ε Subunit of Bacillus subtilis F1-ATPase Relieves MgADP Inhibition

MgADP inhibition, which is considered as a part of the regulatory system of ATP synthase, is a well-known process common to all F1-ATPases, a soluble component of ATP synthase. The entrapment of inhibitory MgADP at catalytic sites terminates catalysis. Regulation by the ε subunit is a common mechanism among F1-ATPases from bacteria and plants. The relationship between these two forms of regulatory mechanisms is obscure because it is difficult to distinguish which is active at a particular moment. Here, using F1-ATPase from Bacillus subtilis (BF1), which is strongly affected by MgADP inhibition, we can distinguish MgADP inhibition from regulation by the ε subunit. The ε subunit did not inhibit but activated BF1. We conclude that the ε subunit relieves BF1 from MgADP inhibition.


Introduction
FoF 1 -ATPase/synthase (FoF 1 ) catalyzes ATP synthesis from ADP and inorganic phosphate coupled with the flow of H + driven by the electrochemical gradient of H + across cellular membranes. FoF 1 consists of a water-soluble ATP-driven F 1 motor (F 1 -ATPase) connected to a membrane-embedded H +driven Fo motor to couple ATP synthesis/hydrolysis and H + flow via a unique rotary mechanism [1][2][3][4]. F 1 -ATPase comprises α 3 , β 3 , γ, δ and ε subunits and its hydrolysis of one ATP molecule drives a discrete 120° rotation of the γε subunits relative to the others [5,6]. In FoF 1 , rotation of the rotor subunits of F 1 (γ and ε) is transferred to the c subunit-ring of Fo to couple ATP synthesis/hydrolysis and flow of H + .
The smallest subunit, ε, is an endogenous inhibitor of the ATPase activity of bacterial and chloroplast F 1 -ATPases and is believed to contribute to the regulation ATP synthase [7][8][9][10]. The mechanism of inhibition by the ε subunit (ε inhibition) varies among species. For example, when F 1 -ATPase is separated from Fo, the ε subunit works as a dissociative inhibitor in Escherichia coli (EF 1 ) and plant chloroplasts (CF 1 ). The ε subunit inhibits ATPase activity, and the enzyme is reactivated when it dissociates from F 1 -ATPase, and the addition of excess ε subunits restores inhibition. In contrast, the ε subunit of F 1 -ATPase from thermophilic Bacillus PS3 (TF 1 ) does not dissociate from the TF 1 complex, and the addition of excess ε subunits does not significantly inhibit activity [10]. Rather, the ε subunit controls the activation state of the enzyme by changing its conformation. Because the dissociation of the ε subunit may not occur within the ATP synthase holo-complex, the ε subunit of EF 1 or CF 1 may also work as a regulator in intact ATP synthase. When in the extended conformation, the C-terminal domain of the ε subunit elongates into the cavity of the α 3 β 3 ring and inhibits ATPase activity [11][12][13][14][15][16]. Upon activation, the C-terminal α helices of the ε subunit are expelled from the α 3 β 3 ring and the ε subunit takes a folded-state conformation in which the C-terminal α helices are folded into a helix-turn-helix conformation, and ATPase activity is not inhibited [17]. We recently demonstrated that, in the case of TFoF 1 , the coupling between ATPase activity and flow of H + is altered when the ε subunit does not bind ATP [18]. F 1 -ATPase is most commonly regulated by MgADP inhibition [19][20][21], which affects all known ATP synthases, and it is caused by the entrapment of MgADP at the catalytic site(s). The recovery from MgADP inhibition is accelerated when ATP binds to non-catalytic sites [22][23][24][25][26]. MgADP inhibition can be observed as pauses of the rotation of the γ subunit [27]. The pause angle of the γ subunit during MgADP inhibition is the same as that of the catalytic dwell (80° from the ATP-binding dwell), which is also the same as that during ε inhibition [28][29][30]. From this and other results, some investigators have proposed that ε inhibition is caused by the stabilization of MgADP inhibition [28,29,31]. Conversely, ε inhibition is prominent even in the presence of the detergent, lauryl dimethyl amine oxide (LDAO) (see supplemental figures of ref [14].), which is known to reduce MgADP inhibition [32]. Further, MgADP inhibition occurs even in the absence of the ε subunit. We demonstrated that the ε subunit greatly reduces the affinity of catalytic sites for MgATP and MgADP [10,33], which counteracts MgADP inhibition rather than stabilizing it. We have shown that the ε subunit relieved MgADP inhibition of a mutant TF 1 unable to bind nucleotides to non-catalytic sites although at low levels [30]. Sekiya et al. reported that the ε subunit does not significantly influence MgADP inhibition of E. coli F 1 -ATPase [34]. Konno et al. proposed the existence of different origins of MgADP inhibition and ε inhibition in cyanobacterial F 1 -ATPase [35]. This discrepancy may be explained by concurrent and indistinguishable MgADP inhibition and ε inhibition.
Although the FoF 1 -ATP synthase from Bacillus subtilis has been studied for decades [36][37][38],, to the best of our knowledge, no detailed kinetic analysis of the purified enzyme has been reported, particularly regarding ε inhibition or MgADP inhibition. To address this question, in the present study, we purified F 1 -ATPase from B. subtilis (BF 1 ) and carried out detailed analyses of the relationship between MgADP inhibition and the function of the ε subunit. Because the activity of BF 1 is strongly affected by MgADP inhibition, we were able to examine the effect of the ε subunit on MgADP inhibition in detail. The results clearly indicate that regulation by the ε subunit is not only distinct from MgADP inhibition but their effects counteract each other.

