Energy Coupling, Turnover, and Stability of the F0F1 ATP Synthase Are Dependent on the Energy of Interaction between gamma  and beta  Subunits*

(Received for publication, October 1, 1996, and in revised form, October 25, 1996)

Marwan K. Al-Shawi , Christian J. Ketchum Dagger and Robert K. Nakamoto §

From the Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Replacement of the F0F1 ATP synthase gamma  subunit Met-23 with Lys (gamma M23K) perturbs coupling efficiency between transport and catalysis (Shin, K., Nakamoto, R. K., Maeda, M., and Futai, M. (1992) J. Biol. Chem. 267, 20835-20839). We demonstrate here that the gamma M23K mutation causes altered interactions between subunits. Binding of delta  or epsilon  subunits stabilizes the alpha 3beta 3gamma complex, which becomes destabilized by the mutation. Significantly, the inhibition of F1 ATP hydrolysis by the epsilon  subunit is no longer relieved when the gamma M23K mutant F1 is bound to F0. Steady state Arrhenius analysis reveals that the gamma M23K enzyme has increased activation energies for the catalytic transition state. These results suggest that the mutation causes the formation of additional bonds within the enzyme that must be broken in order to achieve the transition state. Based on the x-ray crystallographic structure of Abrahams et al. (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), the additional bond is likely due to gamma M23K forming an ionized hydrogen bond with one of the beta Glu-381 residues. Two second site mutations, gamma Q269R and gamma R242C, suppress the effects of gamma M23K and decrease activation energies for the gamma M23K enzyme. We conclude that gamma M23K is an added function mutation that increases the energy of interaction between gamma  and beta  subunits. The additional interaction perturbs transmission of conformational information such that epsilon  inhibition of ATPase activity is not relieved and coupling efficiency is lowered.


INTRODUCTION

The F0F1 ATP synthase links two disparate functions: transport of protons across a membrane and catalysis of ATP synthesis or hydrolysis (for reviews see Refs. 1-5). The fully cooperative mechanism of ATP hydrolysis requires a minimum of three different subunits in a complex containing alpha 3beta 3gamma 1. The transport mechanism is most likely assembled from F0 sector subunits. In the Escherichia coli complex, transport requires three different membrane-spanning subunits, a1b2c~10 (6). In addition, two more soluble subunits, delta  and epsilon , are needed to reconstitute catalytic and transport sectors so that they are coupled to carry out ATP-driven proton pumping or Delta µH+-driven ATP synthesis (7-10).

Catalysis and transport mechanisms most likely communicate indirectly through a series of conformational and electrostatic interactions. Conformational changes relevant to the catalytic state of the enzyme or the presence of a Delta µH+ have been detected by several methods including altered cross-linking patterns, protease susceptibility, environmentally sensitive fluorescent probes, accessibility of epitopes, x-ray diffraction, cryoelectron microscopy, and spectroscopic analyses (reviewed in Refs. 11 and 12). High resolution structural information based on crystals of the bovine mitochondrial F1 has also provided a great deal of information about possible subunit interactions that may be involved in linking transport and catalysis (13).

Mutagenic analysis has also yielded important information about the coupling mechanism (reviewed in Ref. 5). For example, mutations in the single hydrophilic loop of subunit c, an F0 subunit that is involved in proton transport, disrupt coupling between transport and catalytic mechanisms (14-16). Furthermore, genetic and chemical cross-linking results strongly suggest that epsilon  subunit interacts with this portion of subunit c (17, 18).

Likewise, mutations near the catalytic sites have also been found to affect coupling. The most clear example of these mutations is replacement of gamma Met-23 with Arg or Lys (19). These mutations caused greatly reduced ATP-dependent proton pumping and ATP synthesis rates without strongly affecting catalytic or transport functions. The F0 sector was unaffected as were interactions between F0 and F1. Restoration of efficient coupling in the gamma Met-23 right-arrow Lys (gamma M23K) mutant enzyme was conferred by several second site mutations near the carboxyl terminus of the gamma  subunit including the replacement of amino acid gamma Arg-242 and seven different residues between gamma Gln-269 and gamma Ala-280 (20). Furthermore the temperature sensitivity caused by the gamma M23K mutation was suppressed by each of the second site mutations. In the simplest interpretation of these results, direct interactions within the gamma  subunit are perturbed by the gamma M23K mutation, and each of the suppressor mutations is close enough to the gamma M23K residue to directly counteract its influence on structure and function. However, many observations do not coincide with this interpretation (21). Analysis of the x-ray crystallographic structural map of the bovine F1 (13) indicates that gamma Met-23 is a considerable distance from the gamma 269-280 region. Furthermore, the types of amino acid changes that suppress the effects of the gamma M23K mutant are not of any particular functional group, and in some cases multiple changes at a single site result in suppression (20, 21). It is difficult to imagine how multiple and diverse amino acid replacements would be able to compensate for a specific perturbation.

