Báo cáo khoa học: Functional transitions of F0F1-ATPase mediated by the inhibitory peptide IF1 in yeast coupled submitochondrial particles pdf

Functional transitions of F
0
F
1
-ATPase mediated by the inhibitory
peptide IF1 in yeast coupled submitochondrial particles
Mikhail Galkin
1,
*, Rene
´
e Venard
1
, Jacques Vaillier
2
, Jean Velours
2
and Francis Haraux
1
1
Service de Bioe
´
nerge
´
tique & CNRS-URA 2096, Gif-sur-Yvette, France;
2
Institut de Biochimie et Ge
´
ne
´
tique Cellulaires du CNRS,
Bordeaux, France

The mechanism of inhibition of yeast F
0
F
1
-ATPase by its
naturally occurring protein inhibitor (IF1) was investi-
gated in submitochondrial particles by studying the IF1-
mediated ATPase inhibition in the presence and absence
of a protonmotive force. In the presence of protonmotive
force, IF1 added during net NTP hydrolysis almost
completely inhibited NTPase activity. At moderate IF1
concentration, subsequent uncoupler addition unexpect-
edly caused a burst of NTP hydrolysis. We propose that
the protonmotive force induces the conversion of IF1-
inhibited F
0
F
1
-ATPase into a new form having a lower
affinity for IF1. This form remains inactive for ATP
hydrolysis after IF1 release. Uncoupling simultaneously
releases ATP hydrolysis and converts the latent form of
IF1-free F
0
F
1
-ATPase back to the active form. The rela-
tionship between the different steps of the catalytic cycle,
the mechanism of inhibition by IF1 and the interconver-
sion process is discussed.

Keywords: ATP synthase; catalytic state; inhibitory peptide;
latent ATPase; protonmotive force; yeast.
Energy-driven ATP synthesis in energy-transducing mem-
branes is carried out by membrane-bound F
0
F
1
-ATPase
complex or ATP synthase [1]. The extrinsic F
1
subcomplex
is composed of five types of subunits in the stoichiometry
a
3
b
3
cde. Three fast-exchangeable nucleotide-binding sites,
presumably catalytic, and three slow-exchangeable nucleo-
tide-binding sites, certainly noncatalytic, reside in a/b
interfaces [2,3] (an alternative point of view can be found
in [4]). The membranous F
0
subcomplex promotes proton
translocation, energetically coupled to ATP synthesis/
hydrolysis via a stalk connecting F
0
and F
1
.F
1

can be
biochemically separated from F
0
and remains competent for
uncoupled ATP hydrolysis. Numerous data, including
direct observations of the rotation of the central axis of
F
0
F
1
relative to a
3
b
3
crown during ATP hydrolysis [5,6],
indicate that F
0
F
1
is a rotary motor, with an asymmetrical
rotor composed, in mitochondria, of c, d and e subunits,
anchored to a membranous decameric ring of c subunits
thought to compose a proton-driven turbine [7].
Mitochondrial F
0
F
1
is regulated by a naturally occurring
inhibitor protein called IF1 [8]. IF1 is a small acid- and
heat-stable protein, which stoichiometrically binds to the F

1
sector of ATP synthase and inhibits ATP hydrolysis [8–19].
D
~
l
H
þ
is believed to favour the release of IF1 from ATP
synthase [10–17] or, alternatively, to shift this regulatory
peptide from an inhibitory to a silent position on the enzyme
[10,18,19]. The IF1 binding site was proposed to be located
close to the C-terminus proximal DELSEED loop of the
b-subunit [20], close to the catalytic site [21], at the a/b
interface [22], or at the a/c interface [23]. A recent
tridimensional model of the bovine MF
1
–IF1 complex
drawn from radiocristallographic data showed IF1 bound
at the a/b interface, and interacting weakly with c [24].
The F
0
F
1
–IF1 interaction is modulated by a number of
factors such as pH, ionic strength, D
~
l
H
þ
and nucleotides

[8–16,25–31]. Interaction of F
0
F
1
–ATPase with adenine
nucleotides is very complex in itself. In addition to simple
competitive inhibition of ATP hydrolysis, ADP causes
hysteretic inhibition of ATPase activity in contrast to
nonadenylic nucleotides IDP and GDP [32–37]. Likewise,
MgADP-inhibited enzyme can undergo an energy-depend-
ent transformation changing its functional properties
[32–34]. For all these reasons, it is very difficult to
discriminate the roles of D
~
l
H
þ
, nucleotide occupancy and
enzyme turnover in the IF1-related regulatory processes.
However, it is well established that ATPase turnover is
necessary for IF1 binding [12], and a complex relationship
was found between ATP concentration and rate of IF1
binding to isolated bovine MF
1
subcomplex [31].
In this work, we have investigated the influence of D
~
l
H
þ

