(Received for publication, April 10, 1995; and in revised form, July 5, 1995)
From the
Changes in conformation of the -subunit of the bovine heart
mitochondrial F
-ATPase complex as a result of nucleotide
binding have been demonstrated from the phosphorescence emission of
tryptophan. The triplet state lifetime shows that whereas nucleoside
triphosphate binding to the enzyme in the presence of Mg
increases the flexibility of the protein structure surrounding
the chromophore, nucleoside diphosphate acts in an opposite manner,
enhancing the rigidity of this region of the macromolecule. Such
changes in dynamic structure of the
-subunit are evident at high
ligand concentration added to both the nucleotide-depleted F
(Nd-F
) and the F
preparation containing
the three tightly bound nucleotides (F
(2,1)). Since the
effects observed are similar in both the F
forms, the
binding to the low affinity sites must be responsible for the
conformational changes induced in the
-subunit. This is partially
supported by the observation that the Trp lifetime is not significantly
affected by adding an equimolar concentration of adenine nucleotide to
Nd-F
. The effects on protein structure of nucleotide
binding to either catalytic or noncatalytic sites have been
distinguished by studying the phosphorescence emission of the F
complex prepared with the three noncatalytic sites filled and the
three catalytic sites vacant (F
(3,0)). Phosphorescence
lifetime measurements on this F
form demonstrate that the
binding of Mg-NTP to catalytic sites induces a slight enhancement of
the rigidity of the
-subunit. This implies that the binding to the
vacant noncatalytic site of F
(2,1) must exert the opposite
and larger effect of enhancing the flexibility of the protein structure
observed in both Nd-F
and F
(2,1). The
observation that enhanced flexibility of the protein occurs upon
addition of adenine nucleotides to F
(2,1) in the absence of
Mg
provides direct support for this suggestion. The
connection between changes in structure and the possible functional
role of the
-subunit is discussed.
The ATPase (ATP synthase) is the enzyme responsible for ATP
synthesis during oxidative phosphorylation in all energy-transducing
membranes. It is composed of two main parts: F, capable of
proton transport across the membrane and the catalytic part;
F
, bound to F
through a ``stalk''
segment (for reviews see (1, 2, 3, 4, 5) ). F
of eukaryotes and prokaryotes are similar in subunit composition;
they are composed of five different subunits
through
, in
order of decreasing molecular weight, with the stoichiometry 3, 3, 1,
1, and 1(2, 6) . However, these enzymes are not
identical mainly because of differences in two of the subunits;
-
and
-subunits of bacteria (and chloroplasts) are homologous to
oligomycin sensitivity conferring protein and
of beef heart,
respectively. Therefore, the
-subunit of mitochondria lacks a
counterpart in bacteria and chloroplasts. The two major subunits
and
of F
contain at their interfaces a total of six
nucleotide binding sites, which are characterized by different
properties(7, 8, 9, 10, 11, 12, 13) .
Nucleotide sites have been classified as catalytic or noncatalytic
according to their ability to exchange bound ligand rapidly during
hydrolysis of Mg-ATP(13) . According to this definition, there
are three catalytic and three noncatalytic sites. The functional role
of the latter sites has yet to be elucidated. However, several authors
have suggested a regulatory
function(11, 14, 16, 17, 18) .
Only two noncatalytic sites and one catalytic site are fully occupied
on desalted F
; these sites are regarded as tight binding
sites(19) , and this state of occupancy is described by the
symbol F
(2,1) (
)according to Kironde and
Cross(20) . The vacant noncatalytic site is also regarded as
the exchangeable noncatalytic site(21) . Catalytic sites have
been shown to exhibit magnesium dependence and a relatively broad
nucleotide specificity(2, 19, 22) , whereas
noncatalytic sites have a significant preference for adenine
nucleotides(19, 20, 23, 24, 25) .