Because the mechanism of coupling must involve interactions among subunits, we hypothesized that the gamma M23K mutation perturbed interactions between subunits. In this paper, we provide evidence that the gamma M23K mutation is an added function mutation that increases the energy of interaction between gamma  and beta  subunits. The consequences of this increase are destabilization of the alpha 3beta 3gamma complex and inefficient coupling between transport and catalysis. In turn, the suppressor mutations described above counteract by decreasing the energy of interaction.


MATERIALS AND METHODS

Strains and Plasmids

F1 complex and delta  and epsilon  subunits were isolated from strain DK8 harboring plasmid, pBWU13 (22) or pBMU13-gamma M23K (see below). Membranous F0F1 was obtained from strain KF10rA harboring derivatives of plasmid pBWG15 (23).

The gamma M23K mutation was introduced into the uncG gene on plasmid pBWU13 to give pBMU13-gamma M23K. Oligonucleotide-directed mutagenesis with the Stratagene (La Jolla, CA) Chameleon Kit (24) was used to introduce the gamma M23K mutation on plasmid pBWG11 (23) using the synthetic oligonucleotide, 5'-CACTAAAGCGaaaGAGATGGTCGCC-3' (lowercase letters denote the gamma M23K mutation). After sequence verification, the mutation was isolated on the AsuII to RsrII restriction fragment and ligated into pBWU13. Introduction of the mutation into the expression plasmid and presence of the mutation after growth were verified by phenotype and DNA sequencing (25).

Molecular biological manipulations were performed as described (26) or according to the manufacturer's instructions. Restriction enzyme and DNA modifying enzymes were obtained from Amersham Corp., Boehringer Mannheim, Life Technologies, Inc., New England Biolabs, (Beverly, MA), or Promega (Madison, WI).

Isolation of Membranes and Purification of F1 and F1 Subunits

Membranes from KF10rA were prepared as described previously (8). F1 complex was purified as detailed by Duncan and Senior (27) and Al-Shawi and Senior (28). Purified wild-type F1 was used as a source for isolation of delta  and epsilon  subunits (9).

Determination of F1 Content in E. coli Membranes

Determination of F1 in membrane preparations was performed by quantitative immunoblot analysis and comparing results with known amounts of purified F1. Proteins from various amounts of membranes were prepared as in Nakamoto et al. (21), separated on a 12.5% SDS-polyacrylamide gel (29), and transferred to a nitrocellulose filter (30). The filter was immunostained with a 1:1000 dilution of a polyclonal rabbit anti-alpha subunit antibody (obtained from Dr. Alan Senior of the University of Rochester) followed by a secondary anti-rabbit IgG antibody conjugated to fluorescein diluted 1:1000 (Boehringer Mannheim). The fluorescence of the band corresponding to the alpha  subunit was quantified by a Molecular Dynamics FluorImager, and the values were plotted against total protein. By increasing amounts of total membrane protein in adjacent lanes, a linear line was generated for each membrane preparation. The slope of each line was compared with that obtained from a titration of purified E. coli F1 to determine the percentage of total protein that could be attributed to F1. Duplicate experiments were performed, and the values were averaged.

Enzymatic Assays

Protein concentrations were determined by the method of Lowry et al. (31). ATPase activities were measured in the buffers given below and in the figure legends by procedures described in Al-Shawi et al. (32). The experimental conditions detailed in the footnotes to Table II were chosen to optimize for coupling efficiency and enzyme stability. Free Mg2+ and Mg·ATP concentration were determined by the algorithm of Fabiato and Fabiato (33). ATPase reactions were stopped by the addition of 5% sodium dodecylsulfate or 10 mM ice-cold H2SO4. Liberated Pi was determined by the methods of Taussky and Shorr (34) or Van Veldhoven and Mannaerts (35) depending on the sensitivity required. The Van Veldhoven and Mannaerts assay was slightly modified by stopping the final color development reaction with the addition of 0.5 M H2SO4 after 20 min of incubation at room temperature. ATP synthesis was measured as described previously (36). Pyruvate kinase was obtained from Boehringer Mannheim, and hexokinase was from Sigma.

Table II.

