Proc. Natl. Acad. Sci. USAVol. 82, pp. 1886-1890, April 1985Biochemistry

Evidence that the Mg-dependent low-affinity binding site for ATPand Pi demonstrated on the isolated f8 subunit of the FoF1 ATPsynthase is a catalytic site

(HW-translocating ATPase/active site/chemical modification/photophosphorylation/reconstitution)

D. KHANANSHVILI AND Z. GROMET-ELHANAN*Department of Biochemistry, The Weizmann Institute of Science, Rehovot 76100, Israel

Communicated by Martin D. Kamen, November 9, 1984

ABSTRACT Binding sites for one Pi and two ATP or ADPmolecules have been identified on the isolated, reconstitutivelyactive .3 subunit from the Rhodospirillum rubrum Fo-F1 ATPsynthase. Chemical modification of this .8 subunit by thehistidine reagent diethyl pyrocarbonate or by the carboxylgroup reagent Woodword's reagent K results in completeinhibition of Pi binding to (3. The same reagents inhibit thebinding of ATP to a Mg-dependent low-affinity site but not toa Mg-independent high-affinity site on this 13 subunit. Thebinding stoichiometry of ADP to either site is not affected bythese modifications. The /3 subunit modified by either one ofthese reagents retains its capacity to rebind to fl-lesschromatophores but not its ability to restore their photo-phosphorylation. These results indicate that the low-affinity Pibinding site on .3 is located at the binding site of the y-phosphate group of ATP in the Mg-dependent low-afflinitynucleotide binding site. This site contains histidine andcarboxyl group residues, both of which are required for thebinding of Pi and of the V-phosphate group of ATP. The sameresidues must also be involved in the capacity of the isolated 1Bsubunit to restore the catalytic activity of the fl-less ATPsynthase. It is therefore concluded that the low-affinity Mg-dependent substrate binding site identified on the isolated /3subunit of the R. rubrum Fo0F1 ATP synthase is the catalyticsite of this enzyme complex.

The terminal step of ATP synthesis in energy-transducingmembranes is generally considered to be catalyzed by amembrane-bound reversible proton-translocating ATPase(1) present in membranes of mitochondria, chloroplasts, orbacteria and composed of two sectors: Fo and F1 (2-5). Fo isan integral membrane protein that contains at least threesubunits and mediates proton translocation across the mem-brane. F1 is a peripheral membrane protein composed of fivenonidentical subunits-a, /3, y, 8, and E-and is the catalyticportion of the Fo0F1 complex. The molecular mechanism ofATP synthesis and hydrolysis carried out by this Fo-F1complex is still unknown, although a number of mechanismshave been proposed (6, 7). Elucidation of the mechanism ofaction of this enzyme complex requires a detailed descrip-tion of its catalytic site. This includes identification of thecatalytic subunit and characterization of substrate bindingsites and essential amino acid residues on this subunit.A large number of studies using different approaches point

to the p subunit as the one that contains the catalytic site andis involved in substrate binding (3, 4, 8). Thus, variousstudies have shown that the F1 sector has several nucleotidebinding sites that reside in its two larger subunits, a and /3(4,7, 9-11). At least one Pi binding site has also been revealedin the F1 sector (12, 13), and from studies with a Pi analog it

seems to be located on the p subunit (14-16). Attempts toidentify functional amino acids in the catalytic site have beencarried out by using labeled chemical modifiers, known tointeract with specific amino acid residues, that bind to the F1ATPase and inactivate it. After dissociating the labeledenzyme complex to its individual subunits, the label wasdetected mainly on the p subunit (7, 8).A detailed characterization of individual substrate binding

sites and their possible identification with the catalytic site inthe Fl-enzyme complex is difficult because of the complex-ity of its structure and function. It contains two or threecopies of aB pairs, which could be in different confor-mational states in the catalytically active complex (17, 18),and its activity leads to interconversion of the substrates.These difficulties may be overcome if, instead of testing thewhole F1 sector, isolated, purified, and reconstitutivelyactive a and p subunits are used. Such subunits have beenobtained so far only from three bacterial sources: athermophilic bacterium (19), Escherichia coli (20), andRhodospirillum rubrum (21). They are indeed a much sim-pler system for study as they show no subunit-subunitinteractions and no catalytic activity by themselves, al-though they can restore ATP synthesis or hydrolysis (orboth) to preparations that lack them (19-21).