Both ATP hydrolysis and ATP synthesis catalyzed by the F-type
ATPases are cooperative processes now thought to involve ligand-induced
and energy-dependent conformational changes, which modulate the
affinity of catalytic sites for substrates and
products(25, 26) . The mechanism and the particular
domain (or subunit) of F and F
involved in each
individual step of the processes are unknown, in particular the mode of
the transmission of conformational signals between domains of the
protein. Approaches that have been used to monitor conformational
changes in F
and in F
F
-ATPase
preparations, including chemical labeling of certain amino acid
residues of the protein(27, 28) , hydrogen
exchange(29) , binding of inhibitors(30) , and protease
digestion experiments(31, 32) , are invasive, thus
providing information regarding a structurally altered protein. Methods
are needed to selectively monitor conformational changes of specific
domains or subunits of the unmodified protein.
Trp phosphorescence
measured at room temperature has shown considerable potential for the
study of protein structure in solution(33) . The sensitivity of
the triplet-state lifetime of the indole nucleus to the flexibility of
its surrounding matrix (34) has been extremely useful with
respect to revealing subtle conformational changes induced in proteins
by binding of substrates, coenzymes, inhibitors, or interacting
macromolecules(35, 36, 37) . We have recently
used the phosphorescence of the sole tryptophan residue of the
mitochondrial F complex as an internal probe of the
-subunit(38, 39) , and the lifetime (
)
measurements have revealed conformational changes of the
nucleotide-depleted enzyme as a consequence of Mg-ATP binding at low
temperature.
The high complexity of nucleotide binding sites of
F and the existence of temperature-dependent conformational
states of the enzyme prompted us to further investigate on possible
alterations in the dynamic structure of the
-subunit induced by
selective nucleotide-site occupancy. We have analyzed the
phosphorescence decay kinetics of F
at room temperature in
the presence or absence of nucleoside di- and triphosphates associated
with loose or tight, catalytic and noncatalytic nucleotide binding
sites. These noninvasive studies of intrinsic phosphorescence provide
evidence that the conformation of the
-subunit in situ is
affected differently by the binding of nucleoside di- or triphosphates
to the various nucleotide binding sites of F
.
ATP, ADP, GTP, GDP, phosphoenolpyruvate, Hepes, Tris, and NADH were obtained from Sigma as were pyruvate kinase and lactate dehydrogenase in glycerol-containing buffers. Sephadex G-50, Sephacryl S-300, Blue Sepharose CL-6B, and standard marker proteins were obtained from Pharmacia Biotech Inc.
F was prepared from bovine
heart mitochondria according to Penefsky(40) . We have
observed that at this stage the enzyme preparation contained minor
contaminants: a protein with an apparent molecular mass of about 48 kDa
and the ATPase inhibitor protein. All of the contaminants were removed
as follows. By affinity chromatography on Blue Sepharose CL-6B, 16 mg
of protein was loaded onto a column (4 cm
1 cm, inner diameter)
in 0.2 M NaCl, 1 mM EDTA, 1 mM
-mercaptoethanol, 1 mM ATP, and 20 mM Tris-Cl,
pH 8(41) , at a 4 ml/h flow rate. 13 mg of the non-retained
protein were concentrated to 15 mg/ml by ultrafiltration with a Diaflo
XM-300 (Amicon) membrane. The concentrate was then chromatographed on
Sephacryl S-300 (40 cm
1.6 cm, inner diameter) in 25 mM Tris-Cl, 0.25 M sucrose, and 1 mM ATP, pH 8; a
typical elution profile is shown in Fig. 1A. Fractions
containing the 32-40-ml elution volume (9 mg of protein) were
characterized by a constant specific activity, indicating the presence
of a single molecular species that was used in the experiments.
SDS-polyacrylamide gel electrophoresis confirmed this assertion since
even overloading the gel, the typical five-subunit pattern of F
was observed (Fig. 1B). The enzyme solution was
stored at 5 °C as a suspension at 50% ammonium sulfate saturation
in the presence of 4 mM ATP (pH 8). The enzyme activity was
stable for several weeks in this state. Since the technique used in our
experiments evaluates the single Trp of F
, it is extremely
important that no contaminating protein is present in the enzyme
preparation to avoid Trp signals other than that of F
.