Coupling efficiencies for the uncoupling mutation, gamma M23K, with suppressor mutations


Strain ATP synthesisa
ATP hydrolysisb
Coupling ratio
Initial rate % of Wild type Rate % of Wild type Synthesis/hydrolysis Ratio % of Wild type

KF10rA/pBMG15 µmol/min/mg µmol/min/mg
Wild type 0.24 0.76 0.32
 gamma Lys-23 0.029 12 0.29 38 0.10 31
 gamma Cys-242 0.16 67 0.46 61 0.35 109
 gamma Cys-242 + Lys-23 0.039 16 0.38 50 0.10 31
 gamma Arg-269 0.040 17 0.40 53 0.10 31
 gamma Arg-269 + Lys-23 0.047 20 0.11 14 0.43 134

a  ATP synthesis rates were measured at 37 °C with vigorous shaking in a buffer containing 25 mM HEPES-KOH, 200 mM KCl, 5 mM MgSO4 (3.0 mM free Mg2+), 10 mM glucose, 1 mM ADP, 10 mM [32P]Pi, 50 units/ml hexokinase, and 0.1-0.2 mg/ml E. coli membranes at pH 7.5. The reactions were started with the addition of 2 mM NADH. Control blank rates were obtained under the same conditions in the presence of 5 µM CCCP and subtracted from the experimental rates. Time points were taken up to 6 min, and the samples were analyzed as described under "Materials and Methods."
b  ATP hydrolysis rates were measured at 37 °C in a buffer containing 25 mM HEPES-KOH, 200 mM KCl, 8 mM MgSO4 (3.1 mM free Mg2+), 10 mM glucose, 1 mM phosphoenolpyruvate, 5 mM ATP, 5 µM CCCP, 50 µg/ml pyruvate kinase, and the reactions were started with the addition of 0.08-0.2 mg/ml E. coli membranes. Time points were taken up to 20 min, and the samples were analyzed as described under "Materials and Methods."

Arrhenius Analysis and Derivation of Transition State Thermodynamic Parameters

Apparent enzyme activation energies of ATP hydrolysis were calculated from measurements of maximal rates of ATPase activities as a function of temperature. Activation energies and entropic and enthalpic components of the transition state for ATP hydrolysis were calculated from plots of log velocity at saturating ATP (in the presence of an ATP regenerating system) versus the reciprocal temperature as detailed in Al-Shawi and Senior (37). For membrane preparations (0.13-0.3 mg/ml), turnover numbers were calculated using the determined F1 content of the membrane (measured as described above), and assays were performed in the presence of 5 µM carbonylcyanide-m-chlorophenylhydrazone (CCCP).1 For purified F1, enzyme preparations were preincubated in buffer at 23 °C for at least 30 min, and the assay concentration was 65 nM F1. The ATPase buffer, comprised of 0.5 ml of 50 mM HEPES-KOH, 10 mM ATP, 5 mM MgSO4, 5 mM phosphoenolpyruvate, and 32 µg/ml pyruvate kinase, was incubated at the required temperature, and the pH was adjusted to 7.5 using a pH electrode (Sigma) that had been calibrated at that temperature. The linear rate of Pi liberation was determined as described above.


RESULTS

The gamma M23K Mutation Does Not Alter epsilon  or delta  Subunit Interactions with F1

We first assessed if the gamma M23K mutation altered interactions with the single copy F1 subunits, delta  and epsilon . In the wild-type E. coli complex, epsilon  subunit binds tightly to the F1 complex with a KD of approximately 10-8 M (38), and the association constant of isolated gamma  and epsilon  subunits is similar to this value (39). These results suggested that epsilon  interactions with the remainder of the F1 complex are mostly through the gamma  subunit. In turn, the affinity for epsilon  subunit to gamma  subunit can be assessed by the inhibitory properties of the epsilon  subunit on ATPase activity. ATPase activity of the alpha 3beta 3gamma delta complex is inhibited approximately 90% by epsilon  subunit; the KI is very close to the binding constant between isolated gamma  and epsilon  subunits (39).

Sternweis and Smith (38) showed that dilution of the purified F1 complex to a concentration below the KD for epsilon -F1 results in activation of ATPase specific activity. Fig. 1 reproduces this result where the enzyme is activated as F1 is diluted below 100 nM. The same activation is observed for wild-type and gamma M23K enzymes. Titration of the wild-type F1 indicates a half-maximal activation at approximately 10 nM, which is the same value as previously reported (9, 38). delta  subunit is generally believed to bind F1 with lower affinity than epsilon  subunit and is most likely dissociated at the concentration of 10 nM F1 (40).