Direct binding studies with labeled substrates have beencarried out only with the a subunit of E. coli (22) and the 8subunit of R. rubrum (23, 24, 41). These studies haveidentified one nucleotide binding site on the a subunit (22),whereas the p subunit contained two binding sites for ATP(23) or ADP (24) and one binding site for Pi (41). One of thenucleotide binding sites on the p subunit is a Mg-dependenthigh-affinity site, which is very similar to the binding sitelocated on the a subunit. The second is a Mg-dependentlow-affinity site, which has been suggested to contain alsothe binding site for Pi, because both nucleotides have beenfound to be competitive inhibitors of Pi binding (41). Further-more, Pi seems to bind at the site occupied by the y-phosphate group of ATP, since ATP is a much more potentinhibitor of Pi binding to the p subunit than is ADP.

In this study we attempt to identify the amino acidresidues that participate in the substrate binding sites on theisolated p3 subunit and in its reconstitutive activity. For thispurpose we prepared various chemically modified-13 prepa-rations by using a number of reagents that have been shownto inactivate the soluble R. rubrum ATPase (RrF1) (25-28).These modified-,p preparations have been tested for their

Abbreviations: RrFO Fj, proton-translocating ATP synthase-ATPase complex of Rhodospirillum rubrum; RrFj, soluble R.rubrum ATPase; NBD-Cl, 4-chloro-7-nitrobenzofurazan; WRK,Woodword's reagent K; DCCD, N,N'-dicyclohexylcarbodiimide;EEDQ, 1-ethoxycarboxyl-2-ethoxy-1,2-dihydroquinoline; DEPC,diethyl pyrocarbonate; Tricine, N-[tris(hydroxymethyl)methyl]glycine; Mes, 2-(N-morpholino)ethanesulfonic acid.*To whom reprint requests should be addressed.

1886

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Biochemistry: Khananshvili and Gromet-Elhanan

capacity to bind ATP, ADP, and P1 and to restore photo-phosphorylation activity to 8-less R. rubrum chromat-ophores.

MATERIALS AND METHODSR. rubrum cells were grown as outlined in ref. 21. Coupledand ,8-less chromatophores were prepared as described (21,23, 29, 30). The reconstitutively active 8 subunit of proton-translocating ATP synthase-ATPase complex of R. rubrum(RrF0 F1) was extracted from R. rubrum chromatophores byLiCl (21), purified, and stored according to ref. 31. In all ofthe experiments reported here, an electrophoretically pure

subunit, which restored 92-97% of the photophosphoryla-tion or Mg2+-ATPase activities of p-less chromatophores,was used. Before any interaction of the 8 subunit with thevarious chemical modifiers it was freed from the storagebuffer by elution-centrifugation (12) on a Sephadex G-50column preequilibrated with the modification buffer used foreach modifier, as described below.Chemical Modification. Modification of the subunit by

