Mitochondrial coupling factor B

Biochimica etBiophysicaActa, 683 (1982) 39-56

39

Elsevier Biomedical Press

BBA86085

MITOCHONDRIAL

COUPLING

PROPERTIES

ROLE

AND

FACTOR

IN ATP

B

SYNTHESIS

D. RAO SANADI

Department of Cell Physiology, Boston Biomedical Research Institute, 20 Staniford Street, Boston, MA 02114 and Department of Biological Chemistry, Harvard Medical School, Boston, MA 02115 (U.S.A.) (Received December 30th, 1981)

Contents 1.

Coupling factors - an overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

40

II.

F B and its properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Purification and activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Physical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Amino acid composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Antibody to F a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Immunological evidence for F B in H+-ATPase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Quantitation of F a in the H +-ATPase and ETPH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Multiple forms of F B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. Fa in other species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

40 40 40 41 42 42 43 '43 44

II1.

Effects of F a on energy-linked reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

45

IV.

Studies with thiol inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Monothiol inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Dithiol-type inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

49 49 50

V.

Topography of F B in the membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

54

VI.

Atentative mechanism of F B action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

54

VII.

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

55

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

55

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

55

Abbreviations: F~, coupling factor one (or ATPase); Fa, coupling factor B; F0, the membrane sector proteins of the oligomycin-sensitive ATPase; AE particle, submitochondrial particle exposed to amrnonia-EDTA at pH 8.8-9.0; ETPH, electron-transport particles from heavy layer mitochondria; BAL, British Anti-Lewisite (2,3-dimercaptopropanol); oxonol 0304-4173/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

VI, bis-(3-propyl-5-oxoisoxazol-4-yl)pentamethineoxonol; TMPD, N,N,N',N'-tetramethyl-p-phenylenediamine; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; DCCD, N,N'-dicyclohexylcarbodiimide; Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; PCMPS, p-chloromercuriphenylsulfonate; PCMB, p-chloromercuribenzoate.

40

!. Coupling factors-an overview The pioneering work of the laboratories of David Green and Efraim Racker on the resolution and reconstitution of the oxidative phosphorylation system has led to several significant contributions and provided much of our understanding of the complex process. Among these is the recognition that several of the proteins associated with this energy coupling could be detached from the mitochondrial membrane preparations without loss of oxidative activity but with impairment of ADP phosphorylation. Reconstitution of the depleted submitochondrial particles with the purified proteins restored phosphorylation. Several of these coupling factors, defined operationally, have been purified and well characterized [1]. Their properties have been summarized in Table I. It is interesting that all of the coupling factors that have been identified hitherto are present in the purified H +ATPase. 11. F a and its properties

IlA. Purification and activities F B was first recognized in crude preparations of coupling factor A, since these gave greater stimulation of the activity of urea-depleted sub-

mitochondrial particles in energy-linked reactions than the stimulation by highly purified preparations of factor A. Its purification became possible after a suitable assay particle which responded to F B addition with considerable selectivity was devised [9]. The AE particle met the needs for specifity. In the routine assay for F B involving ATPdriven NAD + reduction by succinate, AE particle activity was stimulated several-fold by F B but minimally (less than 15% stimulation) by factor A or F t. The use of the simple ATP-dependent NAD + reduction assay also contributed to rapid progress. The highly purified F B showed more than 100-fold increase in specific activity over the crude extracts from lyophilized, acetone-washed mitochondria. It stimulated the activity of the AE particle in ATPdriven N A D + reduction, A T P - d e p e n d e n t NAD(P) + transhydrogenase, Pi-ATP exchange and ADP phosphorylation coupled to succinate or N A D H oxidation [9-11].

liB. Physical properties Purified F B showed a molecular weight of approx. 32000 by centrifugation in a sucrose density gradient [9]. The activity was clearly separated from horse heart cytochrome c (Fig. !). F a appeared as a single peak in the analytical ultracentrifuge at 6°C (pH 7.5 and 8.0) with a sedimen-

TABLE I M I T O C H O N D R I A L C O U P L I N G F A C T O R S A N D T H E I R PROPERTIES OSCP, oligomycin sensitivity-conferring protein; PAGE, polyacrylamide gel electrophoresis; STA, silicotungstic acid. Factor

Assay particle

Routine assay

Properties

Reference

F~

-

ATPase

Fa

AE particle (pH 8.8)

ATP-driven N A D + reduction by succinate

Cold labile, about 350 kDa. 2 five subunits (53, 40, 33, 13 and 7.5 kDa). Activity is inhibited by ATPase inhibitor subunit. Factor A is a cold-stable, five-subunit form with latent ATPase activity. 3-5 Homogeneous by PAGE, SDS-PAGE and gel This article, filtration. Functionally active -SH 1 and dithiol. Monomer of 14.600 kDa. F a has also been purified from Racker's

OSCP

Ammonia-extracted F0 STA particle ( + F~ + F a + OSCP)

Sensitivity of Fi-ATPase to oligomycin Pi-ATP exchange

F2. F6

Inactivated by trypsin; 18 kDa. Heat stable, 8 kDa by SDS-PAGE, promotes binding of F~ to STA particle.

6, 7 8

41

~,

•

4

80

20

40

3.0

60

C:) (::3

"x 1.5

"~

-

3.0-

c

t

'

-

"~

I~

30 _~

'x

L..)

,~

E::)

I

[

5

I0

60

~1

x

15

20

25

TUBE NUMBER Fig. 1. Separation of Fa activity from cytochrome c by sucrose density gradient centrifugation. The average molecular weight derived from the measurements was 33000 (from Ref. 9). (A)(© ...... C)) Protein, (O O) activity; (B) ( × . . . . . . ×) cytochrome c; (C)(× . . . . . . × ) hemoglobin.

tation constant of 3.11-3.39 S [12], although small amounts of a heavier component were observed occasionally. Iodoacetamide treatment did not alter the sedimentation coefficient. Higher values (4.20-4.65 S) were obtained when the analyses were performed at 25°C or when the samples had been exposed to room temperature. In retrospect, the heavier fractions might have been oligomeric forms of FB; evidence for their existence has since been obtained and some have been partially purified (see later). Conventional disc gel electrophoresis of purified F B at p H 8.8 showed only a single faint band even when a large sample (50 #g) was applied. Gels run in parallel with 1.0 /~g bovine serum albumin were brightly stained (Fig. 2) [12]. The monomeric form of F B also behaved similarly. Poor staining under these conditions (conventional gel electrophoresis) appears to be a feature of F n which is distinct from that of other known coupling factors. In SDS the staining was improved but was significantly less than the staining of the oligomycin sensitivity-conferring protein or the ATPase inhibitor [13].

o

4opg

Io

BSA

Factor B

Fig. 2. Disc gel electrophoresis of FB and bovine serum albumin (BSA). The tubes were from the same experiment (from Ref. 12).

HC. Amino acid composition The amino acid composition of F a showed no unusual features (Table II) [12]. The minimum molecular weight from the amino acid composition was 14600 (Table II), which is one-half the value obtained by sedimentation methods. The obvious conclusion was that this preparation is a dimer. The protein has apparently two cysteine residues per 14650 g, one of which is readily accessible for carboxymethylation and another shielded in a hydrophobic environment. Studies with inhibitors have shown that both -SH groups may be functionally active (see later). F B binding to F0, determined by mixing and centrifugation experiments, increases linearly to levels far beyond that necessary for saturating in the Pi-ATP exchange reaction (Fig. 3). The data suggest that although F B behaves like a typical soluble protein after detachment from the membrane, it might have regions of high hydrophobicity, which is consistent with its tendency to occur in oligomeric forms (see later). It also precipitates in high concentrations. F B has been analyzed for phospholipid (chloroform/methanol-extractable organic phosphate) and carbohydrate (amino sugars during amino acid analysis). We have failed to detect any sign of these (unpublished data).

42 TABLE II A M I N O ACID COMPOSITION OF F B (DIMER) Amino acid

Number of residues/ 29 200 molecular weight

Lysine Histidine Arginine Cysteic acid Carboxymethylcysteine Aspartic acid Threonine Serine Glutamic acid Praline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan

17.4 6.55 10.3 3.99 2.00 25.4 12.7 15.2 27.9 13.4 23.8 24.3 17.0 4.74 13.8 24.8 6.74 8.94 1.90

mol%

6.72 2.53 3.98 1.55 9.80 4.90 5.98 10.75 5.18 9.20 8.60 6.57 1.83 5.33 9.58 2.61 3.46 0.74

l i D . Antibody to F B

An antiserum to F a with a somewhat low titer has been obtained by immunizing rabbits [11]. It produced a single precipitin band in double diffusion with highly purified Fa, partially purified F B o_

£L

50

200

/f.