Construction of a Plasmid to Express the α 3 β 3 γ Complex of BF 1
KOD-Plus DNA DNA polymerase (Toyobo) was used for PCR reactions. The region containing the genes encoding the α, γ, and β subunits of BF 1 was amplified by genomic PCR by using two primers as follows: 5′-CCGAATTCATATGAGCATCAAAGCTGAAGAGATTAGCACG C-3′ contains EcoRI and NdeI sites. The initiation codon of the α subunit (GTG) was replaced with ATG; 5′-GGCTCGAGCTGCAGTTAAACTTCTACACCCATTTCTTTTGC TTTC-3′ contains PstI and XhoI sites and the termination codon for the β subunit. B. subtilis genomic DNA was used as template. The PCR product was cloned into the EcoRV site of the pZero2.1 vector (Invitrogen) to produce pZero-BF1. The initiation codon of the γ subunit was converted from TTG to ATG and the SD sequence of the γ subunit was converted from AAGG to AAGGAGG, as reported for the expression system of TF 1 [39] using overlap-extension PCR [40,41] with the four primers as follows: The mutagenic primers, 5′-AGAGAAAAGGAGGTGAAATCCATGGCCTCATTACG-3′ and 5′-AATGAGGCCATGGATTTCACCTCCTTTTCTCTTC-3′ contain an NcoI site in addition to the modifications described above; flanking primers were 5′-GCTGTCCTTGCTTCTTCGCCGTCCGC-3′ and 5′-TCTTGTGTGATGGCTGCTTGGCGAG-3′. The resulting 1.6kbp fragment, containing segments of the genes encoding the γ and α subunits, was cloned into the EcoRV site of pZero2.1. A 1-kbp BglII fragment containing the initiation codon for the γ subunit was transferred to the cognate site of pZero-BF1 in the correct orientation to generate pZero-BF1ATG. The full-length genes encoding the α, γ, and β subunits were excised from pZero-BF1ATG with NdeI and XhoI and cloned into the respective sites of the pET16b expression vector (Novagen), generating pET16b-BF1 in which a His 10 -tag was introduced at the N-terminus of the α subunit. However, we were unable to express or purify the α 3 β 3 γ complex of BF 1 from this construct as most of the α subunits were expressed as monomers. To introduce the His 6 -tag at the N-terminus of the β subunit, overlap-extension PCR was carried out using the four primers as follows: The mutagenic primers containing the His 6 -tag were 5′-CGATGCATCATCATCATCATCACATGAAGAAAGGACGCGT TAGCCAGG-3′ and 5′-CTTCATGTGATGATGATGATGATGCATCGCTATCCCTCCT GACAAAATC-3′.
The flanking primers were 5′-CAGTTCGGTTTTACGGAGTGCTTATC-3′ and 5′-GCGCCGGGTCAGTGTAGTCATCG-3′. The resulting 1.6-kbp fragment, which contained the region around the initiation codon for the β subunit, was cloned into the EcoRV site of pZero2.1 to generate pZero2.1-βhis. To remove the His 10 -tag at the N-terminus of the α subunit, the NdeI/XhoI-digested fragment of pET16b-BF1, which contains the genes for the α, γ, and β subunits, was transferred to the respective sites of pET21a (Novagen) to produce pET21-BF1nohis. Then, to introduce the His 6 -tag into the β subunit in pET21-BF1nohis, pZero2.1-βhis was digested with BseRI/BssSI. A 0.8-kbp fragment, which contained the N-terminus of the β subunit was isolated and ligated to a 7.2-kbp fragment of pET21-BF1nohisdigested with BseRI/DraIII, and a 1.3-kbp fragment, which contained most of the β subunit gene of pET21-BF1nohis, which was digested with BssSI/DraIII, to obtain pET21-BF1. The final product, pET21-BF1, contained the following modifications of the original genes as follows: the His 6 -tag was introduced at the N-terminus of the β subunit, the initiation codon of the γ subunit was replaced with ATG, the SD sequence of the γ subunit was modified, an NdeI site was introduced at the 5′-terminus of the α subunit gene, and an NcoI site was introduced at the 5′-terminus of the γ subunit gene.