Fig. 1. Effect of dilution on the ATPase activity of wild-type and gamma M23K F1. Various concentrations of F1 indicated were preincubated for 1 h at 37 °C in 50 mM Tris-OH, 50 mM HEPES, 20 mM Na2SO4, and 0.1 mM MgSO4, adjusted to pH 7.5 with H2SO4. These buffer conditions were predetermined to be optimal for coupling efficiency and stability of the F0F1 complex. ATP hydrolysis was started by the addition of a stock solution of Mg·ATP in the above buffer such that the final concentration was 10 mM ATP and 5 mM MgSO4, and the final pH remained constant at 7.5. 50-200-µl samples of reaction mix were quenched at various times with 1 ml of 10 mM H2SO4, and Pi liberation was quantitated as described under "Materials and Methods." The linear time course of the reactions were followed in steady state conditions for 10 s to 60 min as required. The results shown are average values of at least three independent determinations (± standard deviation bars). bullet , Normal wild-type F1; black-triangle, gamma M23K F1.
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In a marked difference from wild type, further dilution of gamma M23K F1 caused inactivation of activity with half-maximal inactivation occurring at 0.2 nM. The inactivation was not detected with the wild-type F1 even at the lowest F1 concentration measured, 0.01 nM. This indicates that the gamma M23K mutation causes destabilization of alpha 3beta 3gamma , the minimum complex capable of ATPase activity (32, 41).

Interestingly, the inactivation of the gamma M23K enzyme does not occur until epsilon  and delta  subunits have dissociated and suggests that binding of epsilon  and delta  subunits may help to stabilize the complex. To test this notion, superstoichiometric amounts of purified epsilon or delta  subunits were added, while gamma M23K F1 was diluted to various concentrations (Fig. 2). In the presence of 72 nM epsilon  subunit alone, the ATPase specific activity of 13 nM gamma M23K F1 (the concentration that gave maximal activation) was decreased as expected due to epsilon  inhibition. At 1.3 nM gamma M23K F1, the ATPase specific activity remained about the same, and at 0.13 nM gamma M23K F1, the activity increased more than 2-fold compared with the absence of added epsilon  subunit. This behavior reflects the balance between the dissociation/association of epsilon  subunit and inactivation of alpha 3beta 3gamma .


Fig. 2. Effect of adding delta  and epsilon  subunits on the ATPase activities gamma M23K F1 at various concentrations. Various concentrations gamma M23K F1 were preincubated as in Fig. 1 with superstoichiometric concentrations of purified delta  and epsilon  subunits as indicated. ATPase activities were determined as in Fig. 1. Each result is the average of at least three independent experiments with the standard deviations indicated.
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Results with 72 nM delta  subunit were even more dramatic because the activity was stabilized even at the most dilute F1 concentrations. The activities at the two lower concentrations were maximal, indicating that the concentration of epsilon  subunit was well below its KI. Clearly, delta  subunit alone binds to the complex independent of the epsilon  subunit and in a manner that stabilizes alpha 3beta 3gamma . With both delta  and epsilon  subunits added, activity was consistent with epsilon -inhibited levels.

The stabilization of activity by delta  subunit alone provided a way to directly assess the KI for epsilon  subunit. gamma M23K F1 was diluted to 0.13 nM in the presence of 72 nM delta  subunit to maintain the stabilized complex. Titration of epsilon  subunit resulted in an apparent KI of 13 nM (data not shown), which is in good agreement with the dilution experiments in Fig. 1.

gamma M23K F1 Does Not Release epsilon  Inhibition upon Binding to F0

In order to more fully analyze the kinetic and thermodynamic properties of the gamma M23K enzyme, we accurately determined the concentration of F0F1 complex in the membrane of E. coli strain KF10rA. This was done by a quantitative immunoblot analysis described under "Materials and Methods." In brief, the amount of immunostaining obtained for each of the membrane preparations was compared with the amount of staining of known amounts of purified F1 loaded on the same gel (data not shown).

Knowing the amount of F1 on the membranes, we were able to derive turnover numbers for native F0F1 in membranes. Fig. 3 compares the turnover numbers for wild-type and gamma M23K enzymes as soluble F1 or membranous F0F1. The wild-type F0F1 turnover was 425 s-1 (at 30 °C) compared with 92 s-1 for soluble F1, which is about 85% replete with the inhibitory epsilon  subunit (Figs. 1 and 2). This result confirms the early work of Sternweis and Smith (38) that epsilon  inhibition is relieved upon binding to F0. Interestingly, the turnover numbers of the gamma M23K F1 and F0F1 are quite similar (132 s-1 and 92 s-1, respectively). Two important observations are to be made from these values. First, the gamma M23K mutation does not affect the catalytic mechanism because wild-type and the gamma M23K enzymes have relatively similar turnover numbers, and second, the epsilon  inhibition of the gamma M23K enzyme is not relieved when bound to F0. The latter results may indicate that the gamma M23K mutation perturbs the functional interaction of F1 with F0 as mediated by the epsilon  subunit.