E ~<

,/

'c

/

E

I00

E

x

x o

2.5

~v -

~v ~ x

3,~ 1.6

200

I00

/

X UJ

E': c I

x

and submitochondrial particles. In immunoelectrophoresis against F B as well as submitochondrial particles, a single band was obtained showing its monospecificity (Fig. 4) [13] and its usefulness in F a identification and functional studies. The coupling activity of Fa is completely inhibited when it is either preincubated with antiserum prior to addition to the AE particle or the antiserum is added after the particle and F B are mixed. Since mixing in this manner led to reassociation of F B with the assay particle [12,13], it is apparent that the bound F B is fully accessible to the antiserum. However, when the intact E T P H is treated with the antiserum, the inhibiton is only about 15%. This is also true if the AE particle activity is stimulated by the addition of a low, maximally activating level of oligomycin. This activity probably involves the residual F B left after a m m o n i a - E D T A extraction. The results support the conclusion that on reconstitution of the AE particle with F B, the latter is bound in a more exposed condition than the endogenous Fu. There is also a difference in the sensitivity of F B in the free and membrane-bound state to dithiol inhibitars, which will be discussed later in Section IV. l i E . Immunological evidence for FB in H + -A TPase

When ETPH or H + - A T P a s e is washed with a m m o n i a - E D T A at p H 8.8, its Pi-ATP exchange activity is reduced greatly but can be restored by addition of F B [9,13]. Parallel with such loss of FB-dependent activities, there is also a decrease in F B levels detectable by Ouchterlony double diffusion with F B antiserum. The washings contained significant amounts of F B.

x

nn

X

I

4~.0

~

2 3

8.0

Factor B added, nmoles x rag-' MP Fig. 3. Binding of F a to F0. Fo was derived from H +-ATPase prepared by fractionation using lysolecithin. F B was added to Fo and centrifuged. Unbound F B was assayed in the supernatant and Fo-FB was assayed after addition of F I for Pi-ATP exchange activity. MP, F0 prepared by 3.5 M NaBr treatment of H ÷-ATPase.

Fig. 4. Immunoelectrophoresis of F a with rabbit antiserum at pH 8.8 (from Ref. 11).

43 When F~ is removed from H + -ATPase by treatment with 3.5 M NaBr, the residual Fo supplemented with F~ shows Pi-ATP exchange activity, which is increased 2-fold by added F a [13,14]. This shows that approx. 50% of the F B of the H ÷ATPase remains tightly bound to the F0, perhaps by hydrophobic bonding. Thus, the assignment of F B as a component of Fo segments which still have energy-transducing potential is supported by several criteria. HF. Quantitation of FB in the H+-ATPase and ETPH The H+-ATPase prepared by lysolecithin extraction shows no significant increase in Pi-ATP exchange activity on addition of F~ or F B, showing that its complement of these factors is intact. F~ content of these preparations has been determined and compared to the FI content. Two independent methods were used for F B quantitation - one involving labeling of available -SH groups in the H + -ATPase with N-[ 3H]ethylmaleimide under dissociating conditions in SDS [14,15] and the other by an enzyme-linked immunosorbent assay (ELISA) [16]. The F B content was 2.2 n m o l / m g H +-ATPase by the labeling technique (assuming that only one -SH g r o u p / m o l is alkylated) and 2.1 n m o l / m g by ELISA with a stoichiometry of F a to F¿ of 1.0. The results are also consistent with the appearance of F a as the monomer in SDS-polyacrylamide gel electrophoresis. HG. Multiple forms of Fe Forms of F a have been isolated with different apparent molecular weights although their isolation and purification procedures were essentially similiar. The form isolated by Lam et al. [9] showed a molecular weight of approx. 32000 by sedimentation criteria and a minimum molecular weight of 14600 by amino acid analysis, indicating it was a dimer. The second well characterized form [13] was eluted slightly earlier in gel filtration and retarded in SDS-polyacrylamide gel electrophoresis, compared to horse heart cytochrome c. The molecular weight corresponded to approx. 13000, but slightly larger than that of cytochrome c. In the Swank-Munkres gel system, the molecular weight was 15000. This form apparently is a monomer. Its specific activity was 100-125

units/mg [13] in pooled fractions from Sephadex filtration and 140 in the peak fractions. The overall purification from the acetone powder extract of mitochondria was over 2000-fold. Earlier, Hatefi and co-workers [ 17,18] described a form of F a which is very close to the monomeric form, if not identical to it. This FB-like protein and F B [13] are similar in (a) ability to restore the activity of AE particles in several energy-linked reactions, (b) sensitivity to mercurials, and (c) immunoreactivity to F B antiserum. The preparations differ in some ways: (a) F B binds to CM-cellulose while FB-like protein did not; (b) the specific activity of the latter, assayed by the same type of AE particle, is less than 10% of the specific activity of FB; (c) both monomeric and dimeric forms of F a stain poorly in conventional polyacrylamide gel electrophoresis while no significant difficulty in staining under quite similar conditions has been noted with the FB-like protein; (d) F a migrates slightly slower than cytochrome c in both Sephadex filtration and SDS-polyacrylamide gel electrophoresis while FB-like protein appeared slightly faster; (e) F B inhibits partially the ATPase activity of AE particles while You and Hatefi [18] observed no such inhibition. The inhibition by F B was relieved by the addition of FCCP. Joshi et al. [13] point out that the differences are minor; however, the possibility that FB-like protein may have been derived from F B, perhaps by proteolysis of a small peptide, needs to be kept open until the comparison is carried out in the same laboratory. F B activity has also been purified from crude preparations of F 2 [19]. Although the fractions derived from F2 by CM-cellulose chromatography showed several bands, its specific activity was about 20 (unpublished data), showing that the purified activity might have been the monomeric form obtained by Joshi et al. [13]. A third form of F B, which was only partially purified, has been reported by Shankaran et al. [20] and Lam et al. [21]. It can be separated from the dimeric F a obtained by Lam et al. [21] on both DEAE-cellulose and CM-celhilose. Its molecular weight by sucrose density gradient centrifugation, one of the steps in its purification, was approx. 47000, unmistakably distinct from the monomeric form. Its specific activity was over 10/~mol N A D + reduced/min per mg in the reversed electron-flow

44

assay. It was far less stable than the dimeric form. Its activity was inhibited by PCMPS and antiserum to F a. Immunodiffusion experiments with preparations of F B and the 47000 dalton protein in adjacent wells resulted in precipitin bands which fused at the corners with no evidence of crossover, showing their immunological identity. In SDSpolyacrylamide gel electrophoresis, three well stained bands were seen, but none was detectable in the 13000 dalton region. Higashiyama et al. [22] isolated a multimeric protein of 330000 molecular weight with a subunit molecular weight of 44000 from bovine heart mitochondria. It stimulated AE particle activity in ATP-dependent N A D + reduction by succinate at extremely low levels, with a specific activity corresponding to over 1000 /~mol e x c h a n g e / m i n per mg. The extent of stimulation, however, was only about 20%, in contrast to F B which gave 4-5-fold stimulation. Moreover, this protein did not crossreact with F B antiserum [23], which led to the conclusion that it was not antigenically related to F B•

A 55 000 dalton protein isolated by gel filtration in SDS-urea on BioGel P-100 had an amino acid composition that seemed practically identical to that of F B and quite distinct from that of F~ or adenine nucleotide translocase [24]. It apparently binds ATP, Pi and the uncoupler, 2-azido-4nitrophenol. Immunological cross-reactivity to F B antiserum has not been tested. Crude extracts of lyophilized, acetone-washed mitochondria were directly fractionated on Sephadex G-75 and the fractions were tested for immunoreactivity to F a antiserum by ELISA. In addition to a peak of reactivity around 13000 daltons, another peak was seen in the region of higher molecular weight (Fig. 5) [25]. The latter decreased progressively during F B purification. No coupling factor activity was detected in the high molecular weight peak. It is not known whether the high molecular weight form is an oligomer of F B or F B associated with another protein. The F B content of ETPH and H +-ATPase determined by N-ethylmaleimide labeling and subsequent SDSpolyacrylamide gel electrophoresis to separate F B and by ELISA, which measures total antigen, gave the same value. The result suggests that all forms of F B are converted to the monomer in SDS.