Construction of plasmids to express mutant proteins
The plasmid expressing the mutant (γ S3C ) α 3 β 3 γ complex of BF 1 was constructed as follows. The sequence of the genes encoding the entire γ subunit and part of the β subunit were amplified by PCR from pET21-BF1 with the primers as follows. The mutagenic primer was 5′-AAATCCATGGCCTGTTTACGCGATATTAAG-3′, which contains a γSer3 to Cys substitution, and the other primer was 5′-ATGTAAGGAGCAAGCAAATCAACAAC-3′. The resulting 1.3-kbp fragment was introduced into the EcoRV site of pZero2.1 to produce pZero-γS3C. Then, pZero-γS3C was digested with NcoI/SalI, and a 1-kbp fragment containing the γ S3C region was recovered and ligated to a 7.4-kbp fragment of pET21-BF1 digested with MunI/SalI, and a 0.9-kbp fragment of MunI/NcoI-digested pET21-BF1, which contained a segment of the gene encoding the α subunit, was ligated to obtain pET21-BF1 (γS3C). The plasmid expressing a mutant BF 1 ε subunit (133C, where a Cys was introduced at the C-terminus) was constructed using the following primers for PCR as follows: 5′-GCCGGCGAAGCTTAACATTTCCCTGCTAC-3′, which contains 133C at the C-terminus of the BF 1 ε subunit and a HindIII site, and 5′-GAAATTAATACGACTCACTATAGG-3′, which corresponds to the upstream sequence of the gene encoding the BF 1 ε subunit (T7 promoter). The expression plasmid for WT BF 1 ε [42] was used as the template. The resulting DNA fragment was cloned into the EcoRV site of pZero2.1; the resulting plasmid was digested with NdeI/HindIII, and the DNA fragment was transferred to the respective cognate sites of the pET21b expression vector to produce pET21-BF1ε(133C).

