Structure of mitochondrial cristae membranes

Biochimica et Biophysica Acta, 344 (1974) 119-155 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 85136

STRUCTURE OF MITOCHONDRIAL CRISTAE MEMBRANES H. J. H A R M O N a, J. D. HALL ~ and F. L. C R A N E •

• Department of Biological Sciences, Purdue University, West Lafayette, Ind. 47907 and b Department of CellPhysiology, Boston Biomedical Research Institute Boston, Mass. 02114 (U.S.A.) (Received October 22nd, 1973)

CONTENTS I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Membrane composition . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Protein-lipid interactions . . . . . . . . . . . . . . . . . . . . . . . . . .

1 !9 120 122

II. Topography . . . . . . . . . . . . . . . . . . . . . A. Location of eytochrome c . . . . . . . . . . . . . . B. Location of ATPase . . . . . . . . . . . . . . . . . C. Location of N A D H dehydrogenase (FpN) . . . . . . . D. Location of succinate dehydrogenase (Fp,) . . . . . . . E. Location of iron-associated components . . . . . . . . F. Location of Complex IIl . . . . . . . . . . . . . . . G. Location of cytochrome b . . . . . . . . . . . . . . H. Location of eytochromes a and aa . . . . . . . . . . . I. Proteolytic digestion of the inner membrane . . . . . .

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125 127 130 132 134 134 135 137 137 141

III. Ultrastructure studies . . . . . . A. Electron microscopy . . . . . . B. Cytoc,hemical staining . . . . . C. Schemes for membrane structure

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142 142 147 148

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150

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

151

Acknowledgements

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I. INTRODUCTION

In this review we will consider the arrangement of lipids and proteins in the mitoehondrial eristae. As with other membranes, ideas about the association of lipid and protein and diversity of protein types in membranes have undergone remarkable Abbreviations: DABS, diazobenzenesulfonic acid; F~, coupling factor 1; Q, ubiquinone; OSCP, oligomycin sensitivity conferring protein; CFo, protein fraction necessary for oligomycinsensitive phosphorylation; PMS, phenazine methosulfate; Fps, succinate dehydrogenase; TMPD, N, N, N', N'-tetramethyl-p-phenylenediamine dihydrochloride; CCCP, earboxyeyanide-m-ehlorophenylhydrazone; DABP-, 4,4'-diamino-2,2'-dipyridyl-; FpN, N A D H dehydrogenase.

120 change in the last 10 years. Evidence has gradually developed that the basic concept of a unit membrane must be modified to accommodate specific organizational patterns of lipid and protein and different types of protein [1 ]. For lipid the principle of a bilayer as the basic organization has gained support, with the possible exception of local special association with protein [2]. On the other hand, the idea that protein acts as a surface coat on the lipid bilayer is only partially supported. Study of the binding of protein to the membrane and of the nature of proteins in the membrane has shown that only 30-40 ~ of the proteins of cristae can be considered to be associated with the polar surface of a lipid bilayer or with proteins in the bilayer. Up to 70 ~ of the cristae proteins can be associated eithei partly or completely with the interior of the lipid bilayers. The proteins associated with the surface have been designated as extrinsic or peripheral proteins. Those in the interior of the hydrophobic part of the membrane have been described as intrinsic or integral proteins [3,4]. Each of these protein classes has an anisotropic distribution on opposite surfaces of the membrane or in the hydrophobic interior, respectively. The availability of right-side-out and inside-out cristae membrane vesicles has facilitated the elucidation of lipid and protein orientation or exposure on the membrane. With these two types of vesicles it has been possible to detect differences in the exposure of lipids and proteins by procedures which include: (1) Selective extraction, (2) reaction with impermeable electron donors or acceptors, (3) exposure to impermeable inhibitors, (4) binding of marker molecules or antibodies to exposed proteins, (5) selective hydrolysis of exposed protein or lipid by enzymes. (6) electron microscopic examination of exterior or interior surfaces.

IA. Membrane composition The inner mitochondrial membrane is composed of approximately 75 ~ protein and 25 ~ lipid by weight with over 90~/o of the latter being phospholipid [5]. The protein fraction can be subdivided into two classes, intrinsic and extrinsic proteins, based upon their ease of extraction from the membrane [3,4]. As defined, intrinsic proteins are of a bimodal nature (containing polar and hydrophobic regions) and are hydrophobically linked to the membrane. Extrinsic proteins, comprising approximately 30-40 ~ of the membrane protein [6-8] have been shown to be removed by treatment with urea, acetic acid [8,9], dilute KC1 (cf. section IIA), high salt concentration [10], and mild sonication [11 ]. Removal of extrinsic protein leaves the remainder of the membrane intact in both physical structure and function. The removal of ATPase, classed as an extrinsic protein, leaves the membrane structurally intact, and still capable of electron transport, but unable to catalyze oxidative phosphorylation or ATP hydrolysis (of. section IIB). Included in the extrinsic proteins are most of the factors necessary for oxidative phosphorylation, whose structure and properties were recently reviewed [12]. The proportions of electron transport components in electron transport particles and beef heart mitochondria are summarized in Table I. The composition of intrinsic and extrinsic protein subunits of the inner mitochondrial membrane, as determined

121 TABLE I COMPOSITION OF M I T O C H O N D R I A A N D K E I L I N - H A R T R E E PARTICLES

Cytochrome aa3 Cytochrome a3 Cytochrome a Cytochrome c Cytochrome c~ Cytochrome b FlavinNFMN FlavinsFAD Non-heme iron Cu Q Reference

Keilin-Hartree (/~moles) Molar ratio

Mitochondria (/~moles) Molar ratio

Mitochondria (nmoles/mg protein)

5.59 2.93 2.66 0.87 1.45 2.85

6.08 3.10 2.98 2.68 1.48 2.96

1.30

4 2 2 0.4 1.08 2.04

15

4 1.96 2.04 1.70 1.02 1.96

15

0.45 0.21 0.68 0.2 0.46 3.3 1.47 3.4-4.0 16

TABLE II PROTEINS O F M I T O C H O N D R I A L CRISTAE OF BEEF H E A R T M I T O C H O N D R I A

Intrinsic proteins Cytochrome a q- a.~ Copper protein Cytochrome cl* Non-heme iron III Cytochrome b* N o n heme iron Ib N o n heme iron Ia N A D H dehydrogenase I N o n heme iron II Succinate dehydrogenase Membranifibril CFo Unknown, complex I Unknown, complex I Unknown, complex I, IV Unknown, complex I Unknown, complex II, III Total intrinsic Extrinsic proteins Cytochrome c ATPase subtmit ATPase subunit OSCP Unknown Unknown Unknown Other unknown Total extrinsic

Molecular weight of subunits

70 of total protein

15 500 12 500 37 000 30 000 26 000 16 000 27 000 42 000 28 000 69 000 30 000 78 000 74 000 34 000 23 000 12 000

6.4 4.6 3.9 2.2 2.8 5.8 0.5 0.9 3.0 1.0 10.5 1.6 3.9 1.0 2.8 4.6 55.5

12 900 50 000 53 000 18 000 47 000 55 000 62 000 -

0.8 6.1 3.5 3.0 5. I 4.2 9.2 12.6 44.5

* Polypeptides of mol. wt 37 000 and 50 000 have been associated with cytochrome b [170] and 30 000 has been associated with the heme subunit of cytochrome cl [I 68].

122 by sodium dodeeylsulfate-acrylamide electrophoresis of fractions obtained by KCl-deoxycholate fractionation of beef heart mitoehondria [13,14], are shown in Table II. The electrophoretic distribution of extrinsic protein extracted with acetic acid, urea, or trypsin-urea [17] on polyacrylamide gels is the same as the distribution of the deoxycholate-insoluble fraction from cytochrome oxidase (green) membrane less two components [18,13]. The extrinsic protein fraction and the deoxycholateinsoluble fraction should also contain similar components as the "structural protein" [7] and membranifibril (CFo) fractions [19], less the cytochromes and respiratory enzymes. In particular, 90-/~ spheres, identified as ATPase, have been observed attached to fibers in membranifibrit fractions (cf. section liB) and denatured ATPase has been shown to comprise up to 35 ~o of the protein of "structural protein" from submitochondrial particles [20,21 ]. The extrinsic proteins of mol. wt 50 000 to 62 000 in the denatured form can account for the bulk of the structural and core proteins found in functional complexes of electron transport derived by detergent fractionation of the membrane [22].