L5

300

Cyt C

"~

i

,

"..~_

--

I.()

.~ - -

2oo E

corb

= g

olii 5 50

:

r t

I

t

T

I

I00

IO0

c.~

150

Eluli0n volume, ml Fig. 5. M i t o c h o n d r i a l protein fractions i m m u n o r e a c t i v e to F a antiserum. F a at the CM-cellulose stage was put through a c o l u m n of Sephadex G-75. B S A , b o v i n e serum a l b u m i n ; carb anhy, c a r b o n i c a n h y d r a s e ; cyt C c y t o c h r o m e c; C R M , cross-reactive material.

Hence, it is clear that F B occurs as a monomer in mitochondria. Evidently, it has a strong tendency to form oligomers or bind tightly to other proteins during its purification, and the experimental conditions to maintain it in the highly active monomer form need to be examined. IIH. F e in other species Until recently, no experiments had been carried out to look for an FB-like protein in other species or sources. The availability of an ELISA has permitted screening of several membrane preparations for immunological cross-reactivity to F B antiserum [16]. F a cross-reactive protein was found in washed membranes from Escherichia coli, as well as in an E. coil H +-ATPase preparation supplied by R. Fillingame, membranes from Paracoccus denitrificans, Thermophilus bacterium PS3 and spinach chloroplasts, all of which have electron transport-linked ATP synthetic activity. No crossreactivity was detected in sarcoplasmic reticulum, purple membrane from Halobacterium halobium or rat liver microsomes. (Table III). Houghton and Sanadi (unpublished data) have developed the procedures for almost quantitative

45 T A B L E III D E T E C T I O N OF P R O T E I N CROSS-REACTIVE W I T H F B A N T I S E R I U M S, high-speed supernatant; P, high-speed pellet. Subfractions

Cross-reactivity (units/mg)

Total cr0ss-reactivity (units) a

Bovine heart mitochondria

S P

23 59

E. coli

S P

155 >2000

393 3571

P. denitrificans

S P

60 2000

22 115

Spinach chloroplast

S P

< 5 6000

0.35 414.0

T. bacterium P S 3

S P

1 280 1493

252 89

Rat liver microsomes

0.45 3.75

0.8

-

Sarcoplasmic reticulum

< 0.5

-

Purple membrane from H. halobium

< O. 1

-

a

Expressed as total units of cross-reactive m a t e r i a l / m g protein of starting material. 1 unit of cross-reactivity is defined as the change in A 490 produced when 1 Atg / m l F a is applied to the microtiter well and the plate processed under defined standard conditions [16].

recovery of the F B cross-reactive protein from E. coli membranes by a selective extraction proce-

dure. They have obtained partial purification of the protein by DEAE-cellulose chromatography. IlL Effects of F B on energy-linked reactions

Since F B has no intrinsic activity and has to be assayed by its effects on a multistep, complex reaction, determination of its specific role in oxidative phosphorylation is difficult and challenging. The effect of F B on the activities of FR-deficient preparations in energy-linked reactions has been a primary probe. Inhibition of F~ activity in intact phosphorylating preparations as well as the H +-ATPase has also provided valuable information. F a stimulates the activity of AE particles in all ATP-dependent reactions (NAD + reduction by succinate, NAD(P) + transhydrogenase and Pi-ATP exchange) [9-12]. Low levels of oligomycin also produce similar, but lower stimulation of these activities [9,26]. With an optimally stimulating level of ofigomycin, F B produces further stimulation. These results led to the conclusion that oligomycin

and F B produce their stimulation by unrelated mechanisms [9]. More recent experiments have provided confirming evidence for this conclusion (Section IV). Of all the activities of Fs-deficient particles stimulated by added FB, Pi-ATP exchange has the least number of components and thus becomes the reaction of choice for studies on its mechanism of action. The reaction can be studied in purified preparations of H÷-ATPase which are free of electron-transfer components. Several H+-ATPase preparations have been described, all of which show quite similar distribution of polypeptides in SDS-polyacrylamide gel electrophoresis [13,27,28]. The H+-ATPase obtained by lysolecithin treatment of ETPH and subsequent purification by sucrose density gradient centrifugation in the presence of asolectin has the highest specific activity (over 1200 nmol Pi exchanged/min per mg) [13], which is approx. 10-times the specific activity observed with preparations made using cholate and ammonium sulfate precipitation [27,28]. It retains full activity throughout the isolation and is stimulated negligibly by added phospholipid, F~ or F B.

46

Electron microscopic examination by negative staining has shown that the preparation has vesicles with the F 1 spheres on the outer surface (unpublished data). Addition of ATP to the preparation results in the formation of a membrane potential, reflected by the binding of the voltage-sensitive dye, oxonol VI. The bound oxonol is discharged by FCCP, oligomycin, valinomycin plus K + and by Cd 2+ (Hughes, J.B., Pringle, M. and Sanadi, D.R., unpublished data). Involvement of F B in Pi-ATP exchange but not in oligomycin sensitivity has been unequivocally established. The Pi-ATP exchange activity of H +ATPase made from FB-deficient AE particles is stimulated 2-3-fold by F B [14]. F0 made from this preparation by NaBr treatment (AE-F0) had no ATPase activity but gained ATPase activity which was fully sensitive to oligomycin on supplementation with F t. However, the Pi-ATP exchange activity was minimal and increased 10-fold on addition of F B. Total loss of exchange activity without loss of ability to bind F ) and reconstitute oligomycinsensitive ATPase activity was obtained on treatment of AE-F 0 with N-ethylmaleimide. Addition of F B to N-ethylmaleimide-treated AE-F0+ F~ induced P~-ATP exchange activity, showing absolute requirement of F B for energy coupling but not for F~ binding or forming an oligomycin-sensitive ATPase. (Table IV) [13]. These results clearly

establish that F B converts an energy-uncoupled, oligomycin-sensitive ATPase into a coupled ATPase capable of energy transduction. Also, F B has no role in the oligomycin-sensitive ATPase activity, at least in purified preparations. It is expected that such a conversion to a system capable of P~-ATP exchange should result in a decrease in ATPase activity, since part of the enzyme complex would become coupled and the ApH would now be utilized at least in part for the Pi-ATP exchange reaction rather than be wasted as an ATPase. Such inhibition of ATPase activity of AE-F 0 + F l does occur [13], as shown in Fig. 6. These experiments were carried out with low levels of F I ; with saturating levels of F), the inhibition is low but reproducible and significant (Table V). There is evidence that F 1 preferentially binds to F0 assemblies potentially capable of energy transduction [29]. In all cases the inhibition is relieved by FCCP (Table V) [13], indicating that the apparent inhibition is in fact redirection of the energy towards H + pumping. The ATPase inhibitor also inhibited the ATPase activity, but this inhibition was not relieved by FCCP showing that the F B effect is specific and related to energy transduction. Similarly, Pi-ATP exchange activity could not be restored to N-ethylmaleimide-treated AE-F 0 by the addition of oligomycin sensitivity-conferring protein, F6 or ATPase inhibitor in place of F B.

TABLE IV F B IS AN ABSOLUTE R E Q U I R E M E N T FOR Pi-ATP EXC H A N G E ACTIVITY BUT IS N O T R E Q U I R E D FOR OLIGOMYCIN-SENSITIVE ATPase ACTIVITY AE-Fo was treated with l0 mM N-ethylmaleimide (NEM) and l0 mM dithiothreitol was added after 30 min. Coupling factors and oligomycin were added to AE-Fo and AE-F0 (NEM) where shown and assayed.

AE-Fo AE-Fo + F t A-Fo ( N E M ) + F l

Exchange (nmol/min per mg)

ATPase (~ m o l / m i n per mg)

- Fa

- oligo-

0 32 0

+ Fa

0 148 74

c~

5O

._o} Z

_ ~°

25

O

I

I

2O

40

Foctor B, nmoles x mg -~ Fo

mycin

oligomycin

0 0.60 0.61

0 0.13 0.16

+

Fig. 6. Inhibition of the ATPase activity of AE-Fo + Ft by F a. Fo was reconstituted with 50 p.g F i / m g by incubation for 10 min at room temperature in 0.25 M sucrose/10 mM Tris-acetate buffer. The unbound F I was removed by centrifugation and t h e pellet was washed by resuspension and centrifugation in the same buffer with l mM dithiothreitol. The protein was incubated with the indicated levels of F a and assayed for ATPase activity.