Protein purification
WT or mutant (γ S3C ) α 3 β 3 γ complexes of BF 1 were prepared as follows: E. coli BL21(DE3) was transformed with pET21-BF 1 and grown in 1-L LB medium containing 100 mg/L ampicillin and 10 µM IPTG at 25° C for 24-36 h with vigorous shaking at 250 rpm in a 3-L baffled flask. Typically, approximately 6 g wet cells was produced. Cells were suspended in buffer A (20 mM Tris-H 2 SO 4 (pH 7.5), 300 mM K 2 SO 4 , and 30 mM imidazole) to 0.1-0.2 g cells/ml and disrupted using a French Press. The rest of the procedures was carried out at 25° C. Cell debris was removed by centrifugation at 2,000 × g for 15 min at 25° C. The supernatant was diluted with the same volume of buffer A and applied to a 5 ml HisTrapFF crude column (GE Healthcare Life Sciences) equilibrated with buffer A at a flow rate at 2 ml/min. The column was washed with buffer A until the absorbance at 280 nm plateaued. The adsorbed proteins were eluted with buffer B (buffer A containing 500 mM imidazole) and collected. Fractions were purified using a gel-filtration column (Superdex 200 10/300 GL; GE Healthcare Life Sciences) equilibrated with buffer C (50 mM Tris-H 2 SO 4 (pH 7.5) and 50 mM K 2 SO 4 ), eluted at 0.5 ml/min, monitored at 280 nm. The peak fractions containing α 3 β 3 γ complex were pooled, adjusted to 65% saturated ammonium sulfate, and stored in suspension at 4° C. Approximately 15 mg of α 3 β 3 γ complex was obtained from a 1-L culture. Purified α 3 β 3 γ complex did not contain bound nucleotides (<0.06 mol/mol) as measured by HPLC [43]. The α 3 β 3 γ complex was collected by centrifugation and dissolved in 50 mM Tris-H 2 SO 4 (pH 7.5) and 50 mM K 2 SO 4 .
The cell lysate was centrifuged at 3,000 × g for 10 min at 4° C to remove cell debris, and the supernatant was centrifuged at 180,000 × g for 1 h at 4° C. The rest of the procedures was carried out at 25° C. The supernatant was applied to a DEAE Toyopearl column (40 ml, Tosoh) equilibrated with buffer D. The flow-through fractions containing the ε 133C subunit were collected and solid ammonium sulfate was added to 65% saturation. The precipitate was stored at 4° C. The protein was collected by centrifugation at 6,000 × g for 15 min at 4° C and dissolved in 30 mL of buffer D containing 10% saturated ammonium sulfate and applied to a butyl Toyopearl column (20 mL; Tosoh) equilibrated and washed with the same buffer. The ε 133C subunit was eluted with buffer D at a flow rate at ~3 ml/min and fractions containing the ε 133C subunit were pooled, and solid ammonium sulfate was added to 65% saturation and stored at 4° C. Approximately 40 mg of ε 133C was obtained from a 1-L culture. The ε 133C subunit was collected for analysis by centrifugation and dissolved in 50 mM Tris-H 2 SO 4 (pH 7.5) and 50 mM K 2 SO 4 .

ATPase assay
ATPase activity was measured spectrophotometrically with an ATP-regenerating system coupled to NADH oxidation at 25 °C [44]. The assay mixture (1.5 ml) consisted of 50 mM Tris-H 2 SO 4 (pH 7.5), 50 mM K 2 SO 4 , 2 mM phosphoenolpyruvate, 2 mM MgSO 4 , 0.2 mM NADH, 50 µg/ml pyruvate kinase, 50 µg/ml lactate dehydrogenase, and the indicated concentration of ATP-Mg (equimolar mixture of ATP and MgSO 4 ) was transferred to a glass cuvette. Absorbance at 340 nm was measured using a V-550 spectrophotometer (JASCO) at 0.5 or 1-s intervals. The α 3 β 3 γ complex with or without ε subunit was added 2 min after starting the measurements. The mixture was stirred with a magnetic stirrer for 5 s before and after the addition of α 3 β 3 γ complex. The rate of ATP hydrolysis was determined from the rate of NADH oxidation. The final concentration of α 3 β 3 γ complex was 30 nM when measuring ATPase activity in the absence of lauryldimethylamine oxide (LDAO). Typically, 15 µl of 3 µM α 3 β 3 γ complex solution was added to 1.5 ml of the assay mixture. When ATPase activity was measured in the presence of LDAO, the final concentration of α 3 β 3 γ complex was reduced to 3 nM. In that case, 0.1 mg/ml bovine serum albumin (BSA) was included in stock α 3 β 3 γ complex solution (450 nM) to avoid the adsorption of α 3 β 3 γ complex on the plastic tube. Ten microliters of α 3 β 3 γ complex solution (450 nM) was added to 1.5 ml of the assay mixture without LDAO. Then, LDAO (final concentration 0.1%) was added and the solution was stirred continuously. When the ATPase activity of α 3 β 3 γε complexes was measured, the ε subunit was included in the α 3 β 3 γ complex stock solution at a 1:10 (3 µM α 3 β 3 γ complex and 30 µM ε in the absence of LDAO) to 1:100 (450 nM α 3 β 3 γ complex and 45 µM ε in the presence of LDAO) molar ratio. Reaction rates were determined at 2-7 s (initial) and 12-13 min (steady-state) after adding BF 1 . The reaction rate in the presence of LDAO was determined 100-150 s after the addition of LDAO.

Preincubation with MgADP
The effect of preincubation with MgADP was determined as follows: BF 1 (10 µM α 3 β 3 γ complex ± 100 µM ε) in 50 mM Tris-H 2 SO 4 (pH 7.5), 50 mM K 2 SO 4 , and 4 mM MgSO 4 was mixed with an equal volume of 2× MgADP (equimolar mixture of ADP and MgSO 4 ) and incubated for more than 10 min at 25° C (Mg 2+ concentration was in 2 mM excess ADP). Nine microliters of the mixture was added to 1.5 ml of ATPase assay mixture containing 2 mM MgATP (30 nM α 3 β 3 γ complex ± 300 nM ε). The initial rate (2-4 s after the addition of BF 1 ) was determined in this experiment.