IB. Protein-lipid interactions The decrease of enzymic activity upon extraction or hydrolysis of phospholipid and the subsequent reconstitution of activity upon addition of phospholipids indicate a strong dependence of mitochondrial function upon their presence. The digestion of sonicated particles and mitochondrial membranes by phospholipases has indicated that the primary dehydrogenases of NADH and succinate are relatively unaffected by phospholipase A and C digestion even though extensive phospholipid digestion (except for cardiolipin) has taken place [23,24]. However, digestion with low levels of phospholipase inhibits electron transport reactions, phosphorylation, and ATPase in sonicated particles, NADH oxidation being more sensitive to phospholipase A than succinate oxidation and succinate oxidation being more sensitive to phospholipase C than NADH oxidation [24-26]. In contrast, phosphorylation but not respiration was inhibited by phospholipases in mitochondria [27-29]. Data from phospholipase A and C digestion are summarized in Table III. These findings support a role for phospholipid in succinate oxidation and oxidative phosphorylation [24]. Reconstitution of respiration and phosphorylation can be achieved by the addition of phospholipids to digested membranes [27,30,31 ]. Suecinate oxidase has been shown to be inhibited by phospholipase D digestion also [32]. Studies of lipid extraction with various solvents have also shown the role of a lipid environment in the activity of electron transport enzymes [5,19,33,34]. Maximum restoration of activities by the addition of phospholipids occurs when the amount of rebound lipid is approximately the same as that originally present. Extraction of lipid from submitochondrial particles and mitochondria with organic solvents has been shown to have no effect on the cross-sectional structure of the membrane [35,36]. However, recent data have been offered showing a breakdown in sonicated particle membrane structure upon acetone extraction [37]. Such

No change in P/O rate [61] Succinate-driven P/O decreased 60 ~ [26] Suceinate oxidase more sensitive than N A D H oxidase [29]

Phospholipase C 40 % inhibition NADH-->Q2 [23,24] 60-75 ~ inhibition N A D H --> duroquinone [23,24] N A D H ~ J u g l o n e unaffected [23,24,26]

60% inhibition of succinate oxidase [26] 65 ~ inhibition of P/O (succinate) [26] N A D H more sensitive than succinate [24]

Phospholipase A 80-907, inhibition N A D H ~ Q 2 or duroquinone [23,21] NADH~Juglone unaffected [23,24]

Inverted particles Lecithin

% hydrolysis

Ca 2+ translocation inhibition [27] restored with bovine serum albumin or lecithin

P/O decrease [61 ] 6070 P/O decrease [27]

Respiration unimpaired [27,29]

90

70 70 P/O inhibition with bovine serum albumin added [27] but no restoration with lecithin 69 Protection of Pi-ATP exchange [27] with bovine serum albumin

Mitochondria

EFFECTS OF PHOSPHOI.,IPASE DIGESTION ON M I T O C H O N D R I A A N D INVERTED PARTICLES

TABLE III

l0

100

Phosphatidylethanolamine

0

0

Cardiolipin

23

23

Ref.

124 evidence suggests that the core of the membrane is protein, not lipid as held in the unit membrane theory, but that lipid is involved in stabilizing the membrane structure. Membrane structure changes little if at all at low levels of lipid digestion by phospholipase even though N A D H dehydrogenase has been solubilized [23,38]. Higher levels of phospholipase A result in thicker membranes, broken vesicles, and the disappearance of 90-A knobs in sonicated particles from both membrane and supernatant [25] even though oligomycin-sensitive ATPase activity is regained upon addition of phospholipid, at which time knobs were still not visible on the membrane surface [39]. Mitochondria treated with phospholipase C exhibit binding characteristics with antibody to coupling factor 1 (F,) similar to those of submitochondrial particles and are seen to contain similarly orientated vesicles [27], indicating a cleavage of membrane to yield inverted vesicles. Conformational and functional changes in response to phospholipid have been observed in the binding and activity of phosphorylation coupling factors [12,17,40]. Similar allotopic changes have been observed in the sensitivity of purified and reconstituted succinate systems to thenoyltrifluoroacetone [41 ]. Phospholipids have been shown to affect both the enzymatic and structural characteristics of purified cytochrome oxidase [42-44]. The lipid-protein interactions and electron paramagnetic resonance findings [45] are summarized in Table IV. Further data on the role of lipid and conformational changes in cytochrome oxidase preparations are given by Crane et al. [46]. In light of evidence for the hydrophobic interaction between membrane proteins and phospholipids [47-49] and physical evidence (infrared spectroscopy, fluorescence, CD, EPR) of hydrophobic interaction between purified cytochrome oxidase [50] and phospholipid [46], a mosaic membrane model for both cytochrome oxidase [50] and intact inner membrane [51] is more attractive than the unit membrane model. Jost et al. [45] have shown that the fluidity of the lipid portion increases with increasing amounts of phospholipid, which also suggests a mosaic arrangement of lipid with protein. This laboratory has advanced the idea of a double lipid bilayer mosaic arrangement [19] on the basis of detergent fractionations [19,37,43]. This view is

TABLE IV LIPID-PROTEIN INTERACTIONS WITH PURIFIED CYTOCHROME OXIDASE mg lipid/ mg protein

Molar ratio

Interaction

Ref.

0.040 mg lipid/mg 1.0-1.3 tightly bound; 44,46 not removed by chloroform; methanol cytochrome oxidase stable complex but removed by chloroform: methanol :NH4+ 5.0-6.5 surface bound EPR immobile; chloroform ; methanol 0.20 mg lipid/rag 45 soluble cytochrome oxidase > 6.5 fluidbilayer > 0.20 mg/mg 43,46 EPR mobile; membranous structures cytochrome oxidase

125 strengthened by the finding that antibodies to the polar headgroups of cardiolipin bind to little cardiolipin in either submitochondrial particles or mitochondria [52]. Were the inner membrane to consist of a single lipid bilayer protein complex, then cardiolipin, which makes up approximately 20 ~ of the phospholipid of the membrane, must have its polar head exposed on one of the membrane surfaces. However, even after heating or exposure to sonic oscillation only 30 ~ of the total cardiolipin can be bound by antibody, a finding consistent with a binary lipid bilayer arrangement, where one or both inner opposing faces contain cardiolipin. Lack of immediate cardiolipin digestion by phospholipase in either sonicated particles or mitochondria supports this arrangement [24]. Since cardiolipin is associated with both cytochrome oxidase and N A D H dehydrogenase [24] and both enzyme activities are found in the green fraction following membrane splitting with deoxycholate [14], cardiolipin may be primarily located in the inner face of the outer (C side) lipid bilayer in the inner mitochondrial membrane.

II. TOPOGRAPHY Study of membrane topography is favored by the availability of right-side-out and wrong-side-out membrane vesicles. The orientation of the inner membrane in mitochondria is right side out and certain preparations called electron transport particles are wrong side out. For clear results the sidedness of vesicles must be carefully established. Using the 90-A spheres of the physiological inner surface of the inner membrane as a marker, Malviya et al. [54], have demonstrated that neither sonicated nor digitonin-treated particles are turned exclusively inside out or right side out (cf. Table V). Visual observation of negatively stained material does not give a conclusive determination of membrane orientation, and a biochemical evaluation of the membrane disposition is desirable since electron transport particles stripped of F1 particles cannot be differentiated from right-side-out membrane fragments but are still capable of catalyzing electron transport (but not ATPase and phosphorylation recreations) [10,11,55]. Crane et al. [56] defined the lack of stimulation of respiratory rate upon addition of cytochrome c as the major criterion for the presence of electron transport particle orientation. The "closed-ness", or inability to react with cytochrome c, of different preparations from the same preparation of mitochondria has been compared in this laboratory, utilizing the stimulation of N A D H oxidase by externally added cytochrome c as the criterion of membrane orientation (cf. Table V). Submitochondrial particles prepared from mitochondria isolated in KC1 by the method of Crane et al. [56] are the least closed and least homogeneous, while electron transport particles prepared with K O H are the most homogeneous [56]. Particles prepared by sonic oscillation [11,57] from this and other laboratories show a variable orientation of membrane surfaces. The best beef electron transport particles show no stimulation of N A D H oxidase or succinate oxidase in the presence of exogenous cytochrome c.

126 TABLE V HETEROGENEITY OF BEEF HEART MITOCHONDRIA AND ELECTRON TRANSPORT PARTICLES AS DETERMINED BY CYTOCHROME c STIMULATION Method of preparation of inverted membranes Sonicated Sonicated Alkaline Alkaline Alkaline KCI KCI KCI

Apparent % ~ Stimulation Assay procedure inverted with cytochrome c** orientation*

Reference

64 74 100 92 88 42 65.6 69.4

29.2 0 8.7 13.4 140 52.9 42.4

electron microscopy NADH--->O2 NADH-*O2 succinate~ 02 NADH--~O2 NADH--+O2 NADH->O2 succinate

54

Mitochondria preparations Digitonin particles 14 Beef heart mitochondria 68 73.8 48.2 Sonicated residue 64

47.4 35.7 100.5 55.8

electron microscopy NADH-~O2 succinate~O2 NADH~O2 NADH-->O2

54 55 55

56 56 55 55 55

* %Inverted orientation deterrnined fromelectron micrographs or from cytochrome c-stimulated respiration = (non-stimulated rate)/stimulated rate × 100%. ** ~oStimulationwithcytochromec -- [(stimulated rate)-(non-stimulatedrate)]/non-stimulated rate × 100%.