Fig. 3. Turnover of wild-type and gamma M23K F1 or F0F1. ATPase assays were performed at 30 °C as detailed under "Materials and Methods" in a buffer containing 50 mM HEPES-KOH, 10 mM ATP, 5 mM MgSO4, pH 7.5, with 5 mM phosphoenolpyruvate and 32 µg/ml pyruvate kinase as an ATP regenerating system. 65 nM F1 or 0.13-0.30 mg of membrane protein/ml was used in each assay. 5 µM carbonylcyanide-m-chlorophenylhydrazone was included with the membrane assays to ensure that there was no back inhibition from Delta µH+. F1 concentration in the membranes was quantitated as described under "Materials and Methods." Turnover numbers were calculated using a molecular mass of 3.82 × 105 Daltons for the F1 complex.
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Altered Transition State Thermodynamic Parameters in the gamma M23K Mutant Enzyme

In order to understand the effects of the gamma M23K mutation, we investigated the results of the mutation on the catalytic mechanism under steady state conditions. We already saw that the turnover of the gamma M23K F0F1 was similar to that of wild type. Furthermore, both enzymes had a pH optimum of around 8.5-9.0 as well as the Km values for Mg·ATP hydrolysis being similar (0.16 and 0.32 mM for wild-type and gamma M23K F0F1, respectively; data not shown). These results reinforce the conclusion that the general reaction schemes and cooperative mechanisms of the mutant enzyme were similar to those of the wild-type enzyme. Additional support for this conclusion can be seen later in the "isokinetic" plots (see Fig. 8) in that the mutant enzyme preparations were close to the regression lines.


Fig. 8. Isokinetic plot of membranous F0F1 and soluble F1 preparations containing various gamma  subunit mutations. The enthalpic term, Delta HDagger of activation for kcat, is plotted against the entropic term, TDelta SDagger of activation for kcat at 30 °C. Filled symbols and a solid line give the results for various wild-type, beta -mutant F1 preparations, bovine mitochondrial F1 (42) as well as gamma  subunit mutant F1 preparations determined in this study. Open symbols and a dashed line illustrate the results from wild-type membranous F0F1, bovine mitochondrial F0F1, and various gamma  subunit mutant F0F1 preparations. Lines were fitted by linear least squares regression analysis.
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However, we have previously demonstrated that the catalytic transition state of the F0F1 ATP synthase was very sensitive to changes in catalytic site conformation and the utilization of binding energy to drive catalysis (42). Thus, in order to probe the effects of the gamma M23K mutation on catalysis and coupling, we measured the thermodynamic parameters of the transition state of ATP hydrolysis by Arrhenius analysis of steady state turnover. In contrast to the results above, the activation energies for the catalytic transition state were strongly affected. Fig. 4 shows an Arrhenius plot of steady state ATPase activity (for clarity, log of the actual ATPase specific activities are plotted instead of turnover). Temperature dependence of maximal velocities were measured with 5 mM Mg·ATP. In the case of membrane-bound enzymes, the protonophore, carbonylcyanide-m-chlorophenylhydrazone, was added to prevent back inhibition from the electrochemical gradient of protons. Purified F1 from wild type and gamma M23K had linear plots from 5 to 45 °C, whereas the membranous F0F1 had a break in the plot around 19 °C. The break in the Arrhenius plot is clearly due to an effect of F0 on F1. This effect is likely a manifestation of the influence of the lipid phase on the function of the F0, which is communicated to the catalytic mechanism through coupling. Significantly, the change in the slope for gamma M23K F0F1 is much less pronounced than that for the wild-type enzyme, suggesting that the influence of F0 on catalysis is decreased in the mutant complex.


Fig. 4. Arrhenius analysis of wild-type and gamma M23K F1 or F0F1. Log V (maximal velocity at saturating ATP in µmol/mg/min for clarity) is plotted against the reciprocal of absolute temperature. ATPase activities were assayed from 5 to 45 °C as detailed under "Materials and Methods." The lines plotted were generated by linear least squares regression of the data. bullet , wild-type F1; black-square, wild-type F0F1; black-triangle, gamma M23K F1; black-down-triangle , gamma M23K F0F1-containing membranes.
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From these plots the enthalpic, entropic, and free energy terms can be calculated for the transition state of the reaction pathway. These values for the wild-type and gamma M23K F1 at 30 °C are listed in Table I and plotted in Fig. 5A. The differences for each parameter between wild type and gamma M23K are plotted in Fig. 5B. Note that the membrane-bound enzyme had the same trends as soluble F1, but the gamma M23K F0F1 had considerably larger differences in both enthalpic and entropic terms. For both soluble and membrane-bound complexes, the gamma M23K enzyme has a more positive Delta HDagger and a less negative Delta SDagger which together add up to a small difference in Delta GDagger . According to transition state theory, these results suggest that the mutation causes the formation of additional bonds between substrate and enzyme or, more likely in this case, bonds within the enzyme that must be broken in order to achieve the transition state.