4-chloro-7-nitrobenzofurazan (NBD-Cl) or Woodword's rea-

gent K (WRK) was carried out by incubating the p subunit at1 mg/ml in TGEN buffer, containing 50 mM N-[tris(hydrox-ymethyl)methyl]glycine (Tricine)/NaOH (pH 8.0), 2 mMEDTA, 50 mM NaCl, and 20% glycerol, with 300 ,MNBD-Cl for 1 hr or with 1 mM WRK for 15 min at 230C (25).For modification by N,N'-dicyclohexylcarbodiimide(DCCD) or 1-ethoxycarboxyl-2-ethoxy-1,2-dihydroquinoline(EEDQ), the p subunit at 1 mg/ml was incubated in MGENbuffer, containing 50 mM 2-(N-morpholino)ethanesulfonicacid (Mes)/NaOH (pH 6.5) instead of the Tricine used in theTGEN buffer, with 100 ,tM of each reagent for 1 hr at 30°C(26). Modification of the subunit by diethyl pyrocarbonate(DEPC) was carried out, as outlined for RrF1 in ref. 27, byfive stepwise additions of 50 ,uM DEPC at 10-min intervals top at 1 mg/ml in MGEN buffer. Each of the above describedmodified-p8 preparations was freed from the unreacted rea-

gent by elution-centrifugation on a Sephadex column thatwas preequilibrated with the buffer used during the modifica-tion.

Substrate Binding. Binding studies were carried out on thenative- and on the various modified-,p preparations with[3H]ATP, [3H]ADP, or 32Pi under the previously establishedoptimal binding conditions (23, 24, 41). at 0.5 mg/ml was

incubated in TGM buffer, containing 50 mM Tricine/NaOH(pH 8.0), 20% glycerol, and 5 mM MgCl2, with the indicatedconcentrations of ATP, ADP, or Pi. The binding was initi-ated by addition of the subunit, and after 1 hr at 23°C theincubation mixtures were subjected to elution-centrifugationon Sephadex columns preequilibrated with TGM buffer butcontaining 20 mM MgCl2 (24). The effluent from eachcolumn was diluted with 1 ml of water and appropriatealiquots were assayed for 3H or 32P radioactivity and proteincontent.

Assays and Materials. Reconstitution of the native-,8 or

modified-p preparation into ,8-less chromatophores was car-

ried out as described in ref. 25. The reconstitutedchromatophores were centrifuged to remove excess unre-

constituted , dissolved in 100 ,ul of 50 mM Tricine/NaOH(pH 8.0), and assayed for photophosphorylation as described(31). Protein was measured according to Lowry et al. (32),and bacteriochlorophyll was determined by using the in vivoextinction coefficient given by Clayton (33). 3H or 32pradioactivity was measured by liquid scintillation counting(12). Binding data were calculated by using a molecularweight of 50,000 for the subunit (34). The number ofmodified histidine residues was calculated by using 3200M-1.cm-l as the molar absorption coefficient forcarbethoxyhistidine (27, 35). [2,8-3H]ATP (24-29 Ci/mmol;

1 Ci = 37 GBq) and [2,83HJADP (26-30 Ci/mmol) werepurchased from New England Nuclear and diluted with thecorresponding nonradioactive nucleotides as described inref. 23. 32p, was obtained from the Nuclear Research Center(Negev, Israel) and purified on Dowex AG 1-X4, so that itcontained <0.001% impurities.

RESULTSEffect of Histidine, Carboxyl, and Tyrosine Modifying

Reagents on the Capacity of the fi Subunit to Bind Pi, ATP,and ADP. The histidine reagent (35) DEPC has been reportedto inhibit the RrFj ATPase activity (27). Complete inactiva-tion required the modification of 2 or 3 histidine residues permolecule of RrF1. Incubation of the isolated, reconstitu-tively active R. rubrum 8 subunit with DEPC, under theconditions described in Materials and Methods, led to themodification of 1.3-1.7 histidine residues per mol of psubunit (not shown).The capacity of this DEPC-modified , subunit to bind Pi,