47

TABLE V F a P A R T I A L L Y INHIBITS T H E ATPase ACTIVITY OF FB-DEFICIENT H+-AT Pase AE-ATPase was assayed after addition of F a and FCCP. Addition to AE-H +-ATPase

Pi-ATP exchange (nmol/min per rag)

ATPase (mol/min per mg)

None F a (2/~g) FCCP ( 1/,M) F a + FCCP ATPase inhibitor (5/.tg) ATPase i n h i b i t o r + FCCP

33 251 0.96 29

4.1 3.0 4.6 4.8 1.3 1.4

The ATPase activity that is not susceptible to inhibition by F B (Table V) is probably the irreversibly uncoupled, damaged ATPase. The presence of such uncoupled ATPase activity complicates the experiments and interpretation of reaction mechanisms. The binding sites for F 1 and F B on F0 are distinct and independent of each other as shown by experiments involving incubation of each of these separately with Fo followed by centrifugation to remove excess factor. The AE-F 0 + F~ thus produced regained exchange activity on addition of F B and the AE-F 0 + F B was reactivated by F 1 (Table VI) [13]. Also, the saturation levels of F~ and F B were independent of how much of the other factor was present together with AE-Fo. The two coupling factors together in solution can be readily separated by filtration on Sephadex G-75.

All these results indicate that FI and F a are not bound to each other, but that each binds F0 separately. Experiments have also been carried out to measure the ApH and membrane potential-dependent binding of oxonol VI in intact ETPH and F Bdepleted membrane particles before and after F B supplementation [30]. The effects of low levels of oligomycin which stimulate energy-linked reactions in FB-depleted particles and high levels which presumably block the H + channel have also been determined. The rate of absorbance change upon energization is probably a more significant parameter than the maximum binding or ApH, since the latter would be influenced by both the rate of uptake and rate of discharge. The rate of respiration-supported increased binding of oxonol to the FB-depleted AE particle is stimulated only slightly in the presence of low oligomycin or F a or both together (Table VII). The steady-state binding is increased significantly by low oligomycin from 2.22 + 0.31 to 3.78-+ 0.4% T but little, if at all, by F B. The discharge of the bound dye is also retarded significantly by low oligomycin (from 2.10 -+ 0.12 to 2.88 - 0 . 1 9 % T ) but is hardly affected by FB, suggesting that if low oligomycin is blocking an H + leak, F a may not be acting by the same mechanism. In fact, F a stimulated the activity of these AE particles in energy-linked reactions far more than did low oligomycin and should show a correspondingly greater increase in t¿/2 if it were acting by a similar mechanism. The rate and extent of ATP-supported binding of oxonol were stimulated substantially (about 50-

TABLE VI B I N D I N G OF F 1 A N D F B I N D E P E N D E N T L Y TO AE-F 0 A E - F o was incubated with F 1 or F B and centrifuged to remove unbound components. The pellets were resuspended for assay with additional F~ or F n or both as shown. Units of exchange are expressed as n m o l / m i n per mg AE-F o. Addition during assay

AE-Fo, centrifuged A E - Fo + F l, centrifuged AE- F o + Fa, centrifuged AE-Fo, not centrifuged

None

F~

FB

Fl + FB

0 21 0 0

22 28 125 21

0 151 0 0

133 146 135 167

48 TABLE VII EFFECT OF F a A N D OLIGOMYCIN ON N A D H - D E P E N D E N T OXONOL BINDING. Aliquots of 200/~g AE particles(AE-P) --+Fa were suspended in 3 ml of 40 mM Tris-HC1 (pH 7.4) containing 6 ~mol MgCl 2 and 6 ~l of 1.5 mM oxonol (OX) VI added. Oxonol binding was initiated by addition of 0.6 mol NADH. Decay rates were determined after addition of l/Lg antimycin A. Low levels of oligomycin represent concentrations which maximally stimulate ATP-supported NAD + reduction by succinate, and high oligomycin represents a level 2-fold greater than that which maximally inhibited the ATP-dependent N A D ÷ reduction. Numbers in parentheses represent standard deviations. T, transmission. Rate OX VI binding (%T603-630) ( s-1 )

Maximum OX VI binding (% T603-630)

Decay rates (/I/2) (S)

AE-P

AE-P + F a

AE-P

AE-P + F a

AE-P

AE-P + F B

0.54 (0.1)

0.64 (0.07)

2.22 (0.31)

2.98 (0.46)

2.10 (0.12)

2.20 (0.26)

+ low oligomycin

0.69 (0.05)

0.64 (0.08)

3.78 (0.40)

4.46 (0.38)

2.88 (0.19)

3.13 (0.18)

+high

0.93 (0.1)

0.86 (0.06)

5.37 (0.38)

5.67 (0.27)

4.97 (0.39)

4.54 (0.24)

oligomycin

100%) by F B or by low oligomycin (Table VIII). However, F B did not change the tl/2 for the discharge of bound dye, again indicating that it does not block a nonspecific H + leak. The results of experiments on H + uptake coupled to ATP breakdown [15,29,30] are consistent with the oxonol binding data (Table IX). Supplementation of AE particles with F B increased reversed electron-flow activity 2-3-fold; however, the effect on H + leak is an apparent small decrease in t~/2, contrary to the expected increase if F B were acting by repairing membrane damage and reducing the leak.

The tl/2 values in the ATP-supported reactions are measured in the presence of inhibitory levels of oligomycin and represent nonspecific leaks through pathways other than the proteolipid-mediated H + channel. The leak rates of ApH generated by substrate oxidation are values with the H + channel intact, i.e., in the absence of inhibitory levels of oligomycin (Table VII), and hence may be more significant for the interpretation of F a function. These results from depletion-reconstitution studies lead to the conclusion that although rebinding of F a to the membrane may influence the tightness of coupling and consequently reduce the H + leakage

TABLE VIII EFFECT OF F n A N D OLIGOMYCIN ON ATP-SUPPORTED OXONOL BINDING The experiments were as in Table VII except 2/.tmol ATP and 0.2 /.tmol ADP were used instead of NADH. Decay rates were determined after addition of inhibiting levels of oligomycin. OX VI, oxonol VI; AE-P, AE particles.

Rate OX VI binding

(% T603_630) (s -1 ) Maximum OX VI binding (% T603_630) Decay rates ( t l / 2 ) (s)

AE-P

AE-P + low oligomycin

AE-P + F a

AE-P + F a + low oligomycin

0.58 (0.12) 2.2 (0.2) 16.0 (0.81)

1.08 (0.15) 5.9 (0.1) -

1.03 (0.01) 3.5 (0.2) 16.8 (0.92)

1.55 (0.08) 6.2 (0.2) -

49 TABLE IX ATP-DRIVEN H + U P T A K E IN SUBMITOCHONDR1AL PARTICLES H + translocation was initiated by addition of 10/~1 of 5 mM ATP in 2 mM MgCI/ (pH 6.1). The pH was followed with a glass electrode. AE-P, AE particles. Uptake

Decay (tl/2) Maximum nequiv. H + / (s) (nequiv. H + ) min ETPH (intact) AE-P AE-P+F a

30.1 16.2 21.5

6.6 2.6 5.2

38 37 31

to a small degree, the major effect is on ATP-dependent H + pumping or ATP synthesis from the electrochemical energy gradient. The data in Table IX also support the hypothesis of Lee and Ernster [26] that low levels of oligomycin stimulate energy-linked reactions in AE-type particles by preventing energy drain. No experimental verification of the hypothesis has been reported previously. A schematic representation of the mode of action of oligomycin and F B is shown in Fig. 7. The AE particle probably contains a heterogeneous population of Fo-FI assemblies in different stages of depletion and disarray. As a result, the ATPase activity is quite high and is no longer stimulated by uncouplers. High levels of oligomycin would tend to bind all available sites regardless of coupling factor deficiency and produce inhibition of all ATP-producing or -utilizing reactions. H + transloc~/tion would be inhibited maximally. Low levels of oligomycin may preferentially bind those complexes that lack F~, reduce their H + leak and increase the electrochemical gradient [26]. In con-

Fig. 7. A schematic representation of the mode of action of F B and oligomycin.

trast, F B probably restores the H +-pumping activity of the complexes which are deficient in only this factor, but has minimal effect on the H ÷ permeability of the complexes that lack F 1 and constitute the major leak. IV. Studies with thiol inhibitors