Table I.

Transition state thermodynamic parameters at 30 °C for purified F1 and membranous F0F1 and comparison between wild-type and gamma M23K enzymes


Preparation  Delta HDagger  Delta Delta HDagger a TDelta SDagger  Delta (TDelta S)Dagger a  Delta GDagger  Delta Delta GDagger a

kJ/mol
Wild-type F1 51.0  -11.8 62.9
 gamma Lys-23 F1 72.7 21.6 10.7 22.5 62.0  -0.9
Wild-type membranes 31.3  -27.8 59.0
 gamma Lys-23 membranes 76.8 45.5 14.0 41.7 62.9 3.8

a  Delta Delta values are differences between parameters for mutant enzyme and the corresponding wild-type preparation. See legend to Fig. 4 for assay conditions.


Fig. 5. Transition state thermodynamic parameters for steady state ATP hydrolysis by wild-type and gamma M23K F1 or F0F1. The activation energy parameters of kcat were calculated at 30 °C from the data of Fig. 4 as described under "Materials and Methods." A illustrates activation energy parameters for wild-type membranes F0F1 (open bars) or wild-type F1 (hatched bars). B illustrates the differences between activation energy parameters for gamma M23K preparations and the corresponding wild-type preparations. Open bars represent F0F1 preparations, and hatched bars represent F1 preparations.
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Suppressor Mutations of gamma M23K Reverse the Effects on Activation Energies

If the changes in transition state thermodynamic parameters are due to the same perturbations that causes the uncoupling phenotype of the gamma M23K mutation, then second site suppressor mutations of gamma M23K (20) would be expected to reverse the altered thermodynamic parameters. This was the case with the suppressor mutation, gamma Q269R.

This mutation was most effective at restoring efficient coupling. The ratio of NADH-driven ATP synthesis versus ATP hydrolysis can be used as a parameter of coupling efficiency (21, 43). Table II shows that the synthesis:hydrolysis ratio of the gamma M23K mutant is restored to wild-type levels in the presence of gamma Q269R. As predicted, the Arrhenius analysis shows that the gamma Q269R mutation counteracted the effects of the gamma M23K mutation on the thermodynamic parameters of the ATPase transition states (Fig. 6).


Fig. 6. Effect of suppressor mutations on transition state thermodynamic parameters of gamma M23K F0F1. Difference activation energy parameters for membranous F0F1 preparations (mutant enzyme minus wild-type enzyme values) were obtained at 30 °C by Arrhenius analysis as detailed in the legend to Fig. 4. Thermodynamic values were calculated as described under "Materials and Methods." Thermodynamic values for F0F1 preparations containing individual gamma  subunit mutations were compared with those for F0F1 preparations containing double gamma  subunit mutations (the original gamma M23K mutation in conjunction with a "suppressor" mutation). Values for membranous F0F1 preparations containing the gamma M23K mutation are represented by the hatched bars.
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In contrast, another suppressor mutation, gamma R242C, did not increase the coupling efficiency even though it genetically suppressed the gamma M23K mutation (20) and resulted in increased ATP synthesis rates (Table II). Instead, the mutation suppressed the effects of the gamma M23K mutation by increasing the turnover rate of the enzyme by 1.44-fold. This result is reminiscent of our observations that overexpression of the gamma M23K F0F1 resulted in increased ATP synthesis rates and increased growth yields on the nonfermentable carbon source, succinate (20).2 With more enzyme present, albeit an inefficient one, there was sufficient ATP synthesis to allow growth of the cell.

Consistent with the lack of recovery of efficient coupling, differences in the transition state thermodynamic parameters of the gamma M23K mutant were only slightly reduced by introduction of gamma R242C (Fig. 6). In this case, it appears that the reduced energy of interaction between gamma  and beta  subunits resulted in a faster turnover rate.


DISCUSSION

We have sought to understand how the gamma M23K mutation perturbs linkage between transport and catalysis to provide information about the mechanism of coupling. We have seen that the mutation does not affect F1 interactions with epsilon  and delta  subunits (this report) nor the F0 sector (19); however, dissociation of delta  and epsilon  subunits leaves a destabilized alpha 3beta 3gamma complex (Fig. 1). Based on chemical cross-linking experiments, epsilon  subunit is believed to interact with gamma , beta , and alpha  subunits (44-47) and apparently does so with a stabilizing effect on the alpha 3beta 3gamma complex. An important property of the epsilon  subunit is its inhibitory activity on the hydrolysis activity of F1. Most significantly, in contrast to the situation in wild type, inhibition by epsilon  subunit in the gamma M23K mutant is not relieved upon binding to F0 (Fig. 3), suggesting a perturbation in communication between transport and catalytic mechanisms. This notion was supported by the decreased change in slope of the Arrhenius plot for the membranous gamma M23K enzyme (Fig. 4). Both of these effects illustrate the impaired coupling between F1 and F0 functions in the mutant enzyme gamma M23K.