ATP, and ADP was compared with that of native-,p (Fig. 1).Incubation of native-p with Pi, ATP, or ADP resulted inbinding of about 0.9 mol of Pi per mol of p (Fig. lA Inset) andabout 2 mol of either ATP or ADP per mol of p (Fig. 1 B andC Inset). A Scatchard plot analysis of the binding datareveals one binding site with a Kd of 270 AuM for Pi (Fig. 1A)and two, very similar, binding sites for each of the nucleo-tides (Fig. 1 B and C). The first is a high-affinity site with aKd of 5 ,uM for ATP and 9 ,uM for ADP (Table 1), which hasbeen shown to be independent of MgCl2 (23). The second isa low-affinity site with a Kd of 200 ,uM for ATP and 90 ,uMfor ADP (Table 1), which has been shown to be absolutelydependent on the presence of MgCl2 (23, 24). Modification ofthe native R. rubrum p subunit by DEPC resulted in com-plete inhibition of the binding of Pi (Fig. 1A) as well as ininhibition of the binding of ATP to the Mg-dependent low-affinity site but not to the Mg-independent high-affinity site(Fig. 1B). The binding stoichiometry ofADP topA, unlike thatof Pi and- ATP, was not affected by this modification (Fig.1C). The only effect of this modification on the binding ofADP was a decrease in the affinity of its binding to thelow-affinity binding site, resulting in an increase in the Kdfrom 90 to 490 AM (Table 1).A very similar effect to that of DEPC on the capacity of

the p subunit to bind Pi, ATP, and ADP was obtained when,p was modified by the carboxyl group modifier WRK (Table1). This modifier, unlike two other carboxyl group modifiers,DCCD and EEDQ, had a very low inhibitory effect on RrFj,when the modification was carried out at pH 6.0 (26), butinhibited the RrFj ATPase activity when incubated with it atpH 7.0 (28). It was shown previously that DCCD and EEDQ,but not WRK, did bind to the isolated R. rubrum p subunitwhen they were incubated at pH 6.0 (26). In light of theseresults the modification of the isolated p subunit by WRKwas carried out at pH 8.0, whereas its modification byDCCD and EEDQ was carried out at pH 6.5 under condi-tions that were shown to lead to the binding of 1 mol of['4C]DCCD per mol of p subunit (26).A marked difference between DCCD or EEDQ and WRK

was also observed when their effect on the capacity of the 8subunit to bind Pi, ATP, and ADP was tested (Table 1).Modification of the 8 subunit with either DCCD or EEDQ,unlike with WRK, had no significant effect on the bindingstoichiometry of any of the tested ligands. Either one,however, decreased the binding affinities of Pi, ATE, andADP to their binding sites, with a 3- to 6-fold increase in theapparent Kd values. These results indicate that the carboxylgroup residue modified by WRK on the isolated p subunit isdifferent from that modified by DCCD and EEDQ.

Proc. Natl. Acad. Sci. USA 82 (1985) 1887

1888 Biochemistry: Khananshvili and Gromet-Elhanan

c.

0_bX

0 0.2 0.6 1.0

Pi bound(mol/molB)

nc.O

W

0 0p

-L a

0 0.5 1.0 1.5 2.0ATP bound(mol/moloI)

T

2

- Qc .O w

.0

0 a

0 0.5 1.0 1.5 2.0ADP bound (mol/mol,8)

FIG. 1. Scatchard plot analysis of the binding of Pi, ATP, and ADP to a DEPC-modified ,B subunit. , was modified by DEPC and freed fromthe unreacted reagent. This modified-,B (o) and native-,3 (A) were incubated with the indicated concentrations of 32Pi (A), [3H]ATP (B), and[3H]ADP (C), freed from the unbound ligand, and assayed for 3H or 32P radioactivity and protein content.

The tyrosine reagent NBD-Cl has been shown to bind toRrF1 and inhibit its ATPase activity. It was also shown tobind to the isolated R. rubrum subunit with a saturatingbinding stoichiometry of 1 mol of ["4C]NBD-Cl bound permol of (25). The capacity of this NBD-p adduct to bind Pi,ATP, and ADP was closer to that of native-p than any of theother types of modified-p8 (Table 1). There was very littlechange either in the binding stoichiometries of the threeligands or in their binding affinities.

