Thiols in oxidative phosphorylation: Thiols in the F0 of ATP synthase essential for ATPase activity

ARCHIVES

OF BIOCHEMISTRY

Vol. 254, No. 1, April,

AND

BIOPHYSICS

pp. 102-109, 1987

Thiols in Oxidative Phosphorylation: Thiols in the F,, of ATP Synthase Essential for ATPase Activity’s* TAKAO YAGI

YOUSSEF HATEF13

AND

Division of Biochemistry, Department of Basic and Clinical Research, Scripps Clinic and Research Foundation, 10666 North Tmeg Pines Road, La Jolla, Cal$%rnin 9.2037 Received October 28,1986

It was shown previously that the ATP synthase complex of bovine heart mitochondria contains an essential set of thiols or dithiols in its membrane sector (FO), whose modification by various reagents results in uncoupling [Yagi, T., and Hatefi, Y. (1934) Bit+ chemistry 23,2449-24551. The sensitivity to modifiers was increased by membrane energization, and the uncoupling was reversed by membrane-permeable thiol compounds when modifiers other than alkylating agents were used to uncouple. The present paper demonstrates that there exists in the F,, of bovine ATP synthase another set of essential thiols, whose modification results in reversible inhibition of ATPase activity. These thiols are most susceptible to modification by mercurials (pchloromercuribenzoate >p chloromercuribenzene sulfonate) and do not appear to be modified by iV-ethylmaleimide. The reversible modification of these thiols by mercurials protects the ATP synthase against irreversible inhibition in F0 by N,N’-dicyclohexylcarbodiimide. The possible location of these two sets of thiols in the F0 of bovine ATP synthase is discussed. 0 1987 Academic

Press. Inc.

The mammalian ATP synthase complex is composed of a minimum of 13 unlike polypeptides (1,2). Among these, the roles of only two polypeptides are known. One of these is the @ subunit of F1-ATPase, which contains the catalytic site (1, 3, 4), and the other is the ATPase inhibitor protein, which under appropriate conditions binds to the /3subunits and inhibits ATPase activity (1, 5, 6). Among the approaches that can be used to investigate the roles of the ATP synthase subunits is identification and localization of essential protein residues. This approach has led to identification of an essential glutamic acid residue i Supported by United States Public Health Service Grants AM 08126 to Y.H. and GM 33712 to T.Y. 2This is Publication Number 4501-BCR from the Research Institute of Scripps Clinic, La Jolla, California. a To whom correspondence should be addressed. 0003-9861/87 33.00 Copyright All rights

0 198’7 by Academic Press, Inc. of reproduction in any form reserved.

102

in the DCCD4-binding protein, and to the suggestion that this protein is a building block of the F,, proton channel (7). In addition to this essential residue and those at or near the active site of the /3 subunits (6, 8, 9), possible essential residues of the mitochondrial ATP synthase are arginyl (10, 11) and tyrosyl (12) residues in F,, of the bovine enzyme, and thiols in the (Yand y subunits of Schizosaccharom~ces pombe Fi-ATPase ((13), see also (14-17)), and in bicarbonate-treated rat liver F1 (18). In an earlier report (19), we showed that ’ Abbreviations used: DCCD, N,N’-dicyclohexylcarbodiimide; F,, membrane sector of the ATP synthase complex; pCMB, p-chloromercuribenzoate; pCMS, p-chloromercuribenzene sulfonate; TMPD, N,N,N’,N’-tetramethyl-p-phenylenediamine; SMP, submitochondrial particles; CCCP, carbonyl cyanide m-chlorophenylhydrazone; OSCP, oligomycin sensitivity-conferring protein; EDTA, ethylenediaminetetraacetic acid; DTT, 2,4-dithiothreitol.