M i t o c h o n d r i a p r e p a r a t i o n s show a p p a r e n t heterogeneity as do s u b m i t o c h o n d r i a l particles as i n d i c a t e d in T a b l e V. D a t a f r o m this l a b o r a t o r y , as well as others, indicate t h a t up to 70 ~/o o f what is considered to be m i t o c h o n d r i a m a y well be s u b m i t o c h o n d r i a l particles, as evidenced by a substantial rate o f o x i d a t i o n o f r e s p i r a t o r y substrates after salt extraction in the absence o f exogenous c y t o c h r o m e c since cytoc h r o m e c is c o n t a i n e d within the s u b m i t o c h o n d r i a l particles present. R a t liver m i t o c h o n d r i a do n o t show such a high rate o f substrate o x i d a t i o n in the absence o f c y t o c h r o m e c as do beef h e a r t m i t o c h o n d r i a a n d are considered to be m o r e intact a n d to c o n t a i n less inverted particles. A f t e r r e p e a t e d washings with KC1, g o o d liver m i t o c h o n d r i a oxidize substrates at 5 ~ the c o n t r o l rate when c y t o c h r o m e c is n o t a d d e d to the assay mixture [58]. Electron t r a n s p o r t particles f r o m rat liver which show high oxidase rates a n d little reactivity with c y t o c h r o m e c have been described [59]. However, n o t all m e m b r a n e s can be described as either exclusively inverted o r n o r m a l l y oriented, or a mixture o f these types. As shown in Fig. 1, three othel situations are possible. Sonication m a y cause a fusion o f inverted a n d n o r m a l l y oriented particles, resulting in a h y b r i d particle reactive with c y t o c h r o m e c a n d with A T P a s e exposed since b o t h M a n d C sides are exposed. F u s i o n o f Escherichia coli m e m b r a n e vesicles by sonication has been r e p o r t e d [172]. S o n i c a t i o n m a y also cause

127

@ A

C

13

~ C D

c

E

Fig. I. Diagrammatic representation of membrane orientations, c represents cytochrome c. A, Mitochondrial oriented particle. B, Electron transport particle of inverted orientation to A. C, Hypothetical disordered particle as a result of sonic irradiation. D, Hypothetical fusion of mitochondrial and inverted orientation particle as a result of sonic irradiation. E, Fragmented particle with both surfaces exposed to the medium.

a displacement of individual proteins within the membrane from their physiological position. A possible result could be to displace a cytochrome across the membrane [80]. Other preparations may be in the form of sheets or ruptured vesicles that have both membrane faces exposed. These three orientations could provide the basis for substantial interactions with cytochrome c but lack of phosphorylation or net proton movement in inverted particles (cf. Table V) [11,57,80,81]. In light of these alternative arrangements diversity of exposure on vesicles may reflect differences in mitochondria or their response to fragmentation procedures.

11,4. Location of cytochrome c The biochemical criterion describing particle homogeneity is allied to the location of cytochrome c in the membrane. That cytochrome c is located on the exterior surface of intact mitochondria membranes and on the interior surface of inverted membranes is indicated by different experimental approaches. Tsou [60] found that cytochrome c could not be extracted from Keilin-Hartree preparations by washing. He also found that exogenous and endogenous cytochrome c were reduced at different rates, as have Lee and others (Table VI) [61,62]. Yet, repeated washing of rat liver mitochondria with dilute KC1 solution yields particles that are cytochrome c deficient and lose almost all respiratory activity [58]. However, as seen particularly in Table V, addition of cytochrome c to mitochondria restores respiratory

128 TABLE VI E F F E C T OF W A S H I N G A N D D E T E R G E N T T R E A T M E N T ON M I T O C H O N D R I A A N D INVERTED PARTICLES Succinate oxidase - cytochrome c + cytochrome c Sonicated particles KCl-washed sonicated particles Sonicated particles Sonicated particles + 2 mg deoxycholate Cytochrome c deficient sonicated particles Cytochrome c deficient sonicated particles + deoxycholate Alkaline particles Alkaline particles q- deoxycholate Alkaline particles Alkaline particles ÷ deoxycholate

0.163 0.173 0.260 0.343 0.060

0.159 0.182 0.277 0.322 0.10l

62* 62 62 62 62

0.029 0.92 0.10 3.90 0.08

0.332

62

1.00 1.21

56**

3.90 2.00

Cytochrome c oxidase stimulation with Lubrol Rat liver mitochondria 12 ~ stimulation Submitochondrial particles (rat) 234 ~/o stimulation Beef heart mitochondria 50 % stimulation Sonicated particles (beef) 300-400% stimulation Oxidase rates:

Reference

56 56 56

NADH-+02 NADH-~02

65 65 93 93

* /~atom/min per mg protein • * /~mole substrate oxidized/rain per mg protein

capability. As shown by Crane et al. [56], addition of cytochrome c has no effect on true electron transport particles since the membrane is impermeable to cytochrome c unless detergents are present to disrupt the structure of the membrane, destroying the intactness of the particle and allowing cytochrome c to be extracted with KC1 [63]. Submitochondrial particles extracted in this way can be reconstituted by incubating the particles deficient in cytochrome c in the presence of KC1 and detergent. "Opening" the inverted membrane by treatment with low levels of detergent makes the sequestered cytochrome c site accessible. Mackler and Green [64] have shown that with increasing amounts of deoxycholate, N A D H oxidase activity decreases, while N A D H cytochrome c reductase and cytochrome oxidase activity increase. Crane et al. [56] have shown a similar situation with succinate oxidase. Treatment of sonicated particles from cytochrome c-deficient mitochondria with deoxycholate elicits a 6-fold increase in succinate oxidase activity upon addition of cytochrome c only after deoxycholate is added [62]. Similarly, addition of Lubrol to submitochondrial particles increases succinate-cytochrome c reductase activity to a level resembling the mitochondrial rate and increases cytochrome c oxidase activity by more than 3-fold while having virtually no effect on rat liver mitochondria [65] (cf. Table VI). Studies on the effect of antibodies specific to cytochrome c complement the findings based on extraction and the membrane opening phenomena that the location

129 TABLE VII EFFECT OF ANTI-CYTOCHROME c ON MITOCHONDRIA AND INVERTED PARTICLES Data from Racker et al. [68]. ETP, electron transport particles. Assay

Ascorbate-PMS NADH oxidase Succinate oxidase

% net inhibition Mitochondria ETP 50 35 71

0 2 7

ETP sonicated with antiserum 100 83

of cytochrome c is on the inside of the inverted membrane, but exposed in mitochondria on the C side [66,67]. As seen in Table VII, addition of anti-cytochrome c antibody substantially inhibits succinate oxidase, N A D H oxidase, and ascorbate-phenazine methosulfate(PMS) activity in mitochondria, but not in submitochondrial particles [68]. Using 35S-labeled diazobenzenesulfonic acid ([asS]DABS)as a reactant in erythrocytes, Berg [69] has shown that virtually all hemoglobin remains unlabeled and that the membrane is impermeable to DABS. Employing this characteristic of DABS, Schneider et al. [70] have observed a difference in the amount of cytochrome c labeled by [asS]DABS in submitochondrial particles, mitochondria and isolated cytochrome c by determining the radioactivity of cytochrome c extracted and purified from the labeled membranes. A labeling ratio of 1 : 0.13: 1 was shown between mitochondria:inverted particles:isolated cytochrome c, indicating that cytochrome c is exposed in mitochrondria. This experiment affords an excellent example of the difficulties of interpreting data involving heterogeneous membranes. Our interpretation of the above data is that the inverted preparation used contained 87 % inverted vesicles and 13 % mitochondria or broken vesicles. Mitchell and Moyle [71] and Klingenberg and Bucholz [72] have shown that ferricyanide and ferrocyanide ions do not penetrate the mitochondrial membrane and can react only with components that are exposed to the medium. It has been shown that electron transport from ferrocyanide to molecular oxygen is restored in 95 % cytochrome c-deficient rat liver mitochondria by the addition of low levels of cytochrome c [73]. Because of a 4-fold stimulation in the rate of Fe(CN)63- reduction of succinate or N A D H upon addition of cytochrome c to rat liver mitochondria, Estabrook [74] similarly concluded that ferricyanide reacts at the level of cytochrome c. This point is illustrated in Table XII (section lIC). Further evidence of ferricyanide interaction with the electron transport chain will be presented in relation to discussion of cytochrome cl. Polylysine, protamine, and other basic proteins have been shown to inhibit the cytoehrome oxidase, cytochrome c reductase, and N A D H dehydrogenase portions of the electron transport chain [23,75-79]. The effect of polycationic proteins on mitochondria and inverted particles are summarized in Table VIII. Neither polylysine