Figure 1:
Purity of the
F complex. A, elution profile of the complex
passed through a Sephacryl S-300 column (see ``Materials and
Methods''). B, the samples were analyzed by
SDS-polyacrylamide gel electrophoresis (45) and stained with
Coomassie Brilliant Blue R-250. LanesA, standard
marker proteins (Pharmacia) are as follows: rabbit muscle phosphorylase b (94.0 kDa), bovine serum albumin (67.0 kDa), egg white
ovalbumin (43.0 kDa), bovine erythrocyte carbonic anhydrase (30.0 kDa),
soybean trypsin inhibitor (20.1 kDa), bovine milk
-lactalbumin
(14.4 kDa); lanesB-D, 4, 6, and 10 µg of
Nd-F
, respectively; lanesE and F, 3 and 9 µg of F
(3,0), respectively; lanes G and H, 15 and 20 µg of F
(2,1),
respectively. The positions of the F
subunits are indicated
at the right side.
F(2,1) was obtained from the above enzyme suspension by
centrifugation, and the sedimented enzyme was dissolved at 4-6
µM in a buffer containing 150 mM sucrose, 1
mM KH
PO
, 1 mM
MgSO
, 10 mM K
-Hepes, pH 8; it was
desalted on a Sephadex G-50 centrifuge column (42) equilibrated
with the same buffer. F
(3,0) was prepared from the ammonium
sulfate suspension removing unbound nucleotide and desalting by passage
through a centrifuge column equilibrated with 150 mM sucrose,
10 mM Hepes, 1 mM MgSO
, pH 8; it was then
followed by a procedure based on the displacement of nucleotides from
the catalytic sites by a brief exposure (1 min) to pyrophosphate and
removal of unbound nucleotides by gel filtration(20) .
F
(3,0) was also prepared from F
(2,1) by
substituting pyrophosphate with Mg-GDP, according to (43) .
Nd-F
was prepared from submitochondrial particles by gel
permeation chromatography in the presence of 50% glycerol (v/v) as
previously described(38) . All of the F
preparations were pure and had the typical subunit stoichiometry
as evidenced from SDS-polyacrylamide gel electrophoresis (Fig. 1B). This is of particular importance, since it
has been hypothesized that the
-subunit might be substoichiometric
with respect to the rest of the protein(12) , which could
result in misinterpretation of the phosphorescence data. The percent
volume calculated for the protein bands of a typical F
,
using the Molecular Analyst PC image analysis software for the Bio-Rad
densitometer (model GS-670) was
+
= 85.81,
= 9.49,
= 3.41, and
= 1.29.
The nucleotide content of each enzyme preparation was determined by
reverse-phase HPLC, following nucleotide extraction according to Di
Pietro et al.(44) . The HPLC analysis of the extracts
was performed on a Nova-Pak C
column (150
3.9 mm, 4-µm Nova-Pak
packing material,
Waters) equilibrated in 30 mM KH
PO
, pH
5.4, containing 3 mM tetrabuthylammonium sulfate. Nucleotides
were eluted with a 0-30% acetonitrile gradient over 25 min at a
flow rate of 1 ml/min. Detection was by absorbance at 260 nm. The
nucleotide/F
molar ratios observed were 0.4 ± 0.1
for Nd-F
, 2.8 ± 0.4 for F
(2,1), and 2.6
± 0.5 for F
(3,0) using a molecular mass of 370 kDa-
for the enzyme (6) in all stoichiometry calculations.
SDS-polyacrylamide gel electrophoresis was carried out according to Laemmli (45) using a polyacrylamide gradient from 14 to 25% containing 0.1% SDS. The procedure has been previously described in detail(46) .