THIOLS

IN OXIDATIVE

treatment of bovine ATP synthase with modifiers of mono- and vicinal dithiols results in uncoupling at the level of FO. Sensitivity to the modifiers was increased by membrane energization and the uncoupling could be reversed by subsequent treatment of the enzyme with membrane-permeable thiol compounds, such as dihydrolipoamide. These results suggested the presence in F,, of a mono- or vicinal dithiol, whose environment is altered by membrane energization and whose structural integrity is essential for proper proton translocation through F0 (see also (20)). The present study shows that, in addition to the above thiols, bovine F0 appears to contain another set of thiols whose modification by mercurials results in reversible inhibition of ATPase activity. The reversible modification of these thiols partially protects the ATP synthase against irreversible modification by DCCD, suggesting a spatial relationship between the DCCD-reactive glutamic acid residue of the DCCD-binding proteolipid and the F0 thiols that are essential for ATPase activity. MATERIALS

AND

103

PHOSPHORYLATION RESULTS

Inhibition of oligomycin-sensitiveATPase activit?( by thiol mod&rs. As was mentioned above, treatment of SMP or ATP synthase preparations (complex V) with a variety of modifiers of mono- and vicinal dithiols resulted in uncoupling at the level of F,, and in an increase of ATPase activity (19). The reagents used were pCMB, pCMS, N-ethylmaleimide, o-phenylenedimaleimide, monobromobimane, phenylarsene oxide, diamide, Cd’+, and CL?+plus o-phenanthroline (19). At higher concentrations however, some of these reagents inhibited the ATPase activity of SMP or complex V. As seen in Fig. 1, pCMB

METHODS

SMP (21), complex V (22), Fi-ATPase (23), F. (24), and OSCP (25) were prepared from bovine heart mitochondria according to the references given. ATP hydrolysis, using an ATP-regenerating system, and ATP-“Pi exchange (22) were assayed at 30°C according to the reference cited. Membrane potential changes were monitored at 30°C by the absorbance change of oxonol VI at 630 nm minus 603 nm as described previously (19), using an Aminco DW-2a dual wavelength spectrophotometer. Any variations from these procedures and other details are described in the figure legends. Protein concentration was determined by the Biuret method (26) in the presence of 0.1% deoxycholate or by the method of Lowry et aL (27). Sodium ascorbate, N-ethylmaleimide, lactic dehydrogenase, pyruvate kinase, phosphoenolpyruvate, lipoamide, pCMB, and pCMS were obtained from Sigma; ATP and oligomycin were from Boehringer; dithiothreitol and NADH were from Calbiochem; triphenyltin chloride was from ICN Pharmaceuticals; DCCD was from K and K Laboratories; 82pi was from ICN, TMPD was from Eastman-Kodak; rotenone was from S.B. Penick. Oxonol VI was a generous gift of Dr. W. G. Hanstein, University of Bochum. Dihydrolipoamide was prepared from lipoamide by the method of Reed et al (28). Other chemicals were reagent grade or of the highest quality available.

FIG. 1. Effect of pCMB on the ATP-“Pi exchange activity, ATPase activity, and respiration-induced membrane potential of SMP. The particles at 10 mg/ (pH 7.5) ml in 0.25 M sucrose and 50 mM Tris-acetate were incubated at 30°C for 30 min with the indicated amounts of pCMB. They were then diluted lo-fold with the sucrose-Tris buffer and kept on ice for assay. The assay for ATP-azPi exchange was carried out at 30°C in a mixture containing 0.25 M sucrose, 50 mM Trisacetate (pH 7.5), 4.23 mM MgC12, 20 mM 82pi (potassium salt), 2 mM ATP, and 49.8 pg of SMP/ml. Membrane potential was monitored by the absorbance change of 1.95 pM oxonol VI at 630 minus 603 nm in a mixture containing 0.25 M sucrose, 50 mM Tris-acetate (pH 7.5), and 39.8 pg of SMP/ml. Membrane potential was induced by the combined addition of 10 mM sodium ascorbate and 0.8 mM N,N,N’,N’-tetramethyl-pphenylenediamine (TMPD). ATPase activity was assayed at 30°C in a mixture containing 0.25 M sucrose, 50 mM Tris-acetate (pH 7.5), 50 mM potassium acetate, 3 mM MgC12, 2 mM phosphoenol pyruvate, 180 pM NADH, 10 units/ml lactic dehydrogenase, 10 units/ml pyruvate kinase, 2 mM ATP, 25 pM rotenone, and 4.89 pg of SMP/ml in the presence (0) or absence (0) of 5 WM CCCP.