130 TABLE VIII EFFECT OF POLYCATIONIC PROTEINS ON ELECTRON TRANSPORT IN MITOCHONDRIA AND INVERTED PARTICLES stimulation = rate with cytochrome c -- rate without cytochrome c × 100 9/oo rate without cytochrome c inhibition Inverted Mitochondria Ref. particles NADH oxidase

0

80

NADH-cytochrome c reductase

85

79

Cytochrome oxidase

85

100

Succinate oxidase

0

78

% stimulation with cytochrome c

0

400

5 nmoles polylysine 76 mg protein 5 nmoles polylysine 76 mg protein 5 nmoles polylysine 76 mg protein 2.5 mg protamine sulfate nag protein 2.5 mg protamine sulfate mg protein

nor protamine sulfate inhibit the overall oxidation of N A D H or succinate in electron transport particles, while a comparable level of basic proteins inhibits the majority of mitochondrial activity. The data in Table VIII for N A D H - c y t o c h r o m e c and cytochrome c oxidase indicate inhibition of these activities in inverted vesicles. The fact that these activities are present at all is evidence for mitochondrial contaminants or broken vesicles being present in this preparation, since evidence presented thus far indicates that cytochrome c is on the outer surface of the inner membrane (the inner surface of electron transport particles) and hence not available to impermeant reactants or inhibitors (including cytochrome c).

liB. Location of ATPase The presence of 90-A spheres on the matrix face of the inner mitochondrial membrane has frequently been used as a visual indication of the presence of wrongside-out particles. It should be remembered that inverted particles without the characteristic knobs can be produced and are seemingly indistinguishable from intact mitochondria [10,11,55,82,83]. Racker et al. [55] have shown that sonicated particles are stripped of projecting 90-A spheres (later denoted as F1) by treatment with trypsin and urea, resulting in a loss of ATPase activity without significant disruption of electron transport activity. Removal of radioactively labeled ATPase with urea accompanied by the disappearance of both ATPase activity and inner membrane spheres [17] and reconstitution of ATPase activity and knob appearance [17,20,21] upon addition of F1, have indicated conclusively that the 90-A spheres on the inner surface represent the ATPase. The use of antibody specific to F1 to inhibit ATPase activity and/or phos-

131 TABLE IX LOCATION OF F1 BY USE OF SPECIFIC ANTIBODY °/o inhibition by antibody

Phosphorylation NADH oxidase NADH-driven phosphorylation Succinate oxidase Succinate-driven phosphorylation Ascorbate-Oz Ascorbate-driven phosphorylation Phosphorylation Succinate oxidase

Ref.

Mitochondria

Washed Mitochondria

Submitoehondrial particles

19

0

60 76

68 84

72 76

84 84

59 33

84 84

87 93 < 1

84 85 85

15 -- 11

phorylation has shown the accessibility of F1 to antibody in inverted particles and its inaccessibility in mitochondria [84,85] (cf. Table IX). Since the inhibition varies with the source of antibody and attachment of F~ to an inverted particle, it is possible that allotopic changes in F1 due to attachment to the membrane may affect the binding of antibody. It is also interesting to speculate that different antibody preparations may not contain the antibodies of all F1 subunits [86]. It has been shown that antibodies to all coupling factor subunits of chloroplasts must be present for maximal inhibition [87,88]. Radioactive labeling with [asS]DABS has shown that only 5.3 ~o of the ATPase of beef heart mitochondria was labeled as compared to submitochondrial particles, which confirms the location of ATPase on the matrix side of the inner membrane. Numerous sources have reported the existence of ATPase inhibitors ranging in molecular weight from 5700 to 15 000 [89-91]. The inhibitory protein should be too large to cross the membrane and thus inhibit ATPase activity only in inverted membranes. Using the ATPase inhibitor prepared from beef heart [92] we have been able to demonstrate a 2.5-fold greater inhibition in electron transport particles than in mitochondria. On the basis of inhibition of succinate oxidase by polycationic protein, the electron transport particles are 86 ~o homogeneous (14 ~o inhibition) and the mitochondria that are not inverted can exist as either intact mitochondria or broken vesicles, the ATPase of broken vesicles being inhibited by the protein. On the basis of data presented in Table X, it is assumed that at best 70 ~o of the mitochondria in this particular preparation were intact, preventing ATPase inhibition. Huang et al. [93], using sonicated beef heart mitochondrial particles separated on a sucrose density gradient, have also shown a difference (3-fold) in ATPase activity between X particles (roughly 75 ~ inverted) and Y particles (approximately 67 non-inverted). Fibers are often seen associated with the 90-A knobs in the mitochondrial or

132 TABLE X EFFECT OF MEMBRANE ORIENTATION ON ATPase ACTIVITY AND INHIBITION BY ATPase INHIBITOR PROTEIN ETP, electron transport particles.

ETP Mitochondria

ATPase activity*

~o inhibition by ATPase inhibitor

Exposure of cytochrome c**

Exposure of cytochrome c***

0.62 0.15

68-77 27

14 79

30--44 70-78

* Expressed in/~moles P~ released/mg protein per rain. ** Determined by percent of suecinate oxidase inhibited by polylysine. *** Determined by percent of antimycin sensitive succinic ferricyanide reductase inhibited by addition of cytochrome c. submitochondrial particles after treatment with detergents and have been termed membranifibrils [94,95]. These fibrous preparations are usually devoid of cytochromes and respiratory enzymes and contain variable amounts of phospholipid. Because of their close association with the 90-A ATPase spheres, it is suggested that the fiber is situated beneath the F1 in the membrane. Racker's group describes similar fibrous material devoid of F1, cytochrome, respiratory enzymes and phospholipid, which is denoted CFo [40,96,97]. CFo can be reconstituted with F~ to give CFo-F1 with a structure similar to that of membranifibrils. Oligomycin-sensitive ATPase activity in CFo-F1 is restored upon addition of phospholipid [12]. CFo proteins or membranifibrils may be the site of attachment of F1 to the membrane.

IIC. Location of NADH dehydrogenase (FpN) The location of N A D H dehydrogenase in rat liver mitochondria was noted by Lehninger [98] who suggested that the inner membrane was impermeable to N A D H since only endogenous N A D H oxidation was coupled to phosphorylation. The noncoupled oxidation was insensitive to antimycin, identifying it as occurring in the outer membrane or microsomal contaminants. A similar observation has been made using beef heart mitochondria, where N A D H is oxidized at a rate comparable to that in submitochondrial particles, but without phosphorylation [57] (use of Krebs' cycle substrates yields phosphorylation). It was assumed that any antimycin-sensitive N A D H oxidation by 02 or cytochrome e was an indication of damaged mitochondria (leaky, broken) that subsequently could not be expected to phosphorylate [99]. As indicated in Table XI, the specific activities of mitochondria and electron transport particles differ greatly even though the composition of the two particle types is essentially the same. A suitable explanation of this finding is that FpN is exposed to the substrate in electron transport particles and more rapidly reduced, whereas FpN is inside the mitochondria and N A D H must cross the relatively impermeable membrane [100]. The rate-limiting step of N A D H oxidation in mitochondria, passage of substrate through the membrane, is time dependent since N A D H in-

133 TABLE XI OXIDATION OF NADH AND SUCCINATE BY 02 IN MITOCHONDRIA AND ELECTRON TRANSPORT PARTICLES Data expressed as/~moles substrate oxidized/mg protein per rain. NADH Electron transport particles 5.29 Mitochondria 2.08

Succinate

NADH*

Succinate*

2.82 1.20

4.15 1.37

0.83 0.2

* From Crane, et al. [56].

corporation into the mitochondrial interior and oxidation occurs at a rate 103 times that of transfer [101,102]. Lee [103] found that pigeon heart mitochondria will reduce only endogenous N A D + by reverse electron transport with a lag period while submitochondrial particles will reduce only exogenous N A D ÷ with no lag period. The location of FpN can also be ascertained by measuring ferricyanide reduction [104]. As shown in Table XII, the addition of rotenone stimulates ferricyanide reduction in inverted particles, but totally inhibits it in intact rat liver mitochondria at 10-3 M Fe(CN)63-, indicating the presence of the primary dehydrogenase on the inside of the membrane. Von Jagow and Klingenberg [105] have used ferricyanide reduction before and after sonication to show N A D H dehydrogenases on both sides of yeast inner membrane. The location of a component involved in duroquinone (2,3,4,6-tetramethyl1,4-benzoquinone) reduction and oxidation has been elucidated with the use of poly(L-lysine). Ruzicka and Crane [23] found 60 ~o inhibition of NADH-duroquinone

TABLE XII FERRICYANIDE REDUCTION IN MITOCHONDRIA AND SUBMITOCHONDRIAL PARTICLES From Grinius et al. [ll2]. A,,t/mg protein per min Submitochondrial particles + NADH Submitochondrial particles + NADH + rotenone + antimycin A Mitochondria -F- glutamate-malate Mitochondria + glutamate-malate + antimycin A Mitochondria + glutamate-malate + rotenone + antimycin A