Effect of Histidine, Carboxyl, and Tyrosine ModifyingReagents on the Capacity of the Subunit to Bind to a-LessChromatophores and Restore Their PhotophosphorylatingActivity. To restore photophosphorylation to p-lesschromatophores, the added subunit must be capable ofrebinding to these chromatophores and, once rebound, ofrestoring their activity (37). Therefore, when testing forpossible reconstitution of p-less chromatophores with vari-ous modified-p preparations three types of effects may beexpected: (i) The modified-p does rebind to 8-less chro-matophores and restores their activity as native-p. In thiscase the modified amino acid residue is not essential forrebinding or for activity. (ii) The modified-p8 does rebind top-less chromatophores but cannot restore their activity. Inthis case the modified amino acid residue is not essential forrebinding but is essential for the reconstitutive activity of the

p3 subunit. (iii) The modified-p cannot rebind to p-lesschromatophores and consequently does not restore theiractivity. In this case the modified amino acid residue isessential for rebinding of the subunit to p-less chro-matophores.Thus, in the last two cases there will be no restoration of

activity, but for different reasons. These two cases can beeasily differentiated by introducing an additional step ofreconstitution with native-p. If the modified-p,. added to thep8-less chromatophores in the first reconstitution step, didrebind to them but could not restore their activity, thesechromatophores will not be able to rebind any native-padded in the second step. Thus, there will be no restorationof activity even after the second step of reconstitution. If,however, the modified-p lost its capacity to rebind to p-lesschromatophores, they will be able to bind the native-p addedin the second reconstitution step and thus regain theiractivity.

Fig. 2 summarizes results of such tests. In the firstreconstitution step (step I) increasing concentrations ofvarious modified-p8 preparations were tested for their capac-ity to rebind to a fixed concentration of p-less chromat-ophores and restore their activity. In reconstitution step II afixed, saturating concentration of native-p was added to

Table 1. Effect of modification of the p subunit by various chemical modifiers on its capacity to bind Pi,ATP, and ADP

Type of Bound 32p,modified mol/mol of (3 Bound [3H]ATP, mol/mol of (3 Bound [3H]ADP, mol/mol of (3f subunit N N N1 N2 N N1 N2Native-f3 0.93 (270) 1.84 0.97 (5) 0.93 (195) 1.88 0.83 (9) 1.05 (87)DEPC-P3 0.01 0.76 0.76 (10) 0 1.76 0.79 (8) 0.97 (490)WRK-(3 0.01 0.85 0.85 (9) 0 1.88 0.90 (10) 0.98 (523)NBD-,B 0.91 (420) 1.81 0.78 (7) 1.03 (220) 1.89 0.86 (12) 1.03 (98)DCCD-,B 0.82 (750) 1.62 0.64 (30) 0.98 (652) 1.83 0.84 (52) 0.99 (505)EEDQ-,B 0.76 (876) 1.79 0.72 (33) 1.05 (678) 1.79 0.81 (62) 0.98 (608)

,3 was modified by the indicated reagents and freed from the unreacted reagents. The modified-/3 preparations wereincubated with 1-4000 AiM [3H]ATP or [3H]ADP or with 10-4000 ,uM 32p;, freed from the unbound ligand, and assayed for3H or 32P radioactivity and protein content as described in the legend to Fig. 1. N is the overall binding stoichiometry ofthe assayed substrates. N1 and N2 represent the binding stoichiometry of each binding site obtained as shown in Fig. 1. Thenumbers in parentheses represent the dissociation constants (in ,.tM) that were obtained by treatment of the Scatchard plotsaccording to Rosenthal (36).

Proc. Natl. Acad. Sci. USA 82 (1985)

Biochemistry: Khananshvili and Gromet-Elhanan

A B C0 100 _ Steps(I+I) Steps(I+ff)

80 _ p

Step I Steps(I+I)0 60 2

040

n

20

Step I Step Iw I BY

0 100 200 0 100 200 0 100 200Native-,8(o) or DCCD-R(o) or DEPC-,3(e) orNBD-,B (s), ELg EEDQ-f3(v),.g WRK-/3 (A), ELg