104

YAGI

AND

at concentrations above 0.25 mM uncoupled SMP, resulting in inhibition of ATP-32Pi exchange activity and collapse of the membrane potential generated by oxidation of ascorbate plus TMPD. Consistent with this uncoupling effect, the ATPase activity of the pCMB-treated SMP increased more than twofold and became comparable to the ATPase activity of another batch of SMP which had been similarly treated with pCMB and assayed in the presence of the uncoupler CCCP. However, as pCMB concentration was increased further, the ATPase activity of the uncoupled particles was also inhibited. pCMS and Cd2+ were less effective (Im - 6 mM for pCMS and >lO mM for CdSO& while Nethylmaleimide at 210 mM caused very little inhibition. As seen in Fig. 2, the addition of dithiothreitol to pCMB-treated particles resulted in nearly complete reversal of inhibition, and the recovered ATPase activity was oligomycin sensitive. When the ATPase activity was inhibited by pCMS, much higher concentrations of dithiothreitol were required to reverse the inhibition, and the reversal of inhibition was incomplete. Moreover, at dithiothreitol concentrations up to about 10 mM, there was an increase in the inhibition of ATPase activ-

HATEFI

Ditiothreitol, mM FIG. 3. Effect of dithiothreitol on the ATPase activity of pCMS-treated complex V. Complex V (at 14.8 mg/ml) in 0.25 M sucrose and 50 mbi Tris-acetate (pH 7.5) was preincubated for 60 min at 30°C in the presence (0, A) or absence (0, A) of 8.3 mM pCMS. It was then diluted lo-fold with the sucrose-Tris buffer, further incubated for 60 min at 30°C with the indicated amounts of dithiothreitol, and kept on ice. ATPase activities were assayed as described in Fig. 1. Where indicated (A, A), the assay mixture contained 2.5 pgcg/ ml of oligomycin.

ity before the reversal of inhibition took place (Fig. 3). Similar results were obtained when dihydrolipoamide was used, instead of dithiothreitol, in conjunction with pCMS-treated SMP (data not shown). The incomplete reversal of pCMS inhibition may be related to our earlier finding that at these high concentrations dithiothreitol and dihydrolipoamide irreversibly inhibit the ATPase activity of F1 (29). Site of inhibition of oligomgcin-sensitive ATPase activity by mercurial. As mentioned above, isolated Fr-ATPase from Schizosacchuromyw

$--*-1-h

---_ 0.5

+ _---,.-.-+---Q/ 1.0 1.5 2.0 Dithiothreital. mM

FIG. 2. Reversal by dithiothreitol of the ATPase activity of complex V inhibited by pCMB. Complex V at 16.9 mg/ml in 0.25 M sucrose and 50 mrd Tris-acetate (pH 7.5) was incubated in the absence (0, A) and presence (0, A) of 2.4 mM pCMB for 60 min at 30°C. It was then diluted 20-fold with the sucrose-Tris buffer, treated for 60 min at 30°C with the indicated amounts of dithiothreitol, and kept on ice. ATPase activity was measured as described in Fig. 1, except that complex V was 3.6 pg/ml. Where indicated (A, A), the assay contained 2.5 *g/ml of oligomycin.

pombe,Sacc~myces

cerevisiae, and rat liver (the latter after treatment with bicarbonate) are susceptible to inhibition by thiol modifiers (1315,18). Light-activated chloroplast ATPase is also inhibited by modification of thiol groups in the y subunit (16,1’7). Therefore, it was of interest to determine whether the inhibition of the bovine enzyme by pCMB or pCMS was the result of modifications in F1 or FO, a point that had not been adequately investigated and clarified previously. It was first shown that under comparable conditions, isolated bovine heart F1 was not inhibited by any of the thiol modifiers used above. Also, unlike the rat liver F1-ATPase

THIOLS

IN OXIDATIVE

(18), the ATPase activity of the bovine enzyme was unaffected when it was incubated for 1 h at 30°C with 4.5 InM pCMB or pCMS in the presence of 20 MM sodium bicarbonate (data not shown). However, these results with the isolated F1-ATPase left open the possibility that the membrane-bound F1 might have a different configuration from the isolated enzyme, and might be susceptible to inhibition by mercurials. Accordingly, two types of experiments were performed to investigate this possibility and to see whether the site of mercurial inhibition is in FO. In the first experiment, SMP were treated with pCMB to inhibit ATPase activity. Then F1 was extracted from the inhibited and control particles and assayed for activity. As seen in Table I, the F1 extracted from the pCMBinhibited particles had essentially the same ATPase activity as that isolated from the control particles. As an added control, Table I also shows that, as expected, the TABLE