0.158 0.187 0.040 0.000 0.000

134 reductase in sonic particles with 3.2 mg/ml poly(L-lysine), indicating either the existence of two poly (L-lysine) sites, one on either side of the duroquinone reaction site with different sensitivities or the inhibition of both oxidation and reduction via a non heme iron protein of mol. wt 16 000 [13].

liD. Location of succinate dehydrogenase (Fps) Indirect evidence for the location of Fps on the outer surface of inverted membranes comes from the fact that almost all reconstitutions of succinate systems utilize particles made deficient in Fps by numerous procedures [106-109]. The rate of oxidation of succinate by rat liver mitochondria has been found to depend upon the concentration of succinate in the reaction mixture and is affected by the degree of ion pumping in the system, with a 5-fold increase in Km in the presence of uncouplers as compared to gramicidin [110]. The difference in specific activity as seen in Table XI can be explained on the basis that succinate need not cross a relatively impermeable membrane to reach the dehydrogenase in inverted particles. The use of ferricyanide reduction by succinate has also indicated the placement of Fp~ on the inside. Purified Fp~ has been shown to catalyze antimycin A-insensitive Fe(CN)6 a- reduction. Ferricyanide reduction in submitochondrial particles has been shown to be partially insensitive to antimycin A while in mitochondria it is almost completely inhibited [72,104,111,112] (cf. Table XIII), thus pointing to the inaccessibility of Fe(CN)63-to Fps in mitochondria. The existence of Fp~ on the matrix face of yeast mitochondria has been shown by Von 3agow and Klingenberg [105].

TABLE XIII ANTIMYCIN INHIBITION OF REDUCTION OF FERRICYANIDE BY SUCCINATE IN RAT LIVER MITOCHONDRIA AND INVERTED PARTICLES inhibition by antimycin A Inverted particles Rat liver mitochondria Reference

10 93 112

0 93 111

66 7

liE. Location of iron-associated components We have observed inhibition of succinate oxidase by bathophenanthroline sulfonate in intact mitochondria but not in alkaline-prepared electron transport particles, indicating the presence of a chelator-sensitive component on the C side. Similar inhibition has been observed in rat liver mitochondria [174]. Bathophenanthroline sulfonate-sensitive sites of N A D H oxidase are located on both the C and M sides. Since duroquinol oxidase is not inhibited in alkaline particles and is greatly stimulated in mitochondria these sites must be located between Fp~ and the common segment of the transport chain on the C side and between FpN and the duroquinol

135 reactive site on both the C and M side. Inhibition of NADH oxidase and stimulation of duroquinol oxidase has been reported in sonicated particles [13]. Substantiation of these conclusions is seen by the inhibition of NADH oxidation to ferricyanide, duroquinone, juglone or cytochrome c (in mitochondria) in normal or inverted particles, but the inhibition of succinate oxidation by ferricyanide and cytochrome c (in mitochondria only) in mitochondrially oriented or detergent treated particles. In contrast to water-soluble bathophenanthroline sulfonate, the lipid-soluble bathophenanthroline inhibits not only NADH, succinate and cytochrome c oxidase but also duroquinol oxidase [175 ] when added from either side of the membrane. The contrast between the effects of a water-soluble and lipid-soluble form of a chelator suggests that chelator-sensitive sites such as certain iron sulfur or copper proteins [176] are buried inside the membrane.

IIF. Location of Complex III Estabrook [74] concluded that, because of the stimulated Fe(CN)6 a- reduction in the presence of cytochrome c, ferricyanide is reduced preferentially at the level of cytochrome c and less efficiently at the level of cytochrome cl, placing both cytochromes c and cx on the C side of the membrane. Similarly, Klingenberg [113] places cytochromes c~ and b more toward the M side or in the middle of the membrane because of their slow reactivity with ferricyanide. Antibodies to cytochrome cl have shown this cytochrome to be on the C side [67]. However, the possible presence of OxF [167] as well as a glycoprotein [168] in purified preparations of cytochrome c~ [167] raises the question of antibody specificity. Data from this laboratory involving ferricyanide reduction by homogeneous electron transport particles indicates the existence of cytochrome c~ (or non-heme iron III) on the inside of the membrane (cf. Table XIV) as shown by substantial antimycin-sensitive ferricyanide reduction. The remaining activity after the addition of antimycin is insensitive to thenoyltrifluoroacetone [111], indicating activity of the primary dehydrogenase. It is of interest that the specific activity of mitochondria, with 84~ of its Fps supposedly sequestered and inaccessible (as determined by cytochrome c activity), to Fe(CN)63is similar to that of inverted particles, yet yields a higher inhibition by antimycin, indicating that, indeed, 80--90 ~ of the membranes are turned right side (C side) out. The high specific activity of mitochondria indicates that ferricyanide can react with cytochrome c~ through the unoccupied site ofcytochrome c. This finding is supported by the study of Estabrook [114] (Tables XII and XIII) where ferricyanide reduction is inhibited by antimycin in inverted particles as well as mitochondria, with a loss in phosphorylation [115]. The presence of antimycin-insensitive, KCN-sensitive ferrocyanide oxidation in electron transport particles indicates that a component between cytochrome c and the antimycin site, presumably cytochrome c~ and/or the iron sulfur protein III, is exposed on the M surface. As seen in Table XV, this oxidation is polylysine insensitive and not stimulated by cytochrome c. In contrast, ferrocyanide oxidation in beef heart mitochondria is greatly stimulated by exogenous cytochrome c and inhibited by

0.17 0.15

2.8

1.37 1.22

2.8

0.14 0.15

2.6

÷ protamine sulfate

16

96-100

3/o ETP

0.45 0.37

0.47 0.4 0.52

0.005 0.007

0.17 0.1 0.23

90 82

64 75 55

Succinate ferricyanide* (/tmoles Fe(CN)3-6/min per mg protein) -- Antimycin A -t- Antimycin A % inhibition

* Assays according to Crane et al., [56] except 0.05/~mole succinate used in place of N A D H and 0.2/~mole KaFe(CN)6 used in 1 ml cuvette ** Assays contained 0.06 mg protein. *** Mitochondria assays contained 0.05 mg protein, 1/~g antimycin A added where indicated to cuvette, 0.2 nag cytochrome c added where indicated, 0.25 mg protamine sulfate, pH 7.0, added where indicated.

Mitochondria***

Electron transport particles **

Succinate oxidase* (t~atom 0/min per mg protein) -- cytoehrome c + cytochrome c

TABLE XIV INHIBITION OF SUCCINATE-FERRICYANIDE REDUCTASE BY ANTIMYCIN A IN ORIENTED PARTICLES

U,

137 TABLE XV FERROCYAN1DE OXIDATION IN BEEF HEART MITOCHONDRIA AND ELECTRON TRANSPORT PARTICLES

Mitochondria Electron transport particles

/~atom 0/rain per mg protein -cytochrome c ÷ cytochrome c

~o inhibition with polylysine*

0.05 0.74

60-86 ~o none

0.8 0.74

* 0.2 mg poly (L-lysine) (mol. wt 70 000) added.

polycationic proteins. These data illustrate that different ferrocyanide-reactive sites are exposed on the M and C sides of the membrane. In addition, the level of ferrocyanide needed to achieve 0.5 V is 2-5 times greater in electron transport particles than in mitochondria, suggesting kinetically different sites on opposite sides of the membrane.

IIG. Location of cytochrome b The location of cytochrome b is unclear. Detergent fractionation of the membrane [14] gives a concentration of cytochromes b and cl in the light membranous fraction stripped off from the membranous pellet which contains cytochromes a and a3. The small amount of cytochrome b associated with the cytochrome a and a3 fraction has an absorption maximum at 565 nm in contrast to the maximum at 562 nm of the cytochrome b associated with cytochrome Cl which suggests distribution of b cytochromes on two sides of a binary membrane [116,117]. Inhibition with hydroxynaphthoquinones [53] also suggests that some site associated with cytochrome b is on the M side of the membrane. However, on the basis of the nonreactivity of cytochrome b in inverted particles or mitochondria with impermeable electron donors such as ferrocyanide, cytochrome b is believed to be located in the center region of the membrane, protected by hydrophobic layers from the exterior environment of the membrane [112]. Recently an arrangement of b cytochromes across the membrane has been suggested [177] that is consistent with our fractionation studies. IIH. Location of cytochromes a and aa It is logical to place cytochrome a on the C side of the mitochondrial membrane where it can react with cytochrome c and have its site of interaction with cytochrome c blocked by a polycationic protein (see Section IIA). However, the location of cytoehrome a3, while placed on the inside of the membrane in terms of Mitchell's chemiosmotic theory, is not easily demonstrated. Studies using specific antibody to cytochrome oxidase have shown that the antibody does not react with cytochrome c [118 ], does precipitate soluble cytochrome oxidase from rat liver mitochondria [119], and inhibits cytochrome oxidase only

138 TABLE XVI LOCATION O F C Y T O C H R O M E POLYCATION CROSS LINKING

OXIDASE

Mitochondria Anti-cytochrome oxidase* Anti-cytoclu'ome oxidase* lasS]DABS label** Km DABS treated K,n control Inhibition of succinate reduction of cytochrome aa3 by cross linking Inhibition of dithionite reduction of cytochrome aa3 by cross linking Inhibition of succinate reduction of cytochrome b by cross linking Inhibition of dithionite reduction of cytochrome b by cross linking

BY

ANTIBODY, DABS LABELING AND

Sonicated particles

Cytochrome oxidase Ref.