From the above data, it seems that the gamma M23K mutation affects the regions of interactions between gamma  and beta  subunits. Based on suppressor mutagenesis results, we earlier concluded that three highly conserved regions of the gamma  subunit, gamma 18-35, gamma 238-246, and gamma 269-280, functionally interact as a domain that mediates energy coupling (21). Upon close inspection of the x-ray crystallographic structural model of Abrahams et al. (13), we observed that each of these three regions is in contact with the surrounding beta  subunits and that at least two of the residues form a hydrogen bond with specific beta  subunit residues, namely gamma Gln-269 with beta Thr-304 and gamma Arg-242 and beta Glu-381 (see below and Fig. 7A). It is apparent that effects of gamma  subunit mutations in these regions on catalysis are due to perturbation of the interactions with the catalytic beta  subunits. Thermodynamic analysis of transition state activation energies for ATP hydrolysis reactions strengthen this notion. Significantly, gamma M23K mutant enzyme had dramatically more positive Delta HDagger and less negative Delta SDagger parameters, suggesting that the amino acid replacement caused an extra bond to form that must be broken in order to achieve the catalytic transition state.


Fig. 7. Regions of interactions between gamma  and beta  subunits. A illustrates the regions of interaction between the gamma  subunit (in blue) with the beta  subunit conformers known as beta DP (in yellow) and beta E (in green). Coordinates for this figure were obtained from x-ray crystallographic data for bovine F1 (13). The braces illustrate the three gamma  subunit helical regions involved in energy coupling (21). The upper asterisk shows the contact between conserved residues gamma Gln-269 and beta Thr-304 (E. coli numbering) near the "hydrophobic sleeve" (13). The lower asterisk shows the contact region between the conserved residue gamma Arg-242 and beta Glu-381 of the conserved beta 380DELSEED386 sequence in beta DP. This region is shown in detail in B along with gamma Met-23 and Van der Waals' contacts.
[View Larger Version of this Image (85K GIF file)]


Fig. 7B shows details of the bovine F1 structural map. gamma M23 is a member of a conserved triad consisting of gamma R242 and one of the three beta E381. The illustrated beta E381 is in the beta  subunit conformer known as beta DP, which has ADP bound in the F1 crystal (13). We note that all three residues are in highly conserved regions of the gamma  and beta  subunits and are identical in all known gamma  subunit sequences; therefore, in the analysis of mutant E. coli complexes, using the positions of these residues as determined from crystals of the bovine enzyme is valid. In turn, we note that the results presented here are entirely consistent with the structural model of Abrahams et al. (13). We propose that when gamma M23 is changed to lysine, the epsilon -amino group forms an additional ionized hydrogen bond with beta Glu-381 during a step of the catalytic cycle, hence the extra bond detected by Arrhenius analysis. Clearly, gamma M23K is an added function mutation. This conclusion is consistent with the similar uncoupling effect of replacing gamma Met-23 with arginine and the lack of effect when substituted by neutral or negatively charged amino acids (19).

This explanation is also consistent with the effects of second site mutations that suppress the effects of the gamma M23K mutation. The gamma Q269R suppressor mutation restored efficient coupling and negated the increase in transition state activation energy. As mentioned before, the amino acid replacement of gamma Gln-269 affected the interactions between gamma  and beta  subunits, causing reduced coupling efficiency (Table II) and stability (20). We propose that the gamma Q269R mutation suppresses the effect of gamma M23K in part by reducing the energy of interaction between gamma  and beta  subunits. It is likely that similar effects were observed by Jeanteur-De Beukelaer et al. (48) with beta  subunit mutations that suppressed the effects of an altered gamma  subunit carboxyl terminus. Related to the effect on coupling is an effect on complex stability. Loss of delta  and epsilon subunits leaves a destabilized alpha 3beta 3gamma with gamma M23K (Fig. 1), and all of the known suppressor mutations of gamma M23K confer temperature stability (20, 21). Clearly, protein-protein interactions between gamma  and beta  subunits are critical for both complex stability and coupling.