The ATPase activity was determined with an ATP regenerating system by following the decrease of NADH absorption at 340 nm in a 7850 model Jasco spectrophotometer. The assay was carried out at substrate-saturating concentration (steady state) as previously reported(38) . The specific activity of the enzyme was 80-100 units/mg protein at 20 °C.
Protein concentrations of enzyme solutions were determined by the method of Lowry et al.(47) .
Phosphorescence spectra and decay
measurements were obtained with a phosphorimeter, constructed in this
institution, as previously described(48) . The photons were
generated by a Cernax xenon lamp (LX 150UV, ILC Technology), and the
wavelength was set with a 250-nm grating monochromator (Jobin-Yvon,
H25). The emission was detected with an EMI 9635 QB photomultiplier.
Phosphorescence decay in fluid solution at room temperature was
monitored with a homemade apparatus suitable for lifetime measurements
in the µs-ms range described in detail
elsewhere(75) . Pulsed excitation( =
292 nm) was generated by a frequency-doubled flash-pumped dye laser
(UV500 M Candela) with a pulse duration of 1 µs and an
energy/pulse typically of 1-10 mJ. The sample, placed in a
vacuum-proof quartz cuvette that allows excitation of the solution from
above, is extensively deoxygenated prior to analysis. The emitted light
is measured at 90° from the excitation light and selected by a
filter combination in the window between 420 and 480 nm. The
photomultipliers are protected from the intense excitation light and
fluorescence pulse by a high speed chopper blade that closes the slits
during laser excitation. The minimum lag time of the apparatus is about
10 µs. The decay signal was digitized by a computerscope system
(ISC-16, RC Electronics) capable of averaging multiple sweeps.
Subsequent analysis of decay curves in terms of the sum of exponential
components was carried out by a nonlinear least squares fitting
algorithm implemented by the program Global Analysis (Global Unlimited,
LFD University of Illinois, Urbana).
For each sample, the phosphorescence decay was measured three times, and samples were prepared at least four times. The standard error of preexponential terms and lifetime components are better than ± 10%. It should be noted, however, that the variability of these parameters can be even somewhat greater when one compares different preparations of the protein. Such variability in the decay kinetics can be traced down to different amounts of quenching impurities present in organic solvents (glycerol), and glasswares(75) . For this reason, comparisons are always made between samples obtained from the same enzyme preparation.
Figure 2:
Decay of phosphorescence intensity of
Nd-F in the presence of Mg-ANP at 293 K. In a is
shown Nd-F
, in b, 1 mM Mg-ATP is added to
Nd-F
, and in c, 1 mM Mg-ADP is added to
Nd-F
. Experimental details are reported in the legend to Table 1.
ADP has an effect opposite that of ATP; it
enhances the protein rigidity since the intrinsic of
the protein increases from 2.8 of Nd-F
to 3.4 ms upon
nucleotide binding.
The opposite effect induced on the -subunit
conformation by ATP and ADP binding suggests that different
conformations of the nucleotide binding sites are induced by the two
nucleotides and that different allosteric effects are then transmitted
to the Trp environment of the
-subunit. This would be consistent
with the idea of several authors who, on the basis of inhibition
studies(18, 51, 52) , speculated that the
F
-ATPase complex may exist into two different
conformations, E
and E
, favored by ADP and ATP
binding, respectively.
When Nd-F is incubated with
stoichiometric amounts of ADP in the presence of Mg
,
it results in inhibition. It has been shown that the inhibitory ADP is
bound in a catalytic site (52, 53, 54) . To
establish whether the binding of ADP to this high affinity catalytic
site affects the conformation of the
-subunit, ADP has been added
stoichiometrically to Nd-F
. The decay parameters (Table 1) do not change significantly with respect to control,
indicating a lack of influence of ADP filling the high affinity
catalytic site on the conformation of the protein at the
-subunit
level. Also, the addition of stoichiometric ATP to Nd-F
(i.e. conditions for unisite catalysis as in (55) ) does not affect the Trp phosphorescence decay.