IVA. Monothiol inhibitors F B preparations lose coupling activity slowly on storage, and the activity is restored on addition of dithiothreitol. F B activity measured by stimulation of succinate-linked N A D + reduction or Pi-ATP exchange is also lost on treatment with PCMPS or iodine and restored substantially by -SH compounds [12]. N-Ethylmaleimide produces irreversible inactivation of F B. Binding of radiolabeled N-ethylmaleimide to F B has been demonstrated [13]. Since F a activity in reconstituted particles shows sensitivity to -SH inhibitors, the effects of these compounds in phosphorylating ETPH have been examined. PCMPS (150 #M) and N-ethylmaleimide also inhibit Pi-ATP exchange as well as other energy-linked reactions in ETPH [10]. In interpreting these data it must also be remembered that the above mercurials are impermeant to membranes [31] and their effect on F B is seen in submitochondrial particles and the H +ATPase which are known to be inverted. In whole mitochondria, mercurials at these concentrations do not cause discharge of the pH gradient [32], presumably owing to their inability to reach the F a thiol site. Membrane-impermeable mercurials also inhibit the energy-dependent binding of bromothymol blue to submitochondrial particle and N A D P + reduction by N A D H energized by ascorbate T M P D oxidation [10]. In the latter reaction, 10 btM PCMPS gave about 90% inhibition. The energy-independent transhydrogenase was inhibited only about 20% and ascorbate-TMPD oxidation was unaffected under the same conditions. Recently, Godinot et al. [33] have also observed that PCMB inhibited Pi-ATP exchange in Complex V, a preparation made by cholate extraction of submitochondrial particles and ammonium sulfate precipitation. They have attempted to identify the -SH protein affected by selective labeling with N-ethylmaleimide and found label in SDS-

50 p o l y a c r y l a m i d e gels in the m o l e c u l a r weight region of 54000 and 8 0 0 0 - 1 0 0 0 0 c o r r e s p o n d i n g to the a - s u b u n i t of F~ a n d p e r h a p s the D C C D - b i n d i n g proteolipid. N o label was d e t e c t a b l e in the 13000 d a l t o n region which is unexpected, since our results (see above) show that mercurials inhibit F B activity. In similar e x p e r i m e n t s carried out in our l a b o r a t o r y , the loss in activity on i n c u b a t i o n with N - [ 3 H ] e t h y l m a l e i m i d e c o r r e l a t e d best with labeling of the F B p e a k although several other peaks were also labeled [15,34]. The d i s c r e p a n c y m a y be e x p l a i n e d b y differences in the H + - A T P a s e m a d e by lysolecithin e x t r a c t i o n and C o m p l e x V. The latter has less than 10% of the exchange activity of the f o r m e r p r e p a r a t i o n . In o u r experience, c h o l a t e - a m m o n i u m sulfate p r e c i p i t a t i o n results in extensive F~ loss which could explain why C o m plex V has low P~-ATP exchange activity a n d fails to show d e t e c t a b l e labeling of the F B peak although the exchange activity is inhibited by mercurial.

1 VB. Dithiol-type inhibitors C a d m i u m , aresenite and organic arsenical comp o u n d s in the presence of excess monothiol* serve * Since the work of Stocken [35] in 1947, it has been recognized that organic arsenicals and arsenite form cyclic dithioarsenical derivatives with vicinal dithiol compounds. These have far greater association constants than the monothioarsenicals owing to their stabilization by cyclization. The inhibition of pyruvate oxidation by low levels of arsenicals and its reversal selectively by BAL suggested involvement of highly sensitive -SH groups [36]. Subsequent work demonstrated the involvement of two dithiols in the oxidative decarboxylation of pyruvate and a-ketoglutarate, viz., lipoic acid [37] and the lipoyl dehydrogenase [38]. Searls et al. [39] found that on reduction of this flavoprotein by NADH, two -SH groups are produced, which bind arsenite as well as Cd 2 ~. Stein and Stein [40] isolated a Cd 2+ derivative of the lipoyl dehydrogenase and characterized its properties. The use of these reagents as probes in unraveling the mechanism of uncoupling of mitochondrial oxidative phosphorylation led Gaber and Fluharty [41] to determined the stability constants of Cd 2+ and arsenite complexes with dithiols. Using a dithiol-substituted polymer (N-dihydrolipoylaminoethoxydextran) the K I value for the 2 -SH/Cd 2+ and 3 -SH/Cd 2+ complexes was 2.7× 1014 M and K 2 was 7.7)< 1013 M. There was also evidence for site cooperativity. Arsenite gave a single 2 -SH/AsO 2- complex with K between 108 and 109 M. For monothiol-substituted dextran, the stability constants for C d 2+ binding were of the order of 106 M.

as relatively specific dithiol g r o u p reagents. C d 2÷ at low levels u n c o u p l e s oxidative p h o s p h o r y l a t i o n in m i t o c h o n d r i a a n d s u b m i t o c h o n d r i a l particles. T h e u n c o u p l i n g is p r e v e n t e d b y dithiols but not by m o n o t h i o l s [42,43]. Similarly, the organic arsenicals, 3 ' - ( p - a r s e n o p h e n y l ) - n - b u t y r a t e [44] and phenylarsine oxide [32] p r o d u c e u n c o u p l i n g which is potentiated by monothiols. Uncoupling by aresenite is seen only in the presence of e q u i m o l a r B A L a n d is reversed by a slight excess of the dithiol [45,46]. M e r c a p t o e t h a n o l and other m o n o thiols were far less effective in p r o d u c i n g these effects. The r e q u i r e m e n t for B A L in p o t e n t i a t i n g the arsenite effect has been a t t r i b u t e d to the need for converting the m e m b r a n e - i m p e r m e a b l e , h y d r o philic arsenite into a h y d r o p h o b i c derivative which has access to the b i n d i n g site. These results suggested that oxidative p h o s p h o r y l a t i o n involves a h y d r o p h o b i c functional group with as high an affinity for C d 2+ , arsenite a n d organic arsenicals as that of dithiols and m u c h higher than that of m e r c a p t o e t h a n o l a n d other m o n o t h i o l s [41-46]. Jacobs et al. [42] a t t e m p t e d to selectively label this dithiol group of rat liver m i t o c h o n d r i a with r a d i o a c t i v e C d 2 ÷ as a means of isolating it. The b i n d i n g of Cd 2+ to m i t o c h o n d r i a increased linearly to a level several times greater than the u n c o u p l i n g concentration, l l s C d was obviously b o u n d to several sites or t r a n s p o r t e d into and a c c u m u l a t e d inside, and the a p p r o a c h could only be used effectively with more purified p r e p a r a t i o n s c a p a b l e of oxidative p h o s p h o r y l a t i o n . It was for this reason that we u n d e r t o o k the f r a c t i o n a t i o n of the m i t o c h o n d r i a l oxidative p h o s p h o r y l a t i o n system. These studies led to the isolation of coupling factors A a n d B, a n d now, after nearly two decades, a r e e x a m i n a t i o n of the p r o b l e m has revealed that F B qualifies as the d i t h i o l - t y p e c o m p o nent that binds A s O 2, Cd 2+ and p h e n y l a r s i n e oxide and as a result, u n c o u p l i n g occurs. W h e n purified F B is i n c u b a t e d with C d 2+, p h e n y l a r s i n e oxide or arsenite-BAL, its activity in s t i m u l a t i n g the P~-ATP exchange and A T P - d e p e n d e n t N A D + reduction reactions of the A E particles is lost [14,47]. A typical e x p e r i m e n t illustrating the effect is shown in Fig. 8. The 150 values for C d 2+ a n d p h e n y l a r s i n e oxide are a b o u t 20 ~ M . Similar inhibition occurs when the particles and F B are p r e m i x e d a n d the inhibitors a d d e d subse-

51 D---~ AE- P + Factor B

,50 ~ T (3.. ~ ~_T

50#M Cdz+

\.

