(Received for publication, October 1, 1996, and in revised form, October 25, 1996)
From the Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908
Replacement of the F0F1
ATP synthase subunit Met-23 with Lys (
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
M23K mutation causes
altered interactions between subunits. Binding of
or
subunits
stabilizes the
3
3
complex, which becomes destabilized by the mutation. Significantly, the inhibition of
F1 ATP hydrolysis by the
subunit is no longer relieved
when the
M23K mutant F1 is bound to F0.
Steady state Arrhenius analysis reveals that the
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
M23K
forming an ionized hydrogen bond with one of the
Glu-381 residues.
Two second site mutations,
Q269R and
R242C, suppress the effects
of
M23K and decrease activation energies for the
M23K enzyme. We
conclude that
M23K is an added function mutation that increases the
energy of interaction between
and
subunits. The additional
interaction perturbs transmission of conformational information such
that
inhibition of ATPase activity is not relieved and coupling
efficiency is lowered.
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
3
3
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,
and
, are needed to
reconstitute catalytic and transport sectors so that they are
coupled to carry out ATP-driven proton pumping or
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 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 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 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
Met-23
Lys (
M23K) mutant enzyme was
conferred by several second site mutations near the carboxyl terminus
of the
subunit including the replacement of amino acid
Arg-242
and seven different residues between
Gln-269 and
Ala-280 (20).
Furthermore the temperature sensitivity caused by the
M23K mutation
was suppressed by each of the second site mutations. In the simplest
interpretation of these results, direct interactions within the
subunit are perturbed by the
M23K mutation, and each of the
suppressor mutations is close enough to the
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
Met-23 is a considerable distance from the
269-280 region. Furthermore, the types of amino acid changes that
suppress the effects of the
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 M23K mutation perturbed interactions between subunits. In this paper, we provide evidence that
the
M23K mutation is an added function mutation that increases the
energy of interaction between
and
subunits. The consequences of
this increase are destabilization of the
3
3
complex and inefficient coupling
between transport and catalysis. In turn, the suppressor mutations
described above counteract by decreasing the energy of interaction.
F1 complex and and
subunits were isolated from strain DK8 harboring plasmid, pBWU13 (22)
or pBMU13-
M23K (see below). Membranous F0F1
was obtained from strain KF10rA harboring derivatives of plasmid pBWG15
(23).
The M23K mutation was introduced into the uncG gene on
plasmid pBWU13 to give pBMU13-
M23K. Oligonucleotide-directed
mutagenesis with the Stratagene (La Jolla, CA) Chameleon Kit (24) was
used to introduce the
M23K mutation on plasmid pBWG11 (23) using the
synthetic oligonucleotide, 5
-CACTAAAGCGaaaGAGATGGTCGCC-3
(lowercase letters denote the
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 SubunitsMembranes 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
and
subunits (9).
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-
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
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.
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.
|
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.
We first assessed if the M23K mutation
altered interactions with the single copy F1 subunits,
and
. In the wild-type E. coli complex,
subunit binds
tightly to the F1 complex with a KD of
approximately 10
8 M (38), and the association
constant of isolated
and
subunits is similar to this value
(39). These results suggested that
interactions with the remainder
of the F1 complex are mostly through the
subunit. In
turn, the affinity for
subunit to
subunit can be assessed by
the inhibitory properties of the
subunit on ATPase activity. ATPase
activity of the
3
3
complex is
inhibited approximately 90% by
subunit; the KI
is very close to the binding constant between isolated
and
subunits (39).
Sternweis and Smith (38) showed that dilution of the purified
F1 complex to a concentration below the
KD for -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
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).
subunit is generally
believed to bind F1 with lower affinity than
subunit
and is most likely dissociated at the concentration of 10 nM F1 (40).
In a marked difference from wild type, further dilution of 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
M23K mutation causes destabilization of
3
3
, the minimum complex capable of
ATPase activity (32, 41).