Therefore, independent of the adenine nucleotide tested, it would
appear reasonable to conclude that filling the high affinity catalytic
site does not result in conformational changes of the protein involving
the N-terminal segment of the
-subunit.
To further investigate
which nucleotide site(s) of F have to be occupied to induce
-subunit conformational changes, preparations of F
at
different levels of nucleotide occupancy of sites have been studied.
Incubation of
F(2,1) with 1 mM Mg-ADP, 1 mM Mg-ATP, or
0.5 mM Mg-GTP at 293 K in 150 mM sucrose, 1
mM MgSO
, 10 mM Hepes, pH 8, induces
consistent changes of the phosphorescence decay (Fig. 3). Table 1shows the phosphorescence decay parameters of a typical
experiment. The data clearly display a significant decrease of the
average lifetime from 4.3 to 3.3 and 3.5 ms when ATP or GTP are bound,
respectively. This indicates an increased flexibility of the
chromophore environment upon occupancy of the vacant nucleotide binding
sites by NTP, whereas ADP has an opposite effect since it enhances
to 4.6 ms (Table 1). Thus, the results are
similar to those observed on Nd-F
, providing further
support to the conclusion that 1) occupation of loose binding site(s)
is responsible for the
-subunit structural change and 2) binding
of the nucleoside triphosphate increases the flexibility of the Trp
environment, whereas the binding of nucleoside diphosphate enhances its
rigidity (Mg
present).
Figure 3:
Decay of phosphorescence intensity of
F(2,1) in the presence of Mg-ANP at 293 K. a,
F
(2,1); b, F
(2,1) in the presence of 1
mM Mg-ATP; and c, F
(2,1) in the presence
of 1 mM Mg-ADP. Experimental details are reported in the
legend to Table 1.
It has been shown that
addition of ATP or ADP plus P in the presence of magnesium
results in a reactivation of the AMP-PNP-inhibited ATP hydrolysis
activity of the enzyme(56) . The similar behavior of ATP or ADP
plus P
has prompted investigation with respect to similar
effect of the ligands on the F
conformation. Thus,
experiments designed to determine whether inorganic phosphate added
together with ADP might have the capability to influence the
-subunit conformation similarly to ATP were initiated. For this
purpose, F
was incubated in the presence of ADP +
Mg
over a range of 0.1-10 mM P
(i.e. the physiological range, according to (57) and (58) ), with care being taken to exclude
adventitious P
from enzyme and buffers. This treatment did
not alter significantly the effect of ADP alone on the phosphorescence
decay parameters of the enzyme (Table 1), indicating that the
activation effect of P
on the Mg-ADP-F
complex
cannot be related to the
-subunit conformation.
Figure 4:
Decay of phosphorescence intensity of
F(3,0) in the presence of Mg-ATP at 293 K. a,
F
(3,0); b, F
(3,0) in the presence of 1
mM Mg-ATP. Experimental details are reported in the legend to Table 1.
The comparison of these data with those obtained on
Nd-F suggests that the effects observed on addition of
nucleotides to both Nd-F
and F
(2,1) are the sum
of two distinct effects; the binding of the exchangeable noncatalytic
site induces an increased flexibility of the
-subunit N-terminal
domain, which likely overwhelms the tightening of the same domain
induced on filling the loose catalytic binding sites.
Addition of ATP in the presence of 2
mM EDTA reduces the value from 4.3 to 3.7 ( Fig. 5and Table 2). This result was expected on the basis
of above results where the filling of the exchangeable noncatalytic
nucleotide binding sites could reduce
, indicating an
enhanced flexibility of the protein. The enhanced flexibility
associated with the filling of the exchangeable noncatalytic site and
the enhanced rigidity associated to the filling of loose catalytic
sites might be additive or not. In fact, it seems they are not since if
they were, one would have expected an even larger increase of the
protein flexibility, for the filling of the catalytic sites under the
present experimental conditions is not favorable. This divergence from
additivity might be explained if one considers that Mg
changes the structure of the nucleotide binding sites as recently
shown (62) and that the binding of sites in the absence of
Mg
might result in structural changes transmitted to
the
-subunit different from those observed in the presence of the
metal.