Crosslinking γ and ε subunits
Crosslinking of the γ subunit to the extended conformation of the ε subunit in α 3 β 3 γ S3C ε 133C was performed as follows. Ammonium sulfate suspensions of α 3 β 3 γ S3C complex and ε 133C were centrifuged individually at 20,000 × g for 15 min at 4° C. Each precipitate was dissolved in 50 mM Tris-H 2 SO 4 (pH 7.5) and 50 mM K 2 SO 4 , and 10 mM DTT was added and incubated for 10 min at 25° C. The α 3 β 3 γ S3C and ε 133C were mixed at a 1: 10 molar ratio and incubated for 15 min at 25° C. Excess ε 133C was removed by ultrafiltration with a centrifugal concentrator (Amicon Ultra, 100-kDa cutoff). The sample was concentrated to approximately 10-fold and ultrafiltration was repeated 3 times after the addition of the same buffer to the original volume. The sample (1 mg/ml) was incubated with or without 4 mM MgATP for 10 min at 25° C; the solution was divided into two tubes, and an equal volume of 100 µM CuCl 2 or the buffer was added. After 1-h incubation at 25° C, 10 mM EDTA was added to terminate the reaction. After 10 min, 0.1% SDS and 15 mM N-ethyl maleimide were added. The samples were analyzed using non-reducing SDS-PAGE (12% acrylamide). Part of the sample without ATP and with CuCl 2 was saved after the addition of 10 mM EDTA for the ATPase assay. A combination of WT α 3 β 3 γ complex and ε 133C served as the control. During the ATPase assays, 50 mM DTT was added to reduce crosslinking between the γ and ε subunits at the time indicated in the figure.

Other methods
Protein concentrations were determined by the method of Bradford [45] using BSA as a standard. DNA sequences for all of the recombinant proteins were confirmed using an ABI 3130xl Genetic Analyzer (Applied Biosystems). Non-reducing PAGE was performed according the method of Laemmli [46]. Chemicals were of the highest grade available. Kinetic data analyses were performed using Spectra Manager (JASCO) and OriginPro 8.5 and 9.0 (OriginLab), and the kinetic parameters are expressed with standard errors.

ATPase activity of BF 1 and the effect of ε subunit
Typical time courses of ATP hydrolysis by α 3 β 3 γ and α 3 β 3 γε complexes of BF 1 are shown in Figure 1. At ATP concentrations ≥ 20 µM, very large initial inactivation was observed, irrespective of the presence of the ε subunit. At ATP concentrations > 50 µM, the inactivation was rapid enough to achieve constant, steady-state ATPase activity within the measurement (13 min), and there were no significant differences between α 3 β 3 γ and α 3 β 3 γε at ATP concentrations > 200 µM ( Figure 1A, B). At lower ATP concentrations, the rate of inactivation slowed and did not reach the steady state ( Figure  1C). Under these conditions, the ATPase activity of α 3 β 3 γε (lower traces in Figure 1) was higher than that of α 3 β 3 γ. Inactivation was diminished at lower ATP concentrations ( Figure 1D). Reaction rates determined at 2-7 s and 12-13 min as a function of ATP concentration are shown in Figure 2. The steady-state ATPase activity of BF 1 exhibited a decrease between 10 and 100 µM ATP possibly due in part to slow inactivation that did not reach the steady-state at low ATP concentrations. The value of k cat (1.83 s -1 for α 3 β 3 γ and 1.80 s -1 for α 3 β 3 γε) for steady-state ATPase activity is very low compared with the F 1 -ATPases from other sources, for example, TF 1 ~60 s -1 and EF 1 ~75 s -1 at 25° C) [39,47]. The ATPase activity increased more than 100-fold by LDAO, which is known to relieve MgADP inhibition ( Figure 2). Because the initial rate of ATP hydrolysis reached only about 80 s -1 ( Figure  2), and 200 mM Pi, which is known to reduce MgADP inhibition [48], activated BF 1 to only ~10-fold (data not shown), the effect of LDAO may not be entirely related to MgADP inhibition. Nevertheless, these findings indicate that the ATPase activity of α 3 β 3 γ and α 3 β 3 γε complexes of BF 1 was highly suppressed by MgADP inhibition. Judging from the activation ratio by LDAO, the degree of MgADP inhibition is low at low ATP concentrations. This could account for the triphasic dependence on ATP concentration dependence of ATPase activity in the absence of LDAO in part. In the presence of LDAO, the concentration-dependence on ATP of ATPase activity followed simple sum of two Michaelis-Menten equations ( Figure 2).
The ε subunit affected the ATPase activity of BF 1 only at low concentrations of ATP (Figures 1 and 2). Surprisingly, no inhibitory effect of the ε subunit was observed, and activation by the ε subunit occurred at ATP concentrations <50 µM ( Figure 1C, D, lower trace). The dissociation of ε subunit from α 3 β 3 γε complex may not account for the equvialent activities of α 3 β 3 γ and α 3 β 3 γε at high ATP concentrations, because the α 3 β 3 γε complex could be isolated by gel-filtration HPLC (Superdex 200 10/300GL) even in the presence of ATP and/or LDAO (data not shown). Further, the addition of up to 30 µM ε subunit to the ATPase assay mixture did not significantly affect steady-state ATPase activity at 2 mM ATP (data not shown). In the presence of LDAO, the ATPase activities of α 3 β 3 γ and α 3 β 3 γε were essentially the same at all ATP concentrations, although the k cat value of α 3 β 3 γε (352 s -1 ) was slightly higher than that of α 3 β 3 γε (268 s -1 ) (Figure 2). The initial rates of ATP hydrolysis were also not significantly different (Figure 2). Therefore, the inhibition by the ε subunit of BF 1 might be very weak, if any.