In contrast, changing gamma R242C resulted in overall higher proton pumping rates and ATP synthesis because the enzyme turned over faster and not because coupling efficiency was restored. Interestingly, the structure suggests that gamma R242C should reduce the energy of interaction between gamma  and beta  subunits because this amino acid change should remove the ionized hydrogen bond between gamma Arg-242 and beta DPAsp-381 (Fig. 7B). It is possible that the cysteine may reduce gamma -beta interactions even more if the environment of gamma R242C induces a lower pK and causes the residue to ionize at neutral pH. The thiolate ion, a strong nucleophile, would form an ion pair with gamma M23K and in addition create a repulsive pair with beta Glu-381. We are analyzing the properties of complexes with additional mutations of both residues to clarify their roles. Not surprisingly, certain amino acid replacements of beta Glu-381 have a similar uncoupling phenotype to the gamma M23K mutation.3 Without question, the gamma -beta interactions involving gamma R242 and beta E381 play an important role in turnover and coupling. The beta  subunit residue is a part of the conserved sequence 380DELSEED386 (E. coli numbering). Residues in this sequence have also been implicated in interactions with epsilon  subunit residue epsilon S108 (45, 49). It seems quite plausible that the perturbations on the conserved beta DELSEED sequence induced by the gamma M23K mutation are communicated to epsilon S108 or nearby residues and perturbs the functional interaction of F1 with F0 as demonstrated by the fact that epsilon  inhibition of F1 ATPase activity in not relieved by F0 binding in the gamma M23K mutant (Fig. 3).

In perturbing the interactions between gamma  and the alpha 3beta 3 complex, the mutation alters thermodynamic parameters of the ATP hydrolysis reaction transition state in both F0F1 and purified F1 complexes (Table I and Fig. 5). Isokinetic plots of Delta HDagger versus TDelta SDagger can be used to investigate perturbations of coupling through the effects of mutations on the structure of the catalytic transition state. Fig. 8 shows isokinetic plots for F1 and F0F1 preparations. It is seen that for various F1 beta  and F1 gamma  mutants, the gamma M23K enzyme preparations lie very close to bovine mitochondrial F1 and F0F1. Al-Shawi et al. (42) suggested that the mitochondrial enzyme is a "better" catalyst than the E. coli enzyme because it binds the substrate in a manner that is closer to the true transition state of pentacoordinate gamma -phosphate and thereby reduces the transition state energy. In order to achieve a lower transition state, however, the enzyme must utilize more binding energy. In the case of the E. coli gamma M23K mutant, the enzyme must also utilize more binding energy to break an extra bond that is created by replacement of the conserved gamma M23 with a positively charged residue. Another interesting feature revealed by the isokinetic plots (Fig. 8) is that the gamma M23K F1 and gamma M23K F0F1 points have very similar values, whereas the wild-type F1 point has a more positive Delta HDagger and TDelta SDagger than the wild-type F0F1 membrane preparations. The origin of this phenomenon was seen in Fig. 3 in that as the wild-type F1 binds to F0 the epsilon  inhibition is relieved on binding F0. As pointed out above, the effect of the gamma M23K mutation perturbs transmission of conformational information, which modulates epsilon  interactions with F0 such that inhibition is not relieved and coupling efficiency is lowered. This is the primary effect of gamma M23K on coupling.

The effect of gamma M23K as well as other mutations in the gamma -beta interface appear to perturb a balance of interactions between the subunits necessary for transmission of coupling information and energy. In addition, the gamma M23K mutation appears to modulate the functional interaction of epsilon  subunit with F0. The effect of the structural perturbation on catalytic mechanism appears to be creation of a branched pathway that either bypasses or skips the coupling step. These results demonstrate the mechanistic linkage between catalysis and coupling that minimally involves beta , gamma , and epsilon  subunits. Furthermore, we suggest that residues gamma Met-23, gamma Arg-242, beta 380DELSEED 386, and epsilon  residues (near epsilon Ser-108) form a common energy coupling domain that transmits conformational energy from F0 to F1 and vice versa at discrete point (times) within the turnover of the enzyme. These suggestions are currently being investigated by further experiments.


FOOTNOTES

*   This work was supported by Public Health Service Grants R01-GM50957 (to R. K. N.) and R01-GM52502 (to M. K. S.) with additional support from National Science Foundation Grant BIR-9216996. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    Recipient of National Institutes of Health Predoctoral Grant HL07284.
§   To whom correspondence should be addressed: Dept. of Molecular Physiology and Biological Physics, University of Virginia, P. O. Box 10011, Charlottesville, VA 22906. Tel.: 804-982-0279; Fax: 804-982-1616; E-mail: rkn3c{at}virginia.edu.
2    R. K. Nakamoto, unpublished observation.
3    C. J. Ketchum, M. K. Al-Shawi, and R. K. Nakamoto, manuscript in preparation.
1    The abbreviation used is: CCCP, carbonylcyanide-m-chlorophenylhydrazone.

Acknowledgments

We thank Dr. Alan Senior of the University of Rochester for the gift of the anti-alpha antiserum as well as many discussions. We would also like to thank Dr. John Walker for immense help with analysis and preparation of the molecular structure figures and for allowing us to visit his laboratory.


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