60% 60% 120 57~ 65% 50~ 68 265 cpm/mg protein 197 cpm/mg protein 1580cpm/mg protein 70

-

0.54

1.19

70

100 %

68 ~

70

37

44 %

70

48 %

48 %

70

26 ~

34%

70

* Percent inhibition of cytochrome c oxidase activity by antibody. ** Oxidase purified from DABS labeled membranes.

about 60 %, either in isolated form or in Keilin-Hartree particles [120]. Experiments performed by Racker et al. [68] provide similar information (Table XVI). Anticytochrome oxidase antibody has been reported to decrease P/O ratios in mitochondria only, and inhibit only the oxidation of added cytochrome c while not inhibiting N A D H or succinate oxidation in mitochondria or submitochondrial particles [66]. A confusing detail is how submitochondrial particles can oxidize reduced cytochrome c at a rate more than twice that of mitochondria [68] if they are indeed submitochondrial particles. Localization of cytochrome oxidase by radioactive labeling provides data that support the antibody findings (Table XV1). Cytochrome oxidase isolated from [35S]DABS-labeled particles or mitochondria exhibited similar amounts of label [70], suggesting that cytochrome oxidase is transmembranous and can be labeled from either side of the membrane [67]. It is important to note that denatured ATPase comprises 20-35 % of "structural protein" from submitochondrial membranes and contains 60-80 % of the bound ATPase [20,21]. This "structural protein" could be present in purified cytochrome oxidase pellets and give higher counts in oxidase purified from [asS]DABS-labeled submitochondrial particles. After electrophoresis of the structural protein from inverted particles reconstituted with 3H-labeled ATPase, 90 % of the radioactivity was found in one protein band. When membranebound F1 was labeled with 3H-labeled acetic anhydride and subjected to electrophoresis, radioactivity was also associated with one major band that migrated

139 similarly to the labeled structural protein band [20]. Such evidence, while circumstantial, suggests that many of the counts associated with cytochrome oxidase may have originated from [35S]DABS-labeled ATPase. DABS was found to affect the Km of cytochrome oxidase for cytochrome c in mitochondria to a greater extent than in submitochondrial particles, strongly suggesting that cytochrome a is on the C side of the membrane where it can bind cytochrome c [70]. Crosslinking polylysine to the surface of submitochondrial particles or mitochondria completely prevents cytochrome aa3 reduction by succinate in mitochondria and prevents of cytochrome aa3 reduction in submitochondrial particles while inhibiting dithionite-reducible cytochrome aa3 by 37 ~ and 44 ~, respectively [70] (cf. Table XVI). If cytochrome a were located on the C side and cytochrome a3 on the M side (assuming homogeneous preparations) crosslinking in mitochondria would result in 50 ~ inhibition of dithionite-reduced cytochrome aa3 but 100 ~ inhibition of succinate-reduced cytochrome aa3. Crosslinking in submitochondrial particles would result in a 50 ~ reduction of dithionite-reduced cytochrome aaz and 50 ~ inhibition of succinate-reduced cytochrome aa3. The experimental data are in fair agreement with theoretical expectations, although minor mitochondrial contamination is present in the inverted membrane preparation. The use of sodium azide to inhibit cytochrome oxidase is another approach to investigate the localization of cytochrome a3. Rat liver submitochondrial particles have been shown to be 20 times less sensitive than mitochondria when both exhibit ion-pumping activity (Table XVII) due to an active concentration of N3- inside the mitochondria [121]. Since azide inhibits cytochrome a3 [122], the latter should be on the M side (knob side), a conclusion supported by the observation that the concentration of N3- needed for 50 ~ inhibition of succinate oxidase in particles is 18 times greater than that needed to inhibit N,N,N',N',-tetramethyl-p-phenylenediamine dihydrochloride (TMPD)-driven cytochrome oxidase or succinate oxidase in mitochondria. However, state-3 or state-4 mitochondria have been found to be able to accumulate azide to a level only twice that of the external medium [123]. Under the conditions used to obtain the data in Table XVII, alkalization of the mitochondrial matrix occurs [124]. Since azide is less inhibitory at alkaline pH (K~ at pH 8.0)/(Kl at pH 7.0) -- 26.2) [125,126] a minimum 260-fold accumulation of N3within the mitochondria is necessary for mitochondria to be 20 times more sensitive to azide than particles, disregarding permeability factors and assuming cytochrome a3 is on the knob side. Nicholls and Kimelberg [122], however, point out that under steady-state conditions, the apparent K~ of N3- in succinate oxidase activity of KeilinHartree particles may be 10 times the real K~. The similarities in inhibition of 02 uptake in inverted particles and uncoupled mitochondria could also be explained by the equilibration of H ÷ between the internal and external phases in the presence of carboxycyanide-m-chlorophenylhydrazone(CCCP) [124]. The situation is confused further by evidence that inverted particles are more sensitive to N3- than mitochondria. Nicholls [127] has noted that cytochrome c-deficient submitochondrial particles show

140 TABLE XVII AZIDE CONCENTRATION REQUIRED TO ACHIEVE 50 ~o INHIBITION OF OXIDASE ACTIVITY Condition

Inverted particles (mM)

Mitochondria (mM)

Cytochrome oxidase Ref. (mM)

Succinic oxidase Succinic oxidase* Ascorbate TMPD**

1.4 0.45 0.18

0.02 0.01

0.1

122 121 121, 122

* TMPD-cytochrome c oxidation. ** Valinomycin present.

increased azide sensitivity. Since this type o f particle was n o n - e n e r g y linked, t r a n s p o r t p h e n o m e n a d o n o t confuse the interpretation, which we s u m m a r i z e in T a b l e X V I I I . The c y t o c h r o m e c - d e p e n d e n t rate (salt inhibited) represents m i t o c h o n d r i a l o r i e n t a t i o n o f the m e m b r a n e a n d the rate unaffected by salt represents the inverted o r i e n t a t i o n which is m o r e sensitive to azide. The greater sensitivity o f inverted particles indicates t h a t c y t o c h r o m e aa is on the matrix (M) side o f the m e m b r a n e . Crosslinking d a t a a n d inhibition o f c y t o c h r o m e c o x i d a t i o n s u p p o r t the placem e n t o f c y t o c h r o m e a on the C side [70], b u t the d a t a cited a b o v e only suggest that c y t o c h r o m e aa is m o r e accessible to the M side o f the m e m b r a n e t h a n c y t o c h r o m e a. A n alternative a r r a n g e m e n t is that c y t o c h r o m e a is on the C side o f the m e m b r a n e while c y t o c h r o m e a3 is on neither. C y t o c h r o m e a 3 could be situated inside a b i n a r y m e m b r a n e a n d n o t exposed to either surface. Such an a r r a n g e m e n t c o u l d possibly a c c o u n t for the azide effects r e p o r t e d by K l i n g e n b e r g [121], since N a - c o u l d l:e a c c u m u l a t e d within the m e m b r a n e to a higher c o n c e n t r a t i o n t h a n observed in the matrix. Mitchell a n d M o y l e [169] have suggested that c y t o c h r o m e a3 is on the inside ( M side) o f the m e m b r a n e because o x i d a t i o n o f external ferrocyanide, p r e s u m a b l y

TABLE XVIII EFFECT OF AZIDE ON SUCC1NATE OXIDASE ACTIVITY OF SUBMITOCHONDRIAL PARTICLES AT HIGH AND LOW SALT C O N C E N T R A T I O N Condition

Succinate oxidase* No azide Plus azide

% inhibition

Active particle orientation

0.02 M phosphate

12

9

25

0.75 7

85 0

normal and inverted inverted normal

0.05 M phosphate Increment dependent on low salt

5 8

* Succinate oxidase with no cytochrome c added. Azide at 1.3 raM. Data from ref. 127.

141 through cytochrome c in mitochondria, shows a very slow decrease of protons in the external media instead of the rapid decrease expected if the oxygen was reduced at the cytochrome a3 level on the exterior of the membrane according to the reaction: 2e- -q- 2H + q- O cytochrome aa H 2 0 I f an uncoupling agent is added during ferrocyanide oxidation by mitochondria the proton decrease (pH increase) in the external media is significantly increased. This p H change is consistent with the idea that uncouplers increase the permeability of the membrane to protons and allow movement of external protons into the interior of the mitochondria to replace protons removed by water formation. Further investigation is necessary to define the location of cytochrome aa in the membrane. A summary of the location of components of the electron transport chain is given in Table XIX.