150/d,M DTT

AEIp+ Factor B

~ ~ AE-P +

AE-P + Factor B ,,,,~ 50, u-M Cdz+ _

13_ ._ -~ E

50/..~M Cd2++

150/z.M OTT'~

E

AE-P ~ /

~"'----....~,~....~

_

"~='AE-P+5OFM CdZ++5OOFM ME i

i

i

50

100

150

//

i

250

/zM Cd z+

Fig. 8. Reversible inactivation of Fa by Cd 2+. (IS3,I ) Dithiothreitol (DTT) added; (A, A) mercaptoethanol (ME) added. Aliquots of 0.5 mg AE particles and/or FBwere incubated with Cd 2+ in 50 ,al for 5 rain on ice. The samples were assayed for PI-ATPexchange activity as described previously[47].

quently. BAL at slightly higher levels (e.g., 2-fold excess) restored the activity but mercaptoethano] failed to do so even in 10-fold excess. In fact, the monothiol potentiated the inhibiton when it was added in amounts up to 10-fold in excess of phenylarsine oxide, e.g., the 150 value was reduced from 21 to 1 3 / t M in one experiment. The potentiation by mercaptoethanol can be attributed to the greater lipophilicity of its complex with phenylarsine oxide than of the arsenical alone. Cysteine, which is much less soluble in lipids, does not cause similar potentiation (unpublished data). The inhibitors did not prevent binding of F B to the assay particle. This was shown by experiments involving exposure of F B to the inhibitor followed by gel filtration to remove unbound inhibitor. This F B was then added to AE particles and centrifuged to remove excess F B. The resulting particle was inactive, and the activity was stimulated by the addition of dithiothreitol to levels comparable to those achieved in the absence of inhibitor [47]. These effects of Cd 2+, arsenite-BAL and organic arsenical on F B activity measured by inhibition of reversed electron flow and Pi-ATP exchange exactly parallel their effects in uncoupling oxidative phosphorylation described earlier. Thus, the hydrophobic dithiol-type functional group seen in the oxidative phosphorylation system may also exist in F B. There is an apparent contradiction in the above data on the extrinsic monothiol readily accessible

to mercurials and the hydrophobic dithiol site, since the amino acid composition shows only two -SH groups per 14600 molecular weight. The data can be reconciled if one assumes that the second -SH group is vicinal but in a hydrophobic environment, and only hydrophobic reagents can reach it. Stiggall et al. [17] have also reported inhibition of F B activity by Cd 2+ , phenylarsine oxide, diamide and p-hydroxymercuribenzoate. No attempts were made to discriminate between monoand dithiol types of functional sites by competiton with external thiols. The mechanism of uncoupling by Cd 2+ , arsenite-BAL and phenylarsine oxide, all of which inhibit F B activity, has been studied in rat liver mitochondria by following their effects on the respiration-dependent p H gradient (ApH). These reagents produce discharge of the ApH in mitochondria respiring with succinate or 3-hydroxybutyrate and N A D + in the absence of added A D P [32]. Fig. 9 illustrates a typical experiment carried out with phenylarsine oxide. The uncoupling is potentiated by a 5-10-fold excess of 2-

H+

FCCP 50ngequiv.

2-M~hgo

~t~=

BAL

5

I

I

t Fig. 9. Discharge of the H ÷ gradient in respiting mitochondria by phenylarsine oxide (PhAsO). Rat liver mitochondria (1 mg/ml) were in 0.25 M sucrose, 0.02 M KCI, 3 mM Hepes buffer at pH 7.1. Succinate (2 mM) and 0.5 ng valinomycin/mg protein were added, followed by the indicated reagents which were 100 `aM 2-mercaptoethanol (2-ME), 20 ,aM BAL, 2.5 ,ag oligomycin (Oligo)/mg, 10 /.tM phenylarsine oxide, and 2 ,aM FCCP. The t]/2 was determined from a log AH + vs. time plot which was linear for over 75% of the response. The pH was monitored with a pH electrode.

52

mercaptoethanol and prevented by a 2-fold excess of BAL over the inhibitor concentration. The experiment in Fig. 9 was carried out in the presence of valinomycin in a K + medium which promotes conversion of the membrane potential energy to ApH. If the above experiment is carried out in the absence of valinomycin, on addition of phenylasine oxide extrusion of H ÷ occurs and the medium p H is first decreased ( A p H increases) to a level close to that obtained in the presence of valinomycin and the ApH is then discharged [32]. The increase is K ÷ dependent and much smaller when K ÷ is replaced by N a n in the medium. Only the increase in ApH, caused by H + extrusion, is produced when the water-soluble, membrane-impermeable PCMPS or mersalyl is used in place of the hydrophobic dithiol-favoring reagents. No discharge of ApH occurs even on prolonged exposure of the mitochondria to these monothiol reagents (e.g., mersalyl), showing that the two sites, one responsible for H + extrusion and the other for decay of ApH, are different. The H ÷ extrusion reaction also may in fact involve a dithiol-type group [32]. The reaction is probably related to the K ÷ -dependent swelling of respiring heart mitochondria documented by Brierley [48] and others [49-51]. The process is reported to be cryptic and activated by membrane-impermeable mercurials as well as by C d 2÷ Uncoupling of oxidative phosphorylation in mitochondria by classical uncouplers such as dinitrophenol or FCCP results from the protonophoric action of these weakly acidic, lipophilic compounds [52]. This H ÷ translocation has been demonstrated in liposomes using a K + diffusion potential [53]. Fig. 10 shows a modified model system where the internal and external K + concentration is the same and the H ÷ translocation is driven by displacement of the external pH. The figure (Polefka, T., unpublished data) shows that the ApH is discharged when both valinomycin and F C C P are present. When Cd 2÷ or phenylarsine oxide is used instead of FCCP, H ÷ translocation does not occur. Since these results exclude t h e possibility that Cd 2+ and phenylarsine oxide are protonophores, it seems likely that the uncoupling or decay of the H ÷ gradient induced by these compounds in rat liver mitochondria results from their interaction with a protein dithiol-like group.

mMKCI 2 mM HEPES 4 mg/ml Liposomes

150

Vol

/

/

/

/

~

'-'

/ /

0.~1pH ~

2

4

l PhAsO I0 ng Vol 5p.M

ng FCCIp"30 Sec-~ 2'/~MCdz+

Fig. 10. Phenylarsine oxide (PhAsO) and Cd 2+ do not have protonophoric activity. The liposomes were prepared by sonication of asolectin (100 mg) in 25 ml of medium containing 150 mM KCI, 2 mM Hepes buffer, pH 7.0. l ml of liposome preparation was taken in the pH electrode compartment and a pH gradient imposed by adding 5 #1 of 0.1 M HCI. The other additions are shown on the tracings. Val, valinomycin.

Experiments have been carried out to determine whether the H ÷ translocation induced by the dithiol inhibitors occurs via the oligomycin-sensitive H ÷ channel. Fig. 9 also shows the effect of oligomycin on the ApH decay. The tl/2 for decay in the presence of phenylarsine oxide is not affected by oligomycin, showing that the H ÷ translocation does not involve the H ÷ channel, at least in its normal, functional state. In these experiments the A p H was generated by substrate oxidation. Similar results were obtained with Cd 2÷ in place of phenylarsine oxide, and with other substrafes. It must be noted also that these decay rates are probably lower (tl/2 values are higher) than the actual values, since respiration is proceeding under these condition and the A p H may be continually recharged. The rate of discharge is apparently faster than the rate at which the pH gradient is being generated. In some recent experiments we have attempted to establish that Cd 2÷ in fact binds to F B in the H + - A T P a s e and submitochondrial particles. 115Cd2+ was added to the H ÷ - A T P a s e [13] at inhibitory levels, excess removed by filtration through Sephadex G-25 and the protein fraction subjected to SDS-polyacrylamide gel electrophoresis. The profile of Coomassie blue-stained protein and zlhCd counts are shown in Fig. 11. The major

53

1500

o 1000

13._ 0 500

0

0

Fig. 11. Cd 2+ binds a 13000 dalton subunit of the mitochondrial H+-ATPase. 115CDC12 (0.2 mM) was added to 5 mg H+-ATPase in 0.3 ml containing 10 mM Tris-sulfate, pH 7.5, 0.25 M sucrose, 1 mM 2-mercaptoethanol and after 15 min was filtered through a 1.5 ml minicolumn of Sephadex G-25. The recovered protein was factionated by SDS-polyacrylamide gel electrophoresis. The figure shows the distribution of protein (stained with Coomassie blue) and radioactivity.

