. The center to center distance between the two M domains is 40,~. Low angle X-ray studies show reflections typical of helices running perpendicular to the lipid bilayer [98] (in the M domains). The model of the arrangement of polypeptides in cytochrome c oxidase given in Fig. 5 is based on a variety of chemical labeling and cross linking studies [91,99-102]. Subunits, I, II, III and V provide the major part of the large C domain.

Fig. 4. Balsawood model of the cytochrome c oxidase monomer reproduced from Fuller et al. [97] with permission.

Subunits I, II and III also contribute to the bilayer intercalated M~ and M 2 domains as judged by labeling with arylazidophospholipids [99,101] and iodonapthylazide [102]. These polypeptides contain 10 [49], 2 [103], and 6 [49] long stretches of hydrophobic amino acid sequence, respectively. Subunits IV, VII Ser and VII Ile are also labeled by arylazidophospholipids [99,101] and each contains a single long stretch of hydrophobic residues [ 104,105]; making 21 such stretches of sequence in the cytochrome c oxidase monomer. These can all be accommodated as closely packed transmembrane helices in the M t and M 2 domains [106]. Heme a and copper a must be located on the C side of the inner membrane in order to accept electrons from cytochrome c. Heme a s and Cu,3 are known to be close together (3.4-7 A) [107-111] but the locus of the oxygen-binding site in the complex is unclear at present.

298 r

m

I

Fig. 5. Arrangement of subunits in cytochrome c oxidase. A cytochrome c oxidase dimer is shown; the shape and positioning of the dimer in the membrane being based on [106]. The approximate position of each subunit is based on chemical labeling and cross-linkingexperiments discussed in the text.

liE. Cytochrome c Cytochrome c is the only inner membrane protein for which high resolutional structural data are available [33,112]. Three dimensional crystals of the protein from several different sources have yielded a structure to better than 2 A resolution for both the oxidized and reduced form of the hemoprotein [ 112]. Cytochrome c binds to complex III [113] and cytochrome ¢ oxidase [114,115] with high affinity ( K d = 10-8M). These sites are localized to cytochrome e 1 of complex III [116] and subunit II of cytochrome c oxidase [117-120]. The high affinity site in bovine heart cytochrome c oxidase is in a cleft probably at the interface between monomers of the oxidase dimer [121-122]. The same domain on cytochrome c binds both reductase (complex III) and oxidase and involves Lys 8, 13, 25, 27, 72, 79, 86 and 87 [113,115,123126] which are arranged in a ring around the heme cleft. Cytochrome c also binds to membranous cytochrome c oxidase in low affinity sites ( K d = 10 -6 M) which have been shown to involve lipid molecules [127,128] tightly bound to the protein. IIF. A TP synthase The ATP synthase, or proton pumping ATPase, is a large complex present not only in mitochondria but in other membranes of eukaryotes e.g. in chromaffin granules [129] and also in chloroplasts [130]

and in the plasma membrane of prokaryotes [ 131 ]. The mitochondrial ATP synthase is much more complicated than its counterpart from prokaryotes, containing as many as 18 different polypeptides [132,133] (Table VIII) as opposed to 8 in E. coli (and chloroplasts) [133-136]. The complex from all sources is readily split by chaotropic salts into a water-soluble fraction, the F1, and a waterinsoluble, lipid-containing fraction, the F0 (reviewed in Refs. 137, 138). The FI part retains ATPase activity but does not make ATP. It is insensitive to oligomycin which inhibits the ATPase activity of the membrane-bound protein in mammalian mitochondria and is not affected by low levels of N, N'-dicyclohexylcarbodiimide (DCCD) which inhibits the activity of the membrane-bound protein in both prokaryotes and eukaryotes [137,138] F~ ATPase from all sources contain five subunits, a / 3 7 o and ~ [137-144]. The sequences of the subunits of the F t of E. coli have been deduced from sequencing of the unc operon [ 145,147]. The/3-subunit of the bovine heart F~ has

TABLE VIII POLYPEPTIDECOMPOSITIONOF BOVINEHEART ATP SYNTHETASE Component no. (Fig. 6)

Mr

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

54000 a 48500 a 33000 a 30500 25000 20800 20500 20000 19500 14700 12300 12300 11800 10700 10200 7627 b 7700 6500

Identification

oscp 8

inhibitor protein

a From WeberSOsborngels. b From sequence data. Remaining values are from SwankMunkres gels. See Table I of Ref. 132.