I

ATPase ACTIVITYOFF~ ISOLATEDFROM PCMB-INHIBITED SMP” ATPase Activity6 Enzyme Control SMP pCMB-treated SMP F1 from control SMP Fr from pCMB-treated SMP

-0ligomycin

+Oligomycin

4.36 0.41 14.7

0.09 0.08 13.7

15.9

15.5

QSMP at 10 mg/ml in 0.25 M sucrose and 50 mM Tris-acetate, pH 7.5, were treated with 2.3 rnM pCMB for 1 h at 3O”C, diluted 12-fold with the same buffer, and centrifuged at 36,000 rpm for 20 min. The pellet was homogenized in 5 ml of 0.25 M sucrose and 10 mM Tris-acetate, pH 7.5, recentrifuged as before, and the pellet was taken up in 1 ml of the second buffer. To 0.8 ml of the particle suspension was added 0.4 ml of chloroform, vigorously mixed for 10 s, and the mixture was centrifuged in a clinical centrifuge at top speed for 2 min. The aqueous layer was withdrawn and centrifuged for 30 min at 36,000 rpm. The supernatant was used as the source of Fi. Control SMP was similarly treated, except that the addition of pCMB was omitted. ’ cmol ATP hydrolyzed (min * mg of protein)-‘.

PHOSPHORYLATION

105

ATPase activity of the particles was sensitive to oligomycin, while the ATPase activity of the extracted F1 was not. In the second experiment, F0 was isolated from complex V preparations (24), and treated with pCMB as described. Then, the pCMB-treated and control F. were reconstituted with F1 and OSCP (24,30), and assayed for ATPase activity. As seen in Experiment 1 of Table II, the ATPase activity of the reconstituted system containing pCMB-treated F,, was only about 25% of the activity of the control. Both reconstituted activities were oligomycin sensitive and, as seen in Experiment 2, the pCMB inhibition could be reversed by treatment of the reconstituted preparation with dithiothreitol. These results show clearly that the site of inhibition by mercurials of the ATPase activity of bovine ATP synthase is not in F1 and resides in F,,. Protection by wm-curiak of the inhibition of oligomycin-sensitive ATPose activity bg DCCD. A finding of considerable interest was that pretreatment of SMP with pCMB appeared to offer considerable protection against irreversible inhibition of ATPase activity by DCCD. As seen in Fig. 4, treatment of SMP for 30 min at 0°C with 10 to 40 PM DCCD (under these conditions, only the DCCD-binding protein was labeled when [14ClDCCDwas used) (31,32) resulted in about 90% inhibition of ATPase activity (Fig. 4, open circles), and subsequent treatment of the particles with dithiothreitol did not reverse the inhibition due to DCCD (Fig. 4, open triangles). However, when the particles were first treated with 4.5 mM pCMB to give -90% inhibition, and then with DCCD as before (Fig. 4, filled circles), subsequent addition of dithiothreitol resulted in considerable reappearance of activity (Fig. 4, filled triangles). These data suggest that pCMB protected the enzyme against irreversible inhibition by DCCD. Consequently, the addition of dithiothreitol reversed the modification by pCMB, resulting in recovery of ATPase activity. Similar results were obtained when triphenyltin chloride was used, instead of pCMB, as the first inhibitor of ATPase activity (data not shown). However, when oligomycin was used, instead of DCCD,

106

YAGI

AND TABLE

HATEFI II

TREATMENT OF F0 WITH pCMB FOLLOWED BY RECONSTITUTION WITH FiO Percentage Experiment 1 2

Enzyme

-0ligomycin

Control Fo + F1 pCMB-Fo + Fi Control F. + Fi, treated with DTT pCMB-Fo + Fi, treated with DlT