FIG. 2. Effect of modification of the 8 subunit by variouschemical modifiers on its ability to rebind to /-less chromatophoresand restore their photophosphorylating activity. The different typesof modified-p were prepared as described in the legend to Table 1.Native-,8 and the various modified-,p preparations were reconsti-tuted, at the indicated concentrations, into p-less chromatophorescontaining 10 utg of bacteriochlorophyll. The reconstituted chro-matophores were centrifuged to remove excess unbound p andeither assayed directly for their photophosphorylating activity (stepI) or subjected to a second reconstitution period with 100 ,ug ofnative-p and then assayed for photophosphorylation (steps 1+II).The control photophosphorylation activity was 847 Amol/hr per mgof bacteriochlorophyll.

such reconstituted chromatophores, after they were washedfree of any excess unreconstituted modified-,p.The various types of modified-,p preparations fall into the

three distinct groups described above. NBD-,B belongs to thefirst group. It restores the photophosphorylating activity ofp-less chromatophores in a concentration-dependent satu-rating curve, which is identical to that obtained with natives(Fig. 2A, step I). When >100 ,ug of either native-p or NBD-,8was used for reconstitution of 8-less chromatophores con-taining 10 ,tg of bacteriochlorophyll, practically all of theirphotophosphorylation capacity was restored. When lowerconcentrations of either native-p3 or NBD-,8 were usedduring the first reconstitution period, the lower restoredactivity could be increased to its maximal level by a secondreconstitution period with 100 ,4g of native-pB (Fig. 2A, stepsI+II).We have earlier shown (37) that [14C]NBD-p did rebind to

p8-less chromatophores while still carrying the [14C]NBDlabel, indicating that the restored activity obtained withNBD-p8 was not due to dissociation of the NBD-,p adductduring the reconstitution period. Thus, the p subunit modi-fied by NBD-Cl is as active as native-p in rebinding to 8-less

Mg Pi

a P ATP a p ATPlG P or R P orY P ADP r P ADP

chromatophores and restoring their activity.DCCD-,p and EEDQ-p3 belong to the third group described

above. This type of modified-,p lost its capacity to rebind tothe p-less chromatophores and, therefore, could not restoretheir activity (Fig. 2B, step I). However, these modified psubunits did not interfere with the binding of natives in thesecond reconstitution period, resulting in full restoration ofphotophosphorylation (Fig. 2B, steps I+II).The most interesting group is that ofDEPC-,B and WRK-p,

which belong to the second type of the above describedgroups. These p adducts, like DCCD-,8 and EEDQ-p3, couldnot restore any activity by themselves (Fig. 2C, step I), butthey inhibited, in a concentration-dependent manner, thebinding of native-p in the second reconstitution period (Fig.2C, steps 1+11). Thus, when 200 ,ug of either DEPC-p orWRK-,p was applied in the first reconstitution period, noactivity could be restored after a second reconstitutionperiod with 100 pug of native-p. These results suggest thatboth DEPC-,3 and WRK-,o can rebind to the 8-lesschromatophores but, once bound, they are incapable ofrestoring any photophosphorylating activity.

DISCUSSIONChemical modification of histidine and carboxyl group resi-dues on the isolated p subunit of the RrF0-F1 ATP synthaseby DEPC and WRK, respectively, was found to affect itscapacity to bind Pi and ATP as well as to rebind to p-lesschromatophores and restore their activity (Figs. 1 A and Band 2C). Thus, when either one of these residues on theisolated p subunit was modified, the modified-,8 lost theactivity of its Pi binding site and of one of its ATP bindingsites, but both its ADP binding sites remained active. Suchmodified-,p can rebind to p-less chromatophores but cannotrestore their activity.The fact that the histidine and carboxyl group residues

that were modified by DEPC and WRK are required for thebinding of ATP and Pi, but not of ADP, suggests that thesemodifications affect the binding of the y-phosphate group ofATP and emphasizes the similarity between the binding ofthis phosphate group of ATP and that of Pi (41). The siteaffected by these modifications is the Mg-dependent low-affinity binding site of ATP, since the high-affinity Mg-independent ATP binding site, also present on the isolated isubunit (Fig. 1B, Table 1, and ref. 23), is not affected at all byeither modifier. These observations have been summarizedin the schematic presentation of Fig. 3.The Mg-independent high-affinity nucleotide binding site

is illustrated as involving mainly the adenosine moiety of thenucleotides, since (i) ATP and ADP have a very similaraffinity to this site (Table 1 and ref. 23), and (ii) it is notaffected by Mg2' at pH 8.0 when all of the phosphate groupsare nearly fully charged. Furthermore, the binding of either