Figure 5:
Decay of phosphorescence intensity of
F(2,1) in the presence of ANP (Mg
absent)
at 293 K. a, F
(2,1) was in 150 mM sucrose, 10 mM K
-Hepes, 2 mM EDTA, pH 8; b, 1 mM was added to
F
(2,1); and c,1 mM ADP was added to
F
(2,1).
Similar considerations could explain the effect of ADP on the
dynamic properties of F in the absence of magnesium ions,
as monitored by Trp phosphorescence. In fact, addition of the
nucleoside diphosphate in the absence of Mg
reduces
from 4.3 to 2.8 ms (Table 2), indicating an
increased flexibility of the protein, whereas an enhancement of
rigidity was observed in experiments in which ADP was added along with
Mg
to the F
-ATPase complex.
Finally,
addition of 0.1-10 mM P along with (or
following) ADP does not change significantly the effect of ADP when
added alone. Therefore, as in the presence of Mg
, in
its absence the addition of inorganic phosphate to the ADP-F
complex is without effect on the
-subunit conformation.
The -subunit of the mitochondrial F
-ATPase
complex is the polypeptide with the lowest molecular mass (5.5 kDa) of
the protein, and its function is unknown. However, it is thought that
it plays a role in the coupling between F
and F
(12, 63) . Consistent with such a role, although
experimental evidence of the
-subunit location is not available,
most of the F
and F
F
models show
the polypeptide located at the interior of the
-
-subunit
core, facing F
, possibly contributing to the stalk
region(3, 5, 12, 51, 64, 65) .
Previously, through the investigation of the intrinsic
phosphorescence of the mitochondrial F-ATPase complex, we
showed that the addition of Mg-ATP to the enzyme bearing vacant
nucleotide binding sites resulted in large conformational changes of
the protein surrounding the N-terminal domain of the
-subunit(38, 39) . However, our experiments were
performed at 273 K or below in the presence of 50% glycerol as a
stabilizing co-solvent to operate under optimal conditions for the
measurement of the phosphorescence signal. Given these conditions, it
is not possible to interpret our results in terms of in vivo function, since the kinetic properties and the conformation of
F
in media characterized by high viscosity and below 16
°C are quite different from those shown between 20 and 37
°C(28, 66) .
In the present study, we have overcome the problem since we were able to carry out the experiments at 20 °C by modifying equipment for phosphorescence measurements, and we extended the investigations analyzing the effect of filling with different nucleotides several enzyme forms characterized by different nucleotide content and configuration.
This research has revealed a
few intriguing features concerning conformational changes of the
-subunit upon binding of nucleotides to the mitochondrial F
complex. 1) The binding of nucleotides to the loose sites is
solely responsible for the conformational changes observed on the
-subunit. This contention is based on the following observations:
first, addition of Mg-ATP or Mg-GTP in large molar excess to both the
Nd-F
and the enzyme containing the tightly bound
nucleotides, F
(2,1), produces a net shortening of the
phosphorescence lifetime, comparable in the two enzyme preparations;
second, incubation of Nd-F
with unistoichiometric Mg-ATP,
which loads a single, high affinity catalytic site, does not affect the
average phosphorescence lifetime of F
; third,
phosphorescence spectra of F
(2,1) are identical to those of
Nd-F
. 2) The comparative analysis of the results obtained
with F
(2,1), F
(3,0), and Nd-F
(this
in the presence of unistoichiometric Mg-ATP) strongly suggests that the
increased flexibility of the Nd-F
and F
(2,1)
forms upon binding of Mg-ATP or Mg-GTP is in fact the result of two
different effects and that the filling of the vacant noncatalytic site
is responsible for the large increased flexibility of the
-subunit. These conclusions are supported by results of the
experiment carried out in the absence of magnesium, a condition
favoring the binding of nucleotides to the noncatalytic sites. Under
this condition, the average phosphorescence lifetime of
F
(2,1) markedly decreases upon incubation with adenine
nucleotides. 3) Mg-ADP addition to the ATPase complex consistently and
greatly enhances the rigidity of the Trp microenvironment, showing an
opposite effect with respect to Mg-ATP. Thus, our results provide
evidence for two opposite conformational changes of the mitochondrial
F
-ATPase
-subunit whether ATP + Mg
or ADP + Mg
is added. Moreover, addition
of P
along with (or following) ADP + Mg
did not significantly alter the effect of ADP +
Mg
only, suggesting that once Mg-ADP is bound,
P
can not influence the ADP-F
structure as
probed at the
-subunit level.