TABLE XIX LOCATION OF INNER MEMBRANE PROTEINS (I), inside (M-side); (0), outside (C-side); (M), middle (is not exposed on either the inner or outer surface); cyt, cytochrome; NHI, non heme iron protein. Well established

Tentative

cyt. c (0) cyt. cl (I) cyt. a (0) NHI III 30M (1) NADH dehydrogenase (I) cyt. az (I) Succinate dehydrogenase (I) F 1 (I)

Suggested

Unknown

NHI~b 16m (I) cyt. b• (I) CFo (I)

cyt. b (M) NHI~[ 28 (0)* NHIla 27 (0)* Transhydrogenase Copper protein

* If bathophenanthroline sulfonate reacts with non-heine iron its inhibitory effects suggest that NHI. and some part of the NHI~ are on the C side.

H L Proteolytic digestion of the inner membrane By following the time-dependent disappearance of protein bands on polyacrylamide electrophoresis gels after mild proteolysis of orientated particles, the location of exposed polypeptides can be determined. We have observed the lability of some protein units to 15-90 min incubation with 0.005 mg trypsin per mg membrane protein. Bands corresponding to the following molecular weights disappear in inverted particles: 57 000; 63 000; 68 000; 75 000; 81 000, and 85 000. In what can be described as fragmented membranes, several polypeptides with molecular weights in excess of 80 000 are labile, as are numerous species in the 60 000-70 000 range. The disappearance of high-molecular-weight bands after trypsin treatment ofsonicated particles has also been observed by Hare [13].

142 III. ULTRASTRUCTURE STUDIES

IliA. Electron microscopy Electron microscopy has been used to elucidate the ultrastructure of mitochondrial membranes. Those techniques which have provided the most information include thin sectioning, negative staining, and freeze-etching. Cytochemistry has yielded supplementary data supporting an asymmetrical arrangement of components in the inner mitochondrial membrane. The ultrastructure of the mitochondrion was first described independently by Palade [128] and SjSstrand [129]. A mitochondrion as seen in thin section is shown in Fig. 2. The two membrane systems and the various compartments are identified. The nature of the electron-dense granules in the matrix has been reviewed recently [130]. The fine structure of the mitochondrial membrane in thin section depends on the fixation technique. Not only do the dimensions and staining properties of the membrane vary but also the arrangement of the cristae membranes with respect to the intracristal space. These differences are illustrated in Fig. 3. With glutaraldehyde fixation the cristae membranes are apposed, thus obliterating the intracristal space (Fig. 3A). The inner membrane measures about I00 A thick. If glutaraldehyde fixation is followed by post-fixation in OSO4, the cristae membranes become separated revealing the intracristal space (Fig. 3B). The membranes have some electron-dense regions in the center and are thinner by about 20 A. In KMnO4-fixed mitochondria the cristae are frequently apposed (Fig. 3C). The membrane thickness varies from 75 to 100 A. This variation is due to the irregularity and poor definition of the membrane edges. In OsO4-fixed mitochondria the cristae membranes are separated and appear almost uniformly electron dense throughout (Fig. 3D). The membranes measure about 75 A thick. The reported dimension of the inner mitochondrial membrane varies from 50 to 150 A, depending upon the fixation procedure used. A summary of these reports is presented in Table XX. One must decide which method of preparation preserves the in vitro structure of the mitochondrial membranes most closely. Conventional fixation procedures result in changes in the circular dichroism spectra of proteins and in the X-ray diffraction patterns of myelin membranes [131,132]. The commonly used dehydrating agents ethanol and acetone denature proteins. SjSstrand and Barajas [133] have devised a method of sample preparation for electron microscopy which preserves the native conformation of protein. This procedure involves perfusion with glutaraldehyde to crosslink proteins, short term dehydration in ethylene glycol, and embedding in Vestopal W. Mitochondrial membranes prepared in this manner appear considerably thicker (150 A) than when conventional fixation procedures are used. Intentional heat denaturation of the membrane protein after perfusion results in a thinner membrane (90 A). The use of acetone as the dehydrating agent in the above procedure likewise results in a thinner

143

Fig. 2. A mitochondrion in the flight muscle of the blowfly. OM, outer membrane; IM, inner membrane; M, matrix; ICS, intracristal space; OC, outer compartment; EDG, electron-dense granule. Glutaraldehyde-OsO4. Note the black particles on the matrix side of the cristae membranes. Particles of this type have been observed in several types of mitochondria and are consistent with the idea that FI projects into the matrix, x 80 000.

144

Fig. 3. Comparison of the cristae membranes of isolated beef heart mitochondria after different fixations. A, Glutaraldehyde. B, Glutaraldehyde-OsO4. C, KMnO4. D, OsO4. A, B, and D post-stained with uranyl acetate and lead citrate; C not stained. Note particles mostly associated with the matrix side of the membrane in B and D. × 200 000.

145 TABLE XX THICKNESS OF THE INNER MITOCHONDRIAL MEMBRANE AS MEASURED IN THIN SECTIONS Thickness in A

Method of preparation and fixative

Ref.

In situ mitochondria 50-60 KMnO4 50 OsO4 70 OsO4 1151,2 Freeze-drying; OsO4 vapors 601 Freeze substitution; OsO4 1401.2 Formaldelayde 60-70 Formaldehyde q- OsO4 1493 Glutaraldehyde 504 Glutaraldehyde

138 138 139 140 141 141 141 133 133

Isolated mitochondria OsO4 70-80 OsO4 49 54 s OsO4 Glutaraldehyde q- OsO4 68 685 Glutaraldehyde q- OsOa lO01 Glutaraldehyde Glutaraldehyde q- OsOa 80 75 OsO4 75-1001 KMnO4

134 35 35 142 142 143 (see Fig. 2) 143 (see Fig. 2) 143 (see Fig. 2) 143 (see Fig. 2)

1 Cristae membranes frequently apposed. Total width of two apposed membranes. 3 Ethylene glycol dehydration. 4 Acetone dehydration. 5 Lipid-extracted mitochondria.

50-A membrane, with or without heat denaturation. The similarity in appearance and dimension of conventionally prepared tissue and intentionally denatured tissue suggests that the images observed in thin sections of conventionally prepared tissues represent denatured membrane proteins. A second point which must be considered in determining the thickness of the inner mitochondrial membrane is the location of the inner membrane particles (FI). By biochemical and immunological techniques the F1 protein has been localized on the matrix surface of the inner mitochondrial membrane [11,20,21,55,87]. The negative-staining technique has revealed the presence of 90-A spheres projecting from the surface of the inner membrane [134], and from the membrane after glutaraldehyde fixation [137]. They have subsequently been identified as the F1 ATPase [78]. The projecting inner membrane particles have been observed repeatedly in thin section [35, 135, 136] (see also Fig. 1). The reality of the FI protein is well established. The question is rather how are these particles associated with the membrane in vivo? Conceivably, the particles could project from the membrane by stalks, be flattened onto the membrane surface,

146 or be embedded into the membrane structure. Both negative staining and conventional fixation techniques are capable of altering the in vivo structure. Assuming a membrane 150 A thick, as determined by Sj6strand and Barajas [133], it is possible for the 90-A F 1 particles to be embedded in or to be part of the membrane continuum. The freeze-etching technique has provided additional information about the organization of mitochondrial membranes because it reveals aspects of membrane structure not visualized with negative-staining or thin-sectioning techniques. In addition, preparation of samples for freeze-etching does not require the use of chemical fixatives, dehydrating agents, or embedding media. Two types of membrane surfaces are revealed by the freeze-etching technique. Fracture faces are exposed as a result of a fracture through hydrophobic regions of the membrane interior. Etched faces are revealed following sublimation of ice from the natural surface of the membrane. Freeze-etching of isolated mitochondria and of purified mitochondrial membranes reveals the presence of particles on both fracture faces of outer and inner mitochondrial membranes [144-147]. The results of these studies are summarized in Table XXI. Both inner and outer membranes have an asymmetrical distribution of particles on the two halves of the fractured membranes. The fracture faces having the greatest particle densities are the matrix half of the inner membrane and the cytoplasmic half of the outer membrane. The size of the observed particles thought to represent membrane proteins range from 50 to 150 A. Packer [148] has found that membrane dehydration modifies the particle distribution in the two halves of the inner membrane. He has also shown that

TABLE XXI CHARACTERISTICS OF F R E E Z E - F R A C T U R E A N D F R E E Z E - E T C H FACES O F ISOLATED M I T O C H O N D R I A A N D M I T O C H O N D R I A L M E M B R A N E S Size, distribution and clustering of fracture face particles is the same in wild type and respiratory deficient yeast [171 ]. + , relative particle density. IMS, intermembrane space. Source of mitochondria Rat liver [144,145] Neurospora [146,147l Fracture faces Outer membrane Cytoplasmic side IMS side Inner membrane IMS side Matrix side Etched faces Outer membrane Inner membrane Matrix Orthodox Condensed

Particulate ( + + + + ) Particulate ( + )

Particulate ( + + ) Particulate ( + )

Particulate ( + + ) Particulate ( + + + + )

Particulate ( + + ) Particulate ( + + + )

-

Smooth Smooth

Granular Fibrous

Fibrous Smooth

147 extraction of membrane lipid from submitochondrial particles disrupts the distribution of particles on the fracture faces and results in clustering of particles. This later observation supports the fact that the particles are protein rather than lipid. The etched faces of submitochondrial particles and inner mitochondrial membranes are smooth, showing no evidence of stalked inner membrane particles [144, 146,147]. This suggests that the F1 particles do not project from the inner membrane in vivo. If the F~ particles were partially embedded in the membrane surface one would expect to see a cobblestone pattern on the etched surface. A smooth surface would indicate that the F~ particles are located entirely within the membrane, are flattened into a continuous layer against the membrane, or are removed from the membrane during preparation of the sample for freeze-etching.