Mg ADP Mg ATP

/3 /

a P ATP/3 P ory P ADP

FIG. 3. A scheme of Pi, ATP, and ADP binding sites demonstrated on the isolated, reconstitutively active /3 subunit of the RrFOF1 ATPsynthase. For explanations, see the text.

Proc. Natl. Acad. Sci. USA 82 (1985) 1889

--n(;.

'A

1890 Biochemistry: Khananshvili and Gromet-Elhanan

ATP or ADP to this site is affected in a very similar mannerby all tested chemical modifying reagents (in Table 1, see thenumbers corresponding to N1) and does not seem to requirethe presence of functional histidine or carboxyl group resi-dues. This Mg-independent site may be located at aninterphase between the v and a subunits of the Fo0F1 ATPsynthase and thus shared by both. Such sharing has beenproposed by a number of investigators (4, 38-40), whosuggested this site to be the catalytic one, whereas our dataindicate that it is not the catalytic site.

Unlike the high-affinity site, the Mg-dependent low-affinity nucleotide binding site is illustrated as involving thephosphate groups in addition to the adenosine moiety. Thebinding of Pi and the y-phosphate group of ATP, but not ofthe other phosphate groups that are present also in ADP, tothis site depends on the presence of functional histidine andcarboxyl group residues. When these are modified, thebinding of Pi as well as that of ATP is eliminated, whereasthe binding affinity of ADP is only slightly decreased.The modification by DEPC and WRK also exerts a similar

effect on the capacity of the /3 subunit to restore thephotophosphorylating activity of p-less chromatophores.This is most likely related to their effect on theMg-dependent low-affinity substrate binding site of the /3subunit. A comparison of the effect of other chemicalmodifiers on the substrate binding properties of / and on itscapacity to reconstitute /3-less chromatophores providesadditional experimental evidence for this suggestion. Thus,both DCCD and EEDQ, which decrease in an almost identi-cal manner the binding affinities of the three tested sub-strates to all of their binding sites (Table 1), exhibit also asimilar effect on the capacity of / to bind to /3-lesschromatophores (Fig. 2B). Also, modification by NBD-Cl,which does not affect the substrate binding properties of ,,shows no effect on its reconstitutive activity.

In light of these results we propose that the low-affinityMg-dependent nucleotide binding site identified on the iso-lated R. rubrum /3 subunit (23, 24) is the catalytic site of theRrF0-F1 ATP synthase. This site is functional in both ATPsynthesis and hydrolysis, in light of our earlier observationson the similar response of both processes in R. rubrumchromatophores to various treatments (21, 25, 26, 31). Therelationship of this site to the nucleotide binding sites thathave been observed in various F1 ATPases (4, 7, 9-11)requires further investigation. On soluble F1 a very high-affinity ATP binding by a catalytic site has been demon-strated (17), whereas we find low-affinity nucleotide bindingby a catalytic site on the isolated /3 subunit. These observa-tions are not in contradiction. They can be explained bysuggesting that the natural state of the catalytic site on theisolated ,3 subunit is a low-affinity one, but incorporation ofthe /3 subunit into the F1 complex causes a drastic shift in thissite that changes it to a high-affinity state. It can then befurther regulated by the cooperative interactions occurringbetween the catalytic sites on the assembled /3 subunits (7,17, 18).

This work was supported in part by the Minerva Foundation ofMunich, F.R.G.

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