299

been sequenced directly (Walker, J., personal communication). It shows considerable sequence homology with the equivalent subunit in the E. coli enzyme. The stoichiometry of subunits in F~ ATPase from mammalian sources has been reported as 3:3:1 : 1:1 and 2 : 2 : 2 : 2 : 2 [137]. The F 1 from rat liver mitochondria has been crystallized [118] and X-ray studies are in progress. Preliminary studies show a globular structure arranged in six domains with a two fold axis of symmetry through the protein. This two-fold symmetry is more consistent with there being two

( y,L II

copies of each subunit than with there being three copies each of a and fl but only one copy of 7, and e. The dimensions of the F 1 ATPase of rat liver are 120 × 110 × 80 A (Pedersen, P., personal communication). This globular protein is the 90A spheres seen in electron microscopy attached at the matrix side of the mitochondrial inner membrane [149]. The F0 portion of the ATP synthase represents the membrane-intercalated part of the complex, involved in the proton-transtocating function [137,138]. The polypeptides of this portion are heavily labeled by arylazidophospholipids [150]. At least one polypeptide (component 12) of the bovine enzyme, is located on the C side of the inner membrane [132]. The three polypeptides present in the F1 from prokaryotes have counterparts in the mammalian enzyme [49,131,151 ] and probably form the core of the F0 segment. These are components 5, 11 and the DCCD-binding proteolipid (component 14). The sequences of both components 5 and 14 from the bovine heart enzyme have been determined [49]. They contain 6 and 2 long hydrophobic stretches of sequence, probably arranged as transmembrane helices. Most of the components of F0 appear to be present in one or two copies [133], the exception being the DCCDbinding proteolipid which has been reported to occur in from 6-12 copies per complex [ 152,154]. II1. A model for the arrangement of proteins in the mitochondrial inner membrane

Fig. 6. Distribution of proteins in the mitochondrial inner membrane. A n area of the inner m e m b r a n e containing one complex III dimer is shown in projection through the membrane. This is equal to approx. 200000~.. The n u m b e r of copies of each protein, their aggregation state and their size are reviewed in the text. This area is about one-third of that containing a complex I dimer. The second part of the figure shows the arrangement of protein across the membrane. Phospholipids, complex III, cytochrome c oxidase, cytochrome c and Fl-ATPase are all drawn approximately to size based on electron microscopy or X-ray crystallographic studies.

The compositional data in Table II and structural data reviewed in the preceding sections provide the needed information to derive a model for the distribution of proteins into the inner membrane. This model is presented in Fig. 6. Most of the components in the membrane appear to be present as dimers. Complex I, [155,156], complex Ill [67], cytochrome c oxidase [157], the transhydrogenase [158,159] and ATP-ADP translocase [160-162] are all dimeric proteins in non-ionic detergents as based on sedimentation studies and in some cases on cross linking experiments. Complex III [67] and cytochrome c oxidase [96] are dimeric in lipid vesicles as seen in electron microscopy and image reconstruction studies. The

300

ADP-ATP translocase is inhibited by carboxyatractylate and bongkrekate in the ratio of 1 M inhibitor per 2 M protein (30-kDa subunit) [37,38]. Only the ATP synthase appears to be a monomer in the inner membrane, as visualized by electron microscopy [149]. Cytochrome c oxidase, complex III and the ATP synthase are each drawn roughly to size and shape and as oriented across the inner membrane in Fig. 6. The structures of complex I and II are less sure. However, labeling studies indicate that the bulk of these proteins is on the M side of the inner membrane. Prosthetic groups are located with respect to the bilayer as described in the previous sections. The electron transfer complexes are shown as randomly distributed in the inner membrane. This is based on the results of several different types of studies. Antibodies to cytochrome c oxidase and to complex III have been shown to aggregate these complexes separately [163]. The lipid content of the mitochondrial inner membrane has been increased up to 8-fold by liposome fusion [164]. This reduces the rate of electron transfer from complex I to complex III, indicating that the distance between these complexes has been increased and that the two proteins are therefore separate in the membrane. Finally, the rotational correlation time of cytochrome c oxidase has proved to be the same whether complex III and cytochrome c are present or not in the same reconstituted membrane [165]. If the two complexes were part of a supramolecular aggregate (with cytochrome c) the motion of cytochrome c oxidase would have been slowed significantly. The area per complex III dimer is around 200000 A 2, as calculated by dividing the surface area of the inner membrane (2 • 106 cmZ/g protein [166] by the number of complex III dimers present (from Table II)). The distance between a complex I and complex III averages 310A and between complex III and cytochrome c oxidase averages 225 A. Cytochrome c is shown in Fig. 6 as bound to complex III and cytochrome c oxidase. There are three high affinity sites for two cytochrome c molecules (contributed by a complex III, and two cytochrome c oxidase monomers) [113-115]. In addition, there are a large number of low affinity