Catalytic properties of mutant BF 1 with its ε subunit fixed in the extended conformation
The activation by the ε subunit was investigated in more detail by examining a mutant α 3 β 3 γε complex of BF 1 , in which the N-terminus of the γ subunit and C-terminus of the ε subunit can be crosslinked via engineering in Cys residues to fix the ε subunit in the extended conformation. Thus, a mutant equivalent to that of TF 1 [13] was prepared. The endogenous Cys residues in the α subunit did not react with the introduced Cys residues in the γ or ε subunits. To determine whether the apparent absence of ε inhibition in BF 1 resulted from the inability of the ε subunit to assume an extended conformation, the presence of γ-ε crosslink formation was determined in the presence or absence of ATP. The γ and ε bands disappeared and a band corresponding to the γ-ε crosslink product appeared only in the absence of ATP (Figures 4, and S2). The distance between the Cα of the residues corresponding to the introduced Cys residues in a recently reported EF 1 structure is 12.9 Å [16]. Although this is a little bit long to form a disulfide bridge, the formation of a disulfide bridge within the mutant α 3 β 3 γ S3C ε 133C complex of BF 1 indicates that the crosslinked structure may reflect the physiological conformation within the range of thermal fluctuation. In the presence of ATP, a dimer of the ε subunit was formed, indicating that ε changed its conformation from the extended to the intermediate or foldedstate in which the C-terminal Cys was accessible on the surface of the molecule. We conclude from these results that the absence of ε inhibition was not caused by the absence of its extended conformation. However, because there were no significant differences in the initial activities of WT α 3 β 3 γ and α 3 β 3 γε complexes, the extended conformation of the ε subunit may readily change upon addition of ATP.

Discussion
ATPase activity of BF 1 is strongly suppressed by MgADP inhibition BF 1 showed high initial ATPase activity, rapid inactivation, and very low steady-state ATPase activity. LDAO dramatically activated the steady-state ATPase activity of BF 1 (Figure 2). The initial ATPase activity was >20-fold higher than the steadystate ATPase activity, 200 mM Pi also activated steady-state ATPase activity by ~10-fold (data not shown), and preincubation with MgADP greatly suppressed the initial ATPase activity (Figure 3). These suggest that the inactivation might be due to strong MgADP inhibition, which could mean that the B. subtilis ATP synthase functions as an ATP synthase that does not hydrolyze ATP, because MgADP inhibition does not inhibit ATP synthesis activity [49].