IIIB. Cytochemical staining Recent developments of cytochemical methods for ultrastructural demonstration of oxidative enzymes has prompted the use of this approach for the investigation of enzyme localization in the inner mitochondrial membrane. The ultrastructural localization of succinate dehydrogenase (Fps) activity in mitochondria has been studied using various electron or hydrogen acceptors including ferricyanide [149, 150] and numerous tetrazolium salts [151-153]. In general, the tetrazolium salts tended to yield reaction products which were deposited on the cristae membranes or accumulated in the intracristal space and outer compartment. Conflicting reports of Fps localization have been obtained with the ferricyanide method. Ogawa et al. [150] observed an accumulation of reaction product on the mitochondrial membrane and in the intermembrane space (intracristal space plus outer compartment). In contrast, Kalina et al. [14] reported the localization of reaction product on the cristal membrane facing the matrix with short incubation. However, with prolonged incubation, the reaction product filled the intracristal space. Originally, they interpreted these contradictory results as arising from differences in the nature of the aeceptors [147]. Later, they dismissed the results obtained with the ferricyanide method because of the soluble nature of the reaction product [153]. Reaction of ferricyanide at sites on both sides of the membrane is consistent with enzyme studies described previously in this paper. The ultrastructural demonstration of cytochrome oxidase activity has utilized diaminobenzidine as the electron donor [154-156]. Several laboratories have observed the reaction product on the surface of the inner mitochondrial membrane facing the intracristal space and the outer compartment. Longer incubation results in reaction product filling the intermembrane space. Occasional staining of the outer membrane has been attributed to cytochrome bs or monoaminoxidase activity [154]. The compound diaminobenzidine is thought to donate electrons to the respiratory chain at the level of cytochrome c based on studies with specific inhibitors of electron transport, thus suggesting the localization of cytochrome c on the outer surface of the inner mitochondrial membrane [154]. The location of cytochromes aa3 are not clearly established by this experiment.

148 The use of diaminobenzidine in the cytochemical demonstration of oxidative enzymes must be interpreted with caution as diffusion artifacts [157] and nonenzymatic staining reactions have been reported with this substrate [158]. Investigation of the cytochrome oxidase activity assayed polarographically with diaminobenzidene as substrate and the resulting ultrastructural staining reaction indicate a direct relationship [155]. The use of 4,4'-diamino-2,2'-dipyridyl (DABP)-ferrous chelate as an electrondense probe for cytochrome c results in electron-dense deposits on both the inner surface of the outer membrane and the cristae membranes [159]. Data indicate that the chelate enters the respiratory chain between cytochromes b and aaa. The cytochemical technique has localized mitochondrial ATPase activity within the matrix [160,161 ]. IIIC. Schemes for membrane structure As seen in Fig. 4, recent models of cristae structure indicate at least one bilayer

A

0

OUTSIDE

B

L INSIDE

H

ATP

yUPLING FACTORS

_ t21 °I H I C'SIDE

MATRIX

I M-SIDE

Fig. 4. Schemes of cristae organization. A, Multi-molecular complex model [133]. B, Internalized ATPase representation [148]. C, Ortho-topographical representation [68]. All of these models would be consistent with membranes 75 to 100 A thick with two or more layers of intrinsic protein. Each would show protein globules on inner freeze fracture surfaces. For scheme B extraction of F1 ATPase would be expected to remove internal globules whereas scheme C would indicate that extraction of F1 would not change the freeze fracture surfaces. See Table XXI.

149 of lipid and at least one bilayer of protein corresponding minimally to a modified unit membrane [3,4]. The molecular complex model of Sj6strand and Barajas [133] (Fig. 4A), while binary in nature, is significantly different from other models in that the membrane proteins are arranged in three dimensions in a way that allows interacting components in Complexes I-III to be separated by a minimal distance and allowing interaction between the discrete complexes. In addition, the width of the membrane is drawn as 150 A while other models assign 80-100-,~ widths. This difference in width may be explained by the fact that phosphorylation complexes (F factors) are not shown in the scheme. Hatase et al. [162] have obtained evidence that 90-A F~ knobs may be extended from the membrane surface by a 50-A stalk or collapsed on the membrane surface, depending on the configurational state of the mitochondrion. If the repeat interval of F~ on membranifibrils (120 A) [94,95] is postulated to exist in three dimension in situ, then collapse of F1 onto the membrane surface could conceivably increase the 90-A intrinsic thickness by at least 30 A to a value comparable to that of Sj6strand and Barajas [133]. However, extrinsic protein (F~) is detached from the membrane by acetone extraction without significant change occurring in the image of sectioned membrane [35,36], indicating that lipid is involved in the attachment of F~ and that extrinsic proteins are not visible in cross-sections of membranes fixed for electron microscopy by conventional means. The fact that the 90-A F1 knobs remain on membranes fixed with glularaldehyde before negative staining suggests that the knobs do project from the membrane [137]. On the other hand, the lack of any projecting particles on the matrix side of the inner membrane exposed by freeze-etching has been presented as evidence that the large ATPase complex (mol. wt 360 000) is inside or closely appressed to the membrane surface. Indeed, the model (Fig. 4B) based largely upon freeze-fracture evidence and published data views the membrane components as being predominantly located near the surface, with a greater density of particles appearing on the M side half-membrane than on the C-side half-membrane [144,145]. A similar distribution of protein has also been suggested on the basis of different density of the inner and outer "railroad tracks" of OsOa-stained thin sections of rat kidney mitochondria [163]. Packer's model [148], by placing the primary dehydrogenases on the M side, Complex III on the C side, and Complex IV as generally transmembranous, provides for directed electron transport and H + transport, but does so in only one transverse "loop" [99], a situation also evident in the model in Fig. 4C. Racker's scheme (Fig. 4C) is based upon antibody and radioactive labeling studies, while that of Grinius et al. [112], a similar model, is based upon the reactivity of electron transport particles and rat liver mitochondria with ferri-(ferro-)cyanide, indicating at least one transverse electron transport loop across the membrane. On the basis of the data presented earlier, in particular the antimycin-sensitive succinate-ferricyanide reductase activity [164], the simplified binary membrane structure in Fig. 5 is proposed, providing three transverse electron transport loops as predicted by the chemiosmotic hypothesis. Since the cytochrome oxidase complex (mol.wt 110000) is extremely large and recent studies of crystalline cytochrome

150

M-SIDE

C-SIDE

Fig. 5. Anisotropic binary arrangement of protein subunits in mitochondrial cristae. Subunit diameter is proportional to its molecular weight. Dashed lines indicate uncertain location. NHI, non heme iron protein. SDH, succinate dehydrogenase. Transmembrane orientation of NADH dehydrogenase complex has been suggested by Wikstr6m [177] and Garland et al, [178]. UQ also functions between NHI 2 and NHI 16,000.

oxidase demonstrate a protein globule 100 A thick in a 45-A lipid bilayer [50], and since the location of cytochrome aa is ambiguous at present, we have tentatively placed cytochrome aa in the middle of the membrane, but closer to the M side. This binary membrane model opens up a possibility that has not been considered in most studies of membrane sidedness, namely that there is an interior region of protein surface exposure which may be also a hydrophilic region. Such a region must be taken into account in translocation studies. For example, the azide transport studies which indicate high sensitivity may be evidence that cytochrome aa is exposed to this interior region of the membrane. Studies on changes of fluoresence enhancement and proton transport in energized membranes should also consider this possibility [165, 166].

ACKNOWLEDGEMENTS Previously unreported work was supported under Grant AMO4663 from the National Institute for Arthritis and Metabolic Diseases. Support for H.J.H. was under training grant T O 1 - G M l 1 9 5 and for F. L. C. under Career Award GMK6-21839 from the Institute for General Medical Science.

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