sites for cytochrome c in the membrane provided by negatively charged phospholipids [127]. It is unlikely, therefore, that cytochrome c exists free in solution in the intracristal space.

IV. Arrangement of prosthetic groups in relation to energy coupling There are three points in the electron transfer chain at which proton movements are coupled to redox changes [167]. These so-called coupling sites occur between N A D H dehydrogenase and cytochrome b (coupling site I), between cytochrome b and cytochrome c (coupling site II) and between cytochrome c and the oxygen binding site (heme a3-Cua3 couple) in cytochrome c oxidase (coupling site III). There is an additional site of coupling of electron transfer to proton translocation off the main respiratory chain pathway and involving the nicotinamide nucleotide transhydrogenase [168,169]. The p H gradient generated by electron transfer is used b y the ATP synthase to make ATP. The ATP synthase is itself a reversible proton pump [137,138]. Mitchell has proposed that proton transfer is effected through the alternate and asymmetric disposition of hydrogen and electron carriers in each coupling site [ 170,171 ]. There is some evidence for such an arrangement of redox components in complex III as described in the Q cycle [172,173] but no evidence of a transmembrane arrangement of redox components or for the presence of a hydrogen carrier in cytochrome c oxidase. Similarly, it is difficult to explain proton translocation through the transhydrogenase [169] by the Mitchell model. An alternative is that these coupling sites are redox linked proton pumps. Proton transfer through cytochrome c oxidase [174,175] and the transhydrogenase [39,40] is inhibited by DCCD, a hydrophobic reagent which modifies glutamic acids and aspartic acids. The same reagent blocks the proton channel function of the ATP synthase [ 176]. For cytochrome c oxidase and the ATP synthase the site of action of DCCD has been determined precisely. In cytochrome c oxidase the reagent reacts predominantly with glutamic acid 90 of subunit III (numbering in the boyine heart enzyme) [175]. This residue is in a sequence of otherwise very hydrophobic amino.acids and is proba-

301

lle

-

Leu

-

Phe

-

lle

-

lle

-

Ser

-

Glu

-

Val

Cyt

oxidase

Leu

-

Gly

-

Phe

-

Ala

-

Leu

-

Ser

-

Glu

-

Ala

ATP

Synthase

Fig. 7. A comparison of the sequences of the DCCD binding site in cytochrome c oxidase subunit III and in the proteolipid protein of the ATP synthetase of bovine heart mitochondria.

bly buried inside the bilayer. Preparations of cytochrome c oxidase have been obtained which are missing subunit III [177]. These do not show a redox linked proton translocation. DCCD reacts with the ATP synthase selectively at a glutamic acid or aspartic acid (depending on the species) in the C terminal half of the proteolipid protein [176-178,179]. Mutants have been obtained in which this residue is altered [180,181] and these are unreactive to DCCD. Fig. 7 presents the sequences around the DCCD reactive glutamic acids in cytochrome c oxidase and in the ATP synthase. The two sequences are remarkably similar. The best characterized protein translocating enzyme in structural terms is bacteriorhodopsin. This intrinsic membrane protein is arranged as a bundle of seven helices across the bilayer [182-184] with the proton channel probably formed at the interface between two or more helices. Cytochrome c oxidase and the ATP synthase also appear to be structured within the membrane as bundles of helices. In bacteriorhodopsin the proton channel is gated by the chromophore retinal [185,186]. In cytochrome c oxidase, heme a is the most likely candidate for the 'gating' function [1871.