100 24.5 78 69

ATPase activityb +Oligomycin 4.1 2.8 3.1 3.4

aFa, isolated from complex V, was suspended at a protein concentration of 7 mg/ml in 0.25 M sucrose and 50 mM Tris-acetate, pH 7.5, and treated with 1.2 mM pCMB for 30 min at 30°C. It was then diluted 25-fold with the same buffer, and centrifuged at 10,000 rpm for 15 min. The precipitate was suspended in the above buffer and adjusted to a protein concentration of about 3 mg/ml. A similar Fo sample not treated with pCMB and control F. was added 2 ~1 of F1 (10 was taken through the same steps.To 20 ~1 each of the pCMB-treated mg/ml), incubated for 4 min at 30°C and then 2 ~1 of OSCP (0.64 mg/ml), and further incubated for 20 min at 30°C. Each mixture was diluted 40-fold with the sucrose-Tris buffer, centrifuged as before, and each pellet was suspended in 50 ~1 of buffer and assayed. Where indicated, 20 ~1 of the reconstituted Fo-Fi suspension was treated with 0.4 ~1 of 100 mM dithiothreitol (DTT) for 30 min at 30°C and then placed on ice until assayed. * 100% ATPase activity of control F,,-F, was 4.68 Mmol (minamg of protein)-’ in Experiment 1, and 2.81 amol (min * mg of protein)-’ in Experiment 2.

pCMB offered no protection, and there was no reversal of inhibition upon subsequent addition of dithiothreitol to particles treated first with pCMB, then with oligomycin (data not shown). The latter results are not inconsistent with the fact that oligomycin also appears to bind to the DCCDbinding protein. Mutational analyses in Saccharomyces cerevisiae have indicated that the oligomycin-binding site on the DCCD-binding protein is a large domain encompassing a stretch of more than 10 residues (33). Therefore, such a large binding domain may not be effectively occluded by pCMB, whereas the reactivity of a single glutamic acid with DCCD might be altered by the close proximity of pCMB whether on the same (Cys-64 in the bovine DCCDbinding protein) or a nearby polypeptide. DISCUSSION

Together, the present work and our previous results (19, 29) indicate that there are at least two sets of essential thiols in the F0 of bovine ATP synthase complex. One set reacts with various modifiers of mono- and vicinal dithiols, its reactivity toward these modifiers is greatly increased by membrane energization, and its modi-

fication results in uncoupling and increase of ATPase activity in SMP (19, 29). The second set, described here, is modified at reagent concentrations higher than those required for uncoupling, is most reactive toward mercurials (pCMB > pCMS) and relatively unreactive toward N-ethylmaleimide. Modification of this set of thiols results in inhibition of ATPase activity. Both the uncoupling and the inhibition of ATPase activity are completely reversible under appropriate conditions, and result in restoration of coupled and oligomycinsensitive ATPase activity. An important question is where in the ATP synthase complex these two sets of thiols are located. Our results have shown clearly that, unlike the yeast and the rat liver enzymes (13-15, X3), the isolated and the membrane-bound Fr-ATPase from bovine heart mitochondria is not susceptible to inhibition under the conditions used in the above references or those used in the studies reported here. Furthermore, coupling factor Fs does not have a cysteine residue (34), and the F0 subunits 6 and A6L appear cysteine-free as judged from their gene sequences(35). OSCP contains a single cysteine residue, which is apparently nonessential (25, 36), and the bovine DCCD-

THIOLS

I

I

0

I

10

20

30

IN OXIDATIVE

I

NIX. pM FIG. 4. Protection by pCMB of the ATPase activity of SMP against inhibition by DCCD. SMP at 9 mg/ (pH 7.5) ml in 0.25 M sucrose and 50 mM Tris-acetate was incubated for 60 min at 30°C in the absence (A, 0) and presence (A, 0) of 4.6 mM pCMB, and then filtered through Sephadex G-25 columns equilibrated in the same buffer to remove unreacted pCMB. The particles were then treated for 30 min on ice with the indicated concentrations of DCCD, diluted lo-fold with the sucrose-Tris buffer, and recentrifuged through Sephadex G-25 columns equilibrated with sucroseTris buffer. Finally, they were incubated for 60 min at 30°C in the absence (0, 0) and presence (A, A) of 360 mM dithiothreitol, and assayed for ATPase activity as described in Fig. 1.