No inhibition by the ε subunit
The ε subunit did not significantly inhibit the ATPase activity of BF 1 but activated at low concentrations of ATP presumably due to the suppression of MgADP inhibition. Further, the ε subunit, fixed in the extended conformation, did not inhibit the mutant enzyme. The ε subunit only inhibited the activity of the extended-state fixed mutant α 3 β 3 γ S3C ε 133C complex of BF 1 in the presence of LDAO ( Figure 5B, S3C after addition of LDAO). In this case, DTT activated the enzyme, indicating that the activity before the addition of DTT was actually suppressed by the extended-state ε subunit. We conclude, therefore, that due to the strong MgADP inhibition, ε inhibition is not evident, Figure 3. Effect of preincubation with MgADP. The α 3 β 3 γ or α 3 β 3 γε (5 µM) was incubated with the indicated concentrations of MgADP for more than 10 min at 25° C. Residual ATPase activity was measured in the presence of 2 mM ATP. The initial rate (2-4 s after the start of the reaction) was measured, and the values relative to the control without incubation with MgADP (82.9 ± 5.4 s -1 and 88.6 ± 3.6 s -1 for α 3 β 3 γ and α 3 β 3 γε, respectively) are plotted. Closed and open circles represent α 3 β 3 γ and α 3 β 3 γε, respectively. Error bars represent standard errors.  because the relief from MgADP inhibition by the ε subunit is more prominent.

Counteraction of MgADP inhibition and ε subunit
As discussed above, the ε subunit suppressed MgADP inhibition. In the absence of LDAO, the mutant α 3 β 3 γ S3C ε 133C complex of BF 1 with an extended-state fixed ε subunit showed considerably higher ATPase activity than the WT, even at 2 mM ATP. LDAO did not activate the ATPase activity ( Figure  5B). Thus, even before the addition of LDAO, the extendedstate fixed α 3 β 3 γ S3C ε 133C complex of BF 1 might be less inhibited by MgADP inhibition. This conclusion is further strengthened by the results when the WT BF 1 was preincubated with MgADP ( Figure 3). In the absence of the ε subunit, preincubation with MgADP suppressed the ATPase activity of α 3 β 3 γ proportionally at a α 3 β 3 γ: MgADP ratio of 1:2, suggesting that binding of MgADP is strong and binding of one or two MgADP is enough to induce MgADP inhibition of α 3 β 3 γ complex. In contrast, greater than 60% of the activity was retained in the presence of 1:2 MgADP and the ε subunit, indicating that binding of MgADP to α 3 β 3 γε was highly suppressed by the ε subunit. This agrees well with our previous observation that the ε subunit of TF 1 significantly suppresses the binding of MgADP [33]. LDAO did not activate the extended-fixed α 3 β 3 γ S3C ε 133C complex of BF 1 ( Figure 5B), indicating that the extended-state ε subunit reduced MgADP inhibition. Considering all of these results, we conclude that ε inhibition is not due to the stabilization of MgADP inhibition [28,29,31], but due to an essentially different and counteracting mechanism. We believe, therefore, that these properties must be common among various F 1 -ATPases, despite the differences in the mechanisms of ε inhibition. It should be noted, however, the ε subunit did not protect mutant α 3 β 3 γ S3C ε 133C complex from MgADP inhibition by the preincubation with ADP ( Figure S1). These apparent contradictory results may be due to the different catalytic site affinity for nucleotides between WT and the mutant (γ S3C ) α 3 β 3 γ complexes, and/or different mode of the action of MgADP during preincubation and ATPase turnover etc. Further experiments, for example, measurement of nucleotide binding to the catalytic sites with WT and mutant α 3 β 3 γ with and without ε subunit will give us a clue to resolve the differences between WT and the mutant in the MgADP preincubation experiment.

Significance of regulation by the ε subunit and MgADP inhibition in vivo
The results presented here indicate that the ATPase activity of BF 1 is very low under normal conditions due to strong MgADP inhibition. Because B. subtilis lives in an aerobic environment and its ATP synthase is primarily used to synthesize ATP but not to hydrolyze ATP, as is the case for bacteria such as E. coli that can grow anaerobically. The ε subunit may not act as an inhibitor of the ATPase activity of B. subtilis ATP synthase. In contrast, its ability to attenuate MgADP inhibition may be its primary role in the regulatory system. Experiments using B. subtilis with mutant FoF 1 to address these questions are underway in our laboratory. Elucidation of the balance and the interplay of these two regulatory systems in different bacteria may be required to understand the regulation of bacterial ATP synthases. Figure S1.