V. Lateriai diffusion of proteins and importance for electron transfer The diffusion rates for electron transfer complexes and the ATP-ADP translocase have been measured in the inner membrane and diffusion constants of from 8.3.10 -1° cmZ/s to 2 . 1 0 9 cmZ/s obtained [188-190]. Cytochrome c has a diffusion constant in the inner membrane of 10 -8 cmZ/s [189] which is close to the diffusion constant for phospholipid in this membrane [191]. From these diffusion constants it can be calculated that a cytochrome c oxidase molecule will take approx. 5 ms to sweep 50000 ~2 while cytochrome

c or ubiquinone (assuming it has the same diffusion constant as lipid molecules) can sweep the same area in 0.5 ms [189,191]. This means that cytochrome c can collide with both the oxidase and reductase several times within the time of a single turnover of the electron transport chain (50-100 m s / e - per cytochrome c oxidase). The fact that the electron transfer complexes can be shown to undergo diffusional and rotational motion under certain experimental conditions does not prove that they do so in vivo. A major fraction of the cytochrome c oxidase and ADP-ATP translocase molecules are immobile in the mitochondrial inner membrane based on rotational correlation measurements [189-190]. This fraction is greatest in intact mitochondria and may be related to the high protein content on the matrix space. Orthodox mitochondria in which the matrix space is 90% of the volume of the organelle [192,193] have a protein content in this inner compartment of between 350 and 700 m g / m l depending on the source of the mitochondria [194,195]. Mitochondria also show a condensed configuration under phosphorylating conditions and if permeant cations are present [ 192,193] where the matrix becomes shrunk (to only 60% of the total volume) and the opposing membranes of the crystal membrane approach closely. The water content of the matrix space of condensed mitochondria is close to that of protein crystals (i.e. around 50% [196]). The diffusion or rotational freedom of intrinsic membrane proteins extending into (and thus a part of) this essentially solid mass would be severely hindered. This would not necessarily slow the rate of electron transfer appreciably because the diffusion rate of ubiquinone (within the bilayer) and cytochrome c (on the C side of the inner membrane) should be independent of the viscosity of the matrix space.

VI. Summary and prospects A picture of the mitochondrial inner membrane is emerging in which proteins, mainly in the form of large multipeptide complexes, are inserted across, and randomly distributed in a fluid lipid bilayer. The major portion of the protein is involved in electron transport driven ATP synthesis. At least 47 different polypeptides make up the

302

electron transport chain, arranged in four functionally integrated complexes. The ATP synthase, in turn, contains as many as 18 different polypeptides. The requirement for so many different components is not clear. Most of electron transfer complexes, the nicotinamide nucleotide transhydrogenase, ADP-ATP translocase and possibly other more minor components in the membrane are present as dimers (or possibly larger aggregates); and this explains the relatively few membrane intercalated particles for such a protein-rich membrane, seen in freeze-fracture studies [191]. The inner membrane particles can in some circumstances diffuse laterally, although it is not clear that they do so in intact mitochondria because of the high protein content in the matrix space. The electron transfer chain, however; contains two components, ubiquinone and cytochrome c, which would be free to diffuse rapidly even if the motion of the larger respiratory chain complexes is limited by the high protein content of the matrix space. The bilayer intercalated parts of the electron transfer complexes and ATP synthase appear to contain bundles of helices composed of long hydrophobic stretches of sequence contributed by around half of the polypeptides present. Other components of these complexes are extrinsic to the membrane and mainly located on the M side of the inner membrane. Progress in detailing the structure of the mitochondrial inner membrane is likely to speed up as more components are obtained in two dimensional crystals suitable for electron microscopy and image reconstruction. Crystals of complex I have recently been described [197]. However this technique has a limited resolution (3.5 A in the case of bacteriorhodopsin, and 12 A in projection for cytochrome c oxidase so far). Inevitably, it will be necessary to use X-ray crystallographic methods to obtain the high resolution data needed to determine the structure-function relationships of the inner membrane proteins. X-ray crystallographic studies of the Fi-ATPase are already in progress and there are preliminary reports of the three dimensional crystallization of complex III and cytochrome c oxidase [198,199].

Acknowledgments I wish to thank my colleagues V. Darley-Usmar, F. Malatesta, F. Millett and G. Georgevich for valuable discussions. This work was supported by grants HL22050 and H L 24526 from the National Institutes of Health and PCM 7826258 from the National Science Foundation. I am the grateful recipient of an Established Investigator Award of the American Heart Association.

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