Interestingly, the inactivation of the M23K enzyme does not occur
until
and
subunits have dissociated and suggests that binding
of
and
subunits may help to stabilize the complex. To test this
notion, superstoichiometric amounts of purified
or
subunits
were added, while
M23K F1 was diluted to various concentrations (Fig. 2). In the presence of 72 nM
subunit alone, the ATPase specific activity of 13 nM
M23K F1 (the concentration that gave
maximal activation) was decreased as expected due to
inhibition. At
1.3 nM
M23K F1, the ATPase specific activity remained about the same, and at 0.13 nM
M23K
F1, the activity increased more than 2-fold compared with
the absence of added
subunit. This behavior reflects the balance
between the dissociation/association of
subunit and inactivation of
3
3
.
Results with 72 nM 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
subunit was well below its KI. Clearly,
subunit
alone binds to the complex independent of the
subunit and in a
manner that stabilizes
3
3
. With both
and
subunits added, activity was consistent with
-inhibited
levels.
The stabilization of activity by subunit alone provided a way to
directly assess the KI for
subunit.
M23K
F1 was diluted to 0.13 nM in the presence of 72 nM
subunit to maintain the stabilized complex.
Titration of
subunit resulted in an apparent KI
of 13 nM (data not shown), which is in good agreement with
the dilution experiments in Fig. 1.
In order to more fully analyze the kinetic and
thermodynamic properties of the 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 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
subunit (Figs. 1
and 2). This result confirms the early work of Sternweis and Smith (38)
that
inhibition is relieved upon binding to F0.
Interestingly, the turnover numbers of the
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
M23K mutation does not
affect the catalytic mechanism because wild-type and the
M23K
enzymes have relatively similar turnover numbers, and second, the
inhibition of the
M23K enzyme is not relieved when bound to
F0. The latter results may indicate that the
M23K
mutation perturbs the functional interaction of F1 with
F0 as mediated by the
subunit.
Altered Transition State Thermodynamic Parameters in the
In order to understand the effects of the 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
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
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.
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 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
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
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.
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 M23K F1 at 30 °C are
listed in Table I and plotted in Fig.
5A. The differences for each parameter
between wild type and
M23K are plotted in Fig. 5B. Note
that the membrane-bound enzyme had the same trends as soluble
F1, but the
M23K F0F1 had
considerably larger differences in both enthalpic and entropic terms.
For both soluble and membrane-bound complexes, the
M23K enzyme has a
more positive
H
and a less negative
S
which together add up to a small difference in
G
. 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.
|
Suppressor Mutations of
If the changes in transition state thermodynamic
parameters are due to the same perturbations that causes the uncoupling
phenotype of the M23K mutation, then second site suppressor
mutations of
M23K (20) would be expected to reverse the altered
thermodynamic parameters. This was the case with the suppressor
mutation,
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
M23K mutant is restored to wild-type levels in the presence of
Q269R. As predicted, the Arrhenius analysis shows that the
Q269R
mutation counteracted the effects of the
M23K mutation on the
thermodynamic parameters of the ATPase transition states (Fig.
6).
In contrast, another suppressor mutation, R242C, did not increase
the coupling efficiency even though it genetically suppressed the
M23K mutation (20) and resulted in increased ATP synthesis rates
(Table II). Instead, the mutation suppressed the effects of the
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
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 M23K mutant
were only slightly reduced by introduction of
R242C (Fig. 6). In
this case, it appears that the reduced energy of interaction between
and
subunits resulted in a faster turnover rate.
We have sought to understand how the 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
and
subunits (this
report) nor the F0 sector (19); however, dissociation of
and
subunits leaves a destabilized
3
3
complex (Fig. 1). Based on chemical cross-linking experiments,
subunit is believed to interact with
,
, and
subunits (44-47) and apparently does so with a
stabilizing effect on the
3
3
complex.
An important property of the
subunit is its inhibitory activity on
the hydrolysis activity of F1. Most significantly, in
contrast to the situation in wild type, inhibition by
subunit in
the
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
M23K enzyme (Fig.
4). Both of these effects illustrate the impaired coupling between
F1 and F0 functions in the mutant enzyme
M23K.
From the above data, it seems that the M23K mutation affects the
regions of interactions between
and
subunits. Based on
suppressor mutagenesis results, we earlier concluded that three highly
conserved regions of the
subunit,
18-35,
238-246, and
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
subunits and that at least two of the residues form a hydrogen bond
with specific
subunit residues, namely
Gln-269 with
Thr-304
and
Arg-242 and
Glu-381 (see below and Fig.