part of the radioactivity was recovered in a band with the peak molecular weight of 13000, which corresponds to F B [13] (Hughes, J.B., and Sanadi, D.R., unpublished data). In similar experiments involving labeling of rat liver mitochondria, followed by preparation and fractionation of the submitochondrial particles, over 60% of the protein-bound l lSCd was recovered in a single band with a molecular weight of 15000. Recently, Godinot et al. [33] observed that carboxypyridine disulfide, a reagent that can be used to distinguish binding to mono from dithiols, inhibits the Pi-ATP exchange activity of Complex V (60% at 2 mM) without affecting ATPase activity. Its inhibitory effect was overcome by the water-soluble cysteine. The reagent itself is readily water soluble and its effects on Complex V were similar to those of PCMB. Thus, although it could react with dithiols, we feel it probably does not react with the hydrophobic thiol group of F B. On the other hand, it could readily react with the extrinsic monothiol group of F B, as does PCMPS, and inhibit Pi-ATP exchange activity. They also

observed little inhibition of exchange activity by N-ethylmaleimide (2 mM, 5 min incubation). In experiments carried out with the more active H ÷ATPase preparation in our laboratory (unpublished data), inhibition was observed with even lower concentrations of the maleimide, but the incubation period to produce inactivation was longer (60 min). Using N-[3H]ethylmaleide, several protein peaks of the SDS-polyacrylamide gel were labeled under these conditions, but the labeling of the F a peak correlated best with the loss of activity. It may be appropriate to point out also that the bifunctional maleimide, dithiobis-(N-ethylmaleimide), which cross-links -SH groups within the y-subunit of chloroplast F l [54] is also not a likely reagent for the F B dithiol. The distance between the reactive maleimide groups may be too large to form a cyclic product in the manner that Cd 2+ or AsO 2 reacts. However, each of the thiols could bind independently to a maleimide derivative. Trialkyl tin compounds are potent inhibitors of oxidative phosphorylation [55]. Their action has been localized to the oligomycin-sensitive ATPase complex [56], but the evidence that they bind dihydrolipoate [57] in the mitochondrial membrane has not been substantiated. However, the observation that the inhibition of oxidative phosphorylation by dibutyltin is reversed by dihydrolipoate and various thiols has been confirmed by Stiggal et al. [17]. The latter authors have speculated that the dibutyltin compound reacts with F B dithiol. However, since inhibition is reversed equally readily with water-soluble glutathione as with hydrophobic dithiols, it is likely that the tin compounds produce their effects primarily by reacting with a hydrophilic monothiol group. Whether the reactive -SH group is in F B or at some other site remains to be explored. If the reaction were with the F B monothiol group, by analogy with the action of PCMB, uncoupling rather than inhibition of oxidative phosphorylation may be expected. Clearly, there are quite a number of unanswered questions regarding the action of these organotin compounds.

54

V. Topography of F a in the membrane

No systematic experiments have been carried out to determine the topography of F B in the membrane. However, the inhibition of P~-ATP exchange but not of oligomycin-sensitive ATPase by membrane-impermeable mercurials and the ready depletion and reconstitution of Fa from submitochondrial particles allow some tentative conclusions. Submitochondrial particles are known to have their membrane polarity reversed compared to intact mitochondria, i.e., they are inside out. Membrane-impermeable mercurials inhibit the P~-ATP exchange activity of ETPH without affecting the oligomycin-sensitive ATPase activity, which is consistent with the suggestion that they act on the monothiol group of F B [10]. These membrane-impermeable inhibitors, however, do not discharge the ApH in intact mitochondria [32], presumably since the inhibitor binding sites are oriented inwards. These data strongly support the interpretation that the monothiol group of F B is extrinsic to the membrane and is on the m-side (matrix side) of the mitochondrial inner membrane. The dithiol-like hydrophobic site that is inhibited by arsenite-BAL, Cd 2+ and phenylarsine oxide is present in purified F B. Pi-ATP exchange activity which has an absolute requirement for F B is inhibited by these compounds in ETPH [14,47]. The above compounds also produce uncoupling

1

2

and discharge the H + gradient in intact mitochondria. If the effect on mitochondria involves F B as the data favor, the dithiol-like binding site of F B is accessible to hydrophobic reagents from both sides of the mitochondrial inner membrane. We suggest that this site may in fact be buried in the hydrophobic region of the membrane. This location is consistent also with the observation that the 150 value for these compounds is much lower in experiments involving isolated F B (10/~M for phenylarsine oxide) compared to those involving ETPH (about 40 ~M) where the F B is already anchored in the F0 segment of the H +ATPase. VI. A tentative mechanism of F a action

A tentative working hypothesis for explaining the role of F B in oxidative phosphorylation consistent with available data is illustrated in Fig. 12. Block 1 attempts to show F a partly buried in the membrane, but mostly outside, with one -SH group outside the membrane phase (extrinsic) and another within the membrane phase. The two thiols are vicinal and capable of forming a bidentate chelate with a Cd or As atom. It is shown in association with the six subunits of the D C C D binding proteolipid. In this configuration, F B participates in the H + - A T P a s e reaction. It must be noted that one monothiol is on the matrix side of the mitochondrial inner membrane.

5

Fig. 12. A t e n t a t i v e m e c h a n i s m for F a f u n c t i o n in the H + - A T P a s e . Block 1 s h o w s a n intact F 0 a s s e m b l y c a p a b l e of H + p u m p i n g . Block 2 r e p r e s e n t s the o p e n i n g o f a n o l i g o m y c i n - i n s e n s i t i v e H + c h a n n e l ( d o t t e d p o r t i o n ) i n d u c e d b y the b i n d i n g of C d 2+ to F B. Block 3 s h o w s a n F a - d e p l e t e d F 0 w i t h a d i s o r g a n i z e d H + c h a n n e l , elliptical i n s t e a d of the c i r c u l a r o n e in b l o c k 1. T h e c o n f i g u r a t i o n is i n c a p a b l e o f H + t r a n s l o c a t i o n , c a n b i n d F I to f o r m a n o l i g o m y c i n - s e n s i t i v e A T P a s e b u t h a s n o i n h i b i t o r - i n d u c e d H + leak as a c o n s e q u e n c e of F B d e p l e t i o n .

55 Block 3 shows FB-depleted F0 with the central H ÷ channel disorganized (elliptical compared to the circular channel in block 1). The Fa-depleted H+-ATPase or submitochondrial particle is no longer capable of pumping H ÷ or making ATP utilizing the proton-motive force. Thus, F B may be regarded as an organizer for the H ÷ channel. An alternative mechanism explaining H ÷ translocation would involve transfer of the H + from one ionizable acidic group to another in a chain from one side of the membrane to another. These acidic groups could conceivably include the two -SH groups of F B and the invariant - C O 0 - of glutamic or aspartic acid at position 65 of the six proteolipid subunits that constitute the H ÷ channel [58]. Binding any of the -SH groups or depletion of F B from the membrane would inactivate H ÷ translocation. Since F 1 would still be bound to the F0, the oligomycin-sensitive ATPase would be unaffected, but instead of the H ÷ being translocated, it may remain on the F) side or m-side of the membrane. Gould [59] has also proposed a role for -SH groups in H ÷ translocation in chloroplasts. Although there is no direct evidence that F B is involved in H ÷ translocation together with the DCCD-binding proteolipid, recent genetic evidence does implicate both subunits b (19000 daltons) and c (proteolipid) of the F0 from E. coli H ÷-ATPase in the reaction [60-62]. Conceivably, the subunit b of E. coli F0 may be the immunological and functional analog of heart mitochondrial F B. It may be emphasized that the effects of F B depletion (block 2) and F B inhibition by mercurials, Cd 2÷ or arsenicals are surprisingly different. In the presence of F B and the inhibitors, all energy-dependent reactions are uncoupled and there is rapid discharge of the proton-motive force from mitochondria and submitochondrial particles (Section IV) as well as discharge of oxonol bound to the purified, ATP-energized H ÷-ATPase (Pringle, M., Hughes, J.B. and Sanadi, D.R., unpublished data). The tl/2 value for the discharge is unaffected by oligomycin. These data indicate the operation of a different oligomycin-insensitive H ÷ channel as a consequence of binding of mercurials and dithiol inhibitors. This reaction is illustrated in block 2 by a second channel next to the proteolipid channel or it could be the same channel that is now disorganized in such a fashion that the H ÷ translocation is no longer sensitive to oligomycin.