binding protein contains a cysteine six residues downstream of the DCCD-reactive GIu-58 (7). Whether the latter cysteine plays an essential role in the bovine F0 is not known. However, the yeast DCCDbinding protein contains a cysteine in a similar position, and mutational exchange of this residue to serine caused neither uncoupling nor inhibition of ATPase activity (37). Aside from these subunits and the ATPase inhibitor protein, with which we need not be concerned here, the bovine ATP synthase appears to contain two other subunits. A polypeptide, designated (b) in Fig. 5, which on Laemmli sodium dodecyl sulfate gels runs immediately behind OSCP, and another (band (d)) which according to Fearnley and Walker (2) comigrates on Laemmli gels with subunit 6. These two polypeptides ((b) and (d)) contain one cysteine each (Dr. J. Walker, private communication), whose modifications

107

PHOSPHORYLATION

could result in uncoupling or inhibition of ATPase activity. However, it is possible that uncoupling by Cd2+,phenylarsene oxide, or Cu2+plus o-phenanthroline is indicative of modification of a vicinal dithiol (see also (20)). In that case, a possible candidate is factor B. Factor B is a water-soluble protein of M, -12,000, whose existence was first discovered by Sanadi and co-workers (38). The protein was subsequently purified by You and Hatefi (39) and was suggested to contain an essential dithiol((40, see also (41)). SMP preparations extracted with NH,OH-EDTA (AE-SMP), presumably to remove factor B (38-41), lose the ability to catalyze energy-linked reactions involving the ATP synthase (i.e., oxidative phosphorylation, ATP-32Pi exchange, ATP-induced reverse electron transfer from succinate to NAD, ATP-induced transhydrogenation from NADH to NADP). Addition of factor B restores all these functions (40). Curiously, however, addition of low levels of oligomycin to AE-SMP also restores these functions (38, 39) whereas addition of low or high levels of oligomycin to SMP uncoupled by treatment with mono- or dithiol modifiers does not restore coupling (20). This difference suggests that the site of uncoupling by thiol modifiers may not be factor B. T ,

i , !

Y 1 III,

0

‘-.

II !I

FIG. 5. Densitometric trace of complex V electrophoresed on a sodium dodecyl sulfate-16% acrylamide slab gel according to the method of Laemmli (43) and stained for protein visualization with Coomassie blue. The amount of complex V placed on the gel was 35 pg. The scanning wavelength was 560 nm. Subunits a, 0, y, and OSCP are designated. The bands flanking OSCP are marked b and ATPase-6,d after Fearnley and Walker (2).

108

YAGI

AND

Finally, we have shown that pretreatment of complex V with pCMB reversibly protects the enzyme against inhibition by DCCD. The extent of this protection is diminished when the DCCD concentration is increased, suggesting that the covalently bound pCMB interferes with the approach of DCCD to its binding site, or that pCMB modification alters the configuration of the DCCD-binding site. Whether the single cysteine of the DCCD-binding protein is modified by pCMB, resulting in occlusion or configuration change of the DCCDbinding site, or the cysteine residues of polypeptides b and d are located near the DCCD-reactive glutamic acid residue of the DCCD-binding protein remains to be seen. As mentioned above, organotin compounds also behave like pCMB in reversibly protecting the ATP synthase against inhibition by DCCD. Delineation of the binding site and mechanism of action of organotin compounds would be particularly interesting, because these compounds inhibit ATP synthesis and hydrolysis, but do not prevent the energy-induced conformation change of F1 as detected by the fluorescence change of F1-bound aurovertin (42).

HATEFI

10.

11. 12. 13. 14. 15. 16. 17.

18. 19. 20.

21. 22.

ACKNOWLEDGMENT The authors thank C. Munoz for the preparation mitochondria.

of

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IN OXIDATIVE

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37. SEBALD, W., WACHTER, E., AND TZAGOLOFF, A. (1979) Eur. J. Biock 100,599-607. 38. SANADI, D. R. (1982) Biochim Biophys. Acta 683, 39-56. 39. You, K.-S., AND HATEFI, Y. (1976) B&him Bie phys. Acta 423,398-412. 40. STIGGALL, D. L., GALANTE, Y. M., KIEHL, R., AND HATEFI, Y. (1979) Arch B&hem Biophys. 196, 638-644. 41. JOSHI, S., AND HUGHES, J. B. (1981) J. Biol Chem 256,11112-11116. 42. MATSUNO-YAGI, A., YAGI, T., AND HATEFI, Y. (1985) Proc. Natl. Acad. Sci. USA 82,7550-7554. 43. LAEMMLI, U. K. (1970) Nature (London) 227,680685.