The markedly different
conformational changes obtained on binding of Mg-ATP, as compared to
Mg-ADP, might have several possible explanations. Thus, F might assume two conformational states, depending whether ADP or
ATP is the ligand. This possibility has been proposed by Boyer (51) on the basis of various experimental observations
reporting significant differences in the ATPase behavior, whether the
enzyme has been examined in the presence of ADP or ATP. Interestingly,
an x-ray crystallographic study of a Ras protein catalyzing GTP
hydrolysis has shown substantial structural differences whether Mg-GTP
or Mg-GDP was the ligand. These differences seemed to be caused by a
different coordination of the active site Mg
ion(67) . Since F
shares with the Ras protein
the conserved phosphate-binding loop(68) , Mg
might have a similar role with F
on binding Mg-ATP or
Mg-ADP. A second possibility is that different types of
metal-nucleotide diastereoisomers could be the true ligands for binding
Mg-ADP or Mg-ATP to F
(69) . It has indeed been
shown that different metal-nucleotide epimers of ADP and ATP are the
substrates for a number of F
-ATPases(70) . The
consequence might be that different structural signals could be
transmitted from the nucleotide binding sites of F
to the
-subunit, depending on the particular type of stereoisomer bound.
If the -subunit is not in close proximity of the nucleotide
binding sites, as it is believed, our results demonstrate that
conformational changes of F
upon substrate binding are
transmitted over long distances. Thus, the
-subunit might be
involved in the propagation of signals to deeper regions of the
F
F
complex.
Finally, observations of certain
similarities between the present study and those carried out on F from other sources should be cited. Evidence for a correlation
between occupation of the nucleotide binding sites, catalysis, and
conformational changes of the
-subunit were obtained in studies
with both Escherichia coli and chloroplast
F
(31, 32, 72, 73) .
The enzyme from the different sources has the functional core
composed of homologous subunits
and has two additional small, single-copy subunits, which appear to
play a role in the energy-coupling mechanism and are physically close
to one another(15, 64, 71, 74) . The
mitochondrial F
-subunit, which has no counterpart in
other species, might share with the
-subunit of F
from
other sources the involvement in the coupling mechanism and/or in its
regulation. Nevertheless, the molecular events involving conformational
changes of the
-subunit of F-type ATPases from different
energy-transducing membranes might differ from one another, as it can
be envisaged if one considers several different effects observed on
small subunits of mitochondria and E. coli F
upon
filling of the nucleotide sites. Mendel-Hartvig and Capaldi (31) found a direct relationship between P
binding
and
-subunit conformation of E. coli F
,
whereas it was not possible to find any such relationship with the
mitochondrial enzyme. Moreover, both Mg-ADP and Mg-ATP could induce a
less tight structure of the
-subunit of E. coli, whereas
in our study Mg-ADP and Mg-ATP could increase or decrease,
respectively, the tightness of the mitochondrial protein.
In
conclusion, our results provide information on dynamic aspects of the
enzyme structure and function and provide the first noninvasive,
extensive experimental evidence for a change in the conformation of the
mitochondrial F-ATPase
-subunit in situ in
response to the filling of different classes of nucleotide binding
sites with several substrates.
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