7A). It is apparent that effects of
subunit mutations in these regions on catalysis are due to perturbation
of the interactions with the catalytic
subunits. Thermodynamic
analysis of transition state activation energies for ATP hydrolysis
reactions strengthen this notion. Significantly,
M23K mutant enzyme
had dramatically more positive
H
and less negative
S
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. 7B shows details of the bovine F1
structural map. M23 is a member of a conserved triad consisting of
R242 and one of the three
E381. The illustrated
E381 is in the
subunit conformer known as
DP, which has ADP bound
in the F1 crystal (13). We note that all three residues are
in highly conserved regions of the
and
subunits and are
identical in all known
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
M23 is changed to lysine, the
-amino
group forms an additional ionized hydrogen bond with
Glu-381 during
a step of the catalytic cycle, hence the extra bond detected by
Arrhenius analysis. Clearly,
M23K is an added function mutation.
This conclusion is consistent with the similar uncoupling effect of
replacing
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 M23K mutation. The
Q269R suppressor mutation restored efficient coupling and negated
the increase in transition state activation energy. As mentioned
before, the amino acid replacement of
Gln-269 affected the
interactions between
and
subunits, causing reduced coupling efficiency (Table II) and stability (20). We propose that the
Q269R
mutation suppresses the effect of
M23K in part by reducing the
energy of interaction between
and
subunits. It is likely that
similar effects were observed by Jeanteur-De Beukelaer et al. (48) with
subunit mutations that suppressed the effects of
an altered
subunit carboxyl terminus. Related to the effect on
coupling is an effect on complex stability. Loss of
and
subunits leaves a destabilized
3
3
with
M23K (Fig. 1), and all of the known suppressor mutations of
M23K
confer temperature stability (20, 21). Clearly, protein-protein
interactions between
and
subunits are critical for both
complex stability and coupling.
In contrast, changing 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
R242C should reduce the energy of interaction between
and
subunits because this amino acid change should remove the ionized hydrogen bond between
Arg-242 and
DPAsp-381 (Fig. 7B). It is possible that the
cysteine may reduce
-
interactions even more if the environment
of
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
M23K and in addition create a repulsive pair with
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
Glu-381 have a
similar uncoupling phenotype to the
M23K
mutation.3 Without question, the
-
interactions involving
R242 and
E381 play an important role in
turnover and coupling. The
subunit residue is a part of the
conserved sequence 380DELSEED386 (E. coli numbering). Residues in this sequence have also been implicated in interactions with
subunit residue
S108 (45, 49).
It seems quite plausible that the perturbations on the conserved
DELSEED sequence induced by the
M23K mutation are communicated to
S108 or nearby residues and perturbs the functional interaction of
F1 with F0 as demonstrated by the fact that
inhibition of F1 ATPase activity in not relieved by
F0 binding in the
M23K mutant (Fig. 3).
In perturbing the interactions between and the
3
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
H
versus T
S
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
and F1
mutants, the
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
-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
M23K
mutant, the enzyme must also utilize more binding energy to break an
extra bond that is created by replacement of the conserved
M23 with
a positively charged residue. Another interesting feature revealed by
the isokinetic plots (Fig. 8) is that the
M23K F1 and
M23K F0F1 points have very similar values, whereas the wild-type F1 point has a more positive
H
and T
S
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
inhibition is relieved on binding
F0. As pointed out above, the effect of the
M23K
mutation perturbs transmission of conformational information, which
modulates
interactions with F0 such that inhibition is
not relieved and coupling efficiency is lowered. This is the primary
effect of
M23K on coupling.
The effect of M23K as well as other mutations in the
-
interface appear to perturb a balance of interactions between the subunits necessary for transmission of coupling information and energy.
In addition, the
M23K mutation appears to modulate the functional
interaction of
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
,
, and
subunits. Furthermore, we suggest that residues
Met-23,
Arg-242,
380DELSEED 386, and
residues (near
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.
We thank Dr. Alan Senior of the University of
Rochester for the gift of the anti- 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.