VII. Conclusions The results summarized in this review have led to a tentative mechanism for F a action as an organizer protein for the H + channel or as a participant via its thiol groups in the chemical reactions of H + transmission across the membrane. Mercurials, C d 2+ and phenylarsine oxide have proved to be valuable probes for studying its function. Even more important, the results raise the question as to whether the thiols could play a role in regulating the proton-motive force of mitochondria, since inactivation of the dithiol by disulfide interchange or oxidation would yield a disulfide similar to the chelating with C d 2+ o r AsO 2 and produce a controlled leak for regulating Aq P levels. The proposed role of the -SH groups in the chemical reactions of H + translocation may provide an impetus to experiments testing the validity of the hypothesis of Williams [63] regarding the importance of localized H + in energy transduction.

Acknowledgements This work was supported by grants from the national Institutes of Health (GM 13641, GM26764 and ES02167). I am deeply indebted to Dr. S. Joshi for suggestions and criticisms, and to Ms. Angela J. Colombo for patient assistance with the manuscript.

References 1 Beechey,R.B. and Cattell, K.J. (1973) Curr. Top. Bioenerg. 5, 306-307 2 Penefsky,H.S. (1974) The Enzymes 10, 375-394 3 Andreoli, T.E., Lam, K.W. and Sanadi, D.R. (1965) J. Biol. Chem. 240, 2644-2653 4 Warshaw, J.B., Lam, K.W., Nagy, B. and Sanadi, D.R. (1968) Arch. Biochem. Biophys. 123, 385-396 5 Joshi, S., Murfitt, R.R. and Sanadi, D.R. (1979) FEBS Lett. 98, 13-17 6 McLennan, D.H. and Tzagoloff,A. (1968) Biochemistry 1, 1603 7 Senior, A.E. (1971) J. Bioenerg. 2, 141-150 8 Kanner, B.I., Serrano, R., Kandrach, M.A. and Racker, E. (1976) Biochem. Biophys. Res. Commun. 69, 1050-1055 9 Lam, K.W., Warshaw, J.B. and Sanadi, D.R. (1967) Arch. Biochem. Biophys. 119, 477-484 10 Sanadi, D.R., Lam, K.W. and Kurup, C.K.R. (1968) Proc. Natl. Acad. Sci. U.S.A. 61,277-283

56 11 Lam, K.W. and Yang, S.S. (1969) Arch. Biochem. Biophys. 133, 366-372 12 Lam, K.W~, Swann, D. and Elizinga, M. (1969) Arch. Biochem. Biophys. 130, 175-182 13 Joshi, S., Hughes, J.B., Shaikh, F. and Sanadi, D.R. (1979) J. Biol. Chem. 254, 10145-10152 14 Hughes, J.B., Joshi, S., Murfitt, R.R. and Sanadi, D.R. (1979) in Membrane Bioenergetics (Lee, C.P., Schatz, G. and Ernster, L., eds.) pp. 81-95, Addison-Wesley, Reading, MA 15 Hughes, J.B., Joshi, S.J. and Sanadi, D.R. (1979) Fed. Proc. 38, 573 16 Joshi, S., Hughes, J.B., Houghton, R.L. and Sanadi, D.R. (1981) Biochim. Biophys. Acta 637, 504-511 17 Stiggall, D.L., Galante, Y.M., Kiehl, R. and Hatefi, Y. (1979) Arch. Biochem. Biophys. 196, 638-644 18 You, K. and Hatefi, Y. (1976) Biochim. Biophys. Acta 423, 398-412 19 Racker, E., Fessenden-Raden, J.M., Kandrach, M.A., Lam, K.W. and Sanadi, D.R. (1970) Biochem. Biophys. Res. Commun. 41 1474-1479 20 Shankaran, R., Sani, B.P. and Sanadi, D.R. (1975) Arch. Biochem. Biophys. 168, 394-402 21 Lam, K.W., Karunakaran, M.E. and Sanadi, D.R. (1970) Biochem. Biophys. Res. Commun. 39, 437-443 22 Higashiyama, T., Steinmeier, R.C., Serrianne, B.C., Knoll, S.L. and Wang, J.H. (1975) Biochemistry 14, 4117-4121 23 Sharma, S. and Sanadi, D.R. (1976) Biochem. Biophys. Res. Commun. 71,347-353 24 Blondin, G.A. (1980) Biochem. Biophys. Res. Commun. 96, 587-594 25 Joshi, S., Hughes, J.B. and Sanadi, D.R. (1980) Fed. Proc. 39, 1845 26 Lee, C.P. and Ernster, L. (1965) Biochem. Biophys. Res. Commun. 18, 523-528 27 Serrano, R., Kanner, B.L. and Racker, E. (1976) J. Biol. Chem. 251, 2453-2461 28 Stiggall, D.L., Galante, Y.M. and Hatefi, Y. 0978) J. Biol. Chem. 253, 956-964 29 Sanadi, D.R., Joshi, S. and Shaikh, F.M. (1978) in Molecular Biology of Membranes (Fleischer, S., Hatefi, Y., MacLennan, D.H. and Tzagoloff, A., eds.), pp. 263-273, Plenum Press, New York. 30 Hughes, J.B., Joshi, S. and Sanadi, D.R. (1980) J. Biol. Chem. 257, 6697-6701 31 Gaudemer, Y. and Latruffe, N. (1975) FEBS Lett. 54, 30-34 32 Sanadi, D.R., Hughes, J.B. and Joshi, S. (1981) J. Bioenerg. Biomembranes 14, 425-431 33 Godinot, C., Gautheron, D.C., Galante, Y. and Hatefi, Y. (1981) J. Biol. Chem. 256, 6776-6782

34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63

Hughes, J.B. (1981) Fed. Proc. 40. 1733 Stocken, L.A. (1947) J. Chem. Soc. 592-595 Peters, R.A. (1949) Symp. Soc. Exp. Biol. 3, 36-58 Reed, L.J. (1957) Adv. Enzymol. 18, 335-382 Searls, R.L. and Sanadi, D.R. (1959) Proc. Natl. Acad. Sci. U.S.A. 445, 697-701 Searls, R.L., Peters, J.M. and Sanadi, D.R. (1961) J. Biol. Chem. 236, 2317-2322 Stein, A.M. and Stein, J.H. (1971) J. Biol. Chem. 246, 670-676 Gaber, B.P. and Fluharty, A.L. (1972) Bioorg. Chem. 2, 135-148 Jacobs, E.E., Jacob, M., Bradley, L. and Sanadi, D.R. (1956) J. Biol. Chem. 223, 147-156 Fluharty, A.L. and Sanadi, D.R. (1962) Biochemistry 1, 276-281 Fluharty, A.L. and Sanadi, D.R. (1963) Biochemistry 2, 519-522 Fluharty, A.L. and Sanadi, D.R. (1960) Proc. Natl. Acad. Sci. U.S.A. 46, 608-616 Fluharty, A.L. and Sanadi, D.R. (1961) J. Biol. Chem. 236, 2772-2778 Joshi, S. and Hughes, J.B. (1981) J. Biol. Chem. 256, 11112-11116 Brierley, G.P. (1976) Mol. Cell. Biochem. 10, 41-62 Azzone, G.F. and Massari, S. (1973) Biochem. Biophys. Acta 301, 195-226 Diwan, J.J., Maroff, M. and Lehrer, P.H. (1977) Indian J. Biochem. Biophys. 14, 342-346 Garlid, K.D. (1979) Biochem. Biophys. Res. Commun. 87, 842-847 Mitchell, P. (1976) Biol. Rev. 41,445-502 Okamoto, H., Sone, N., Hirata, H., Yoshida, M. and Kagawa, Y. (1977) J. Biol. Chem. 252, 6125-6131 Moroney, J.V. and McCarty, R.E. (1979) J. Biol. Chem. 54, 89-8955 Aldridge, W.N. and Street, B.W. (1964) Biochem. J. 91, 287-297 Cain, K. and Griffiths, D.E. (1977) Biochem. J. 162, 575-580 Griffiths, D.E., Hyanes, R.L. and Bertoli, E. (1977) FEBS Lett. 74, 38-42 Sebald, W. and Hoppe, J. (1982) Curr. Top. Bioenerg., in the press Gould, J.M. (1978) FEBS Lett. 94, 90-94 Friedl, F., Bienhaus, G., Hoppe, J. and Schairer, H.U. (1980) Hoppe Seyler's Z. Physiol. Chem. 36, 4578 Friedl, P., Bienhaus, G., Hoppe, J. and Schairer, H.U. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6643-6646 Loo, T.W. and Bragg, P.D. (1981) Biochem. Biophys. Res. Commun. 103, 52-59 Williams, R.J.P. (1978) Biochim. Biophys. Acta 505, 1-44