on IF1-mediated inhibition, using yeast IF1 and coupled
submitochondrial particles (SMP) with ATP and GTP as
substrates. Unexpectedly, in the presence of D
~
l
H
þ
,IF1was
found to promote the conversion of active F
0
F
1
–ATPase
to a latent form which remains inactive in ATP hydrolysis
even after IF1 release. Finally, we propose a scheme where
Correspondence to F. Haraux, Service de Bioe
´
nerge
´
tique & CNRS-
URA 2096, DBJC, CEA Saclay, F91191 Gif-sur-Yvette, France.
Fax: + 33 1 69 08 87 17, Tel.: + 33 1 69 08 98 91,
E-mail:
Abbreviations: IF1, inhibitor peptide of mitochondrial ATPase;
SMP, submitochondrial particles; FCCP, carbonyl-cyanide
p-(trifluoromethoxy)phenylhydrazone.
Enzymes: mitochondrial ATP synthase complex (EC 3.6.3.14).
*Present address: Southern Methodist University, Department of
Biological Sciences, PO Box 750376, Dallas TX 75275-0376, USA.
(Received 3 February 2003, revised 18 March 2004,

accepted 25 March 2004)
Eur. J. Biochem. 271, 1963–1970 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04108.x
specific microscopic states and catalytic steps play an
explicit role in IF1 locking and in the interconversion
between active and latent ATPase.
Materials and methods
Preparation of phosphorylating SMPs
Frozen mitochondria (10–20 mg proteins) prepared as
described by Gue
´
rin et al. [38] were thawed [39] and diluted
to 5–8 mgÆmL
)1
in 10 m
M
Tris/HCl, 100 l
M
EDTA pH 8.1.
The suspension was saturated with argon for 2 min, then
sonicated on ice using a W-225R tip sonicator (Ultrasonics
Inc.). Two 20 s cycles (output control 2; 60% duty cycle)
separated by 30 s interval were applied. Then the suspension
was centrifuged at 23 000 g for 20 min at 4 °C. The
supernatant containing SMP was centrifuged at 100 000 g
for 40 min at 4 °C. The pellet of SMP was suspended in
0.65
M
mannitol. ATPase and ATP synthase activities by
these SMP were stable for at least 1–2 days when stored at
room temperature. SMP could also be rapidly frozen in

liquid nitrogen for future use. Protein content was deter-
mined according to Lowry et al. [40] in the presence of 5%
(w/v) SDS using BSA as a standard. The average yield of
SMP was 10–20% of the total mitochondrial protein.
Conditioning of phosphorylating SMP
Before an experiment a suspension of SMP was thawed
rapidly (at about 85 °C), then diluted (to 1 mgÆmL
)1
)ina
medium containing (final concentrations) 0.65
M
mannitol,
10 m
M
Hepes, 5 m
M
potassium phosphate pH 8.0, 100 l
M
EDTA, 2 m
M
malonate, and a substoichiometric amount
of oligomycin (0.1 lgÆmg
)1
protein). The suspension was
incubated for 1 h at room temperature before the assays for
complete activation of succinate dehydrogenase [41] and
ATP synthesis [32]. Standard SMP preparations coupled by
substoichiometric oligomycin had the following activities
(lmolÆmin
)1

Æmg protein
)1
): coupled ATP hydrolysis, 1–1.5;
uncoupled ATP hydrolysis, 2–3; succinate-mediated ATP
synthesis, 0.3–0.5; coupled succinate oxidation, 0.3; coupled
NADH oxidation, 0.37; uncoupled NADH oxidation, 1.5
(respiratory control with NADH, % 4).
ATP hydrolysis measurements
The reaction was monitored continuously at 25 °Cina
stirred cuvette as H
+
release [42], with phenol red as pH
indicator (A
557
) in 1 or 2 mL of the standard mixture
containing 0.65
M
mannitol, 10 m
M
phosphate pH 8.0,
10 m
M
succinate, 100 l
M
EDTA (potassium salts), 17 m
M
KCl, 5 m
M
MgCl
2

,60 l
M
Phenol red, and 1 mgÆmL
)1
BSA.
(Mg)ATP and (Mg)GTP were added as indicated in the
figures. NTP concentrations > 0.8 m
M
were practically
saturating. Carbonyl-cyanide p-(trifluoromethoxy)phenyl-
hydrazone (FCCP; 5 l
M
)or2.5lgÆmL
)1
gramicidin D was
used as an uncoupler. The same results were obtained with
either uncoupler. The different additions (nucleotides, IF1,
uncouplers) had negligible effects on the pH of the medium
containing no SMP or SMP treated with excess of
oligomycin. First-order kinetics of inhibition by IF
1
[39]
was fitted to the equation:
yðtÞ¼V
1
tþ½ðV
0
ÀV
1
Þ=k

app
ð1Àe
Àkt
app
Þþy
0
Eqn ð1Þ
where y
0
and y(t)areA
557
at zero time and t time after IF1
addition, respectively, V
0
is the initial rate of A
557
change (at
zero time), V
1
is the final rate of A
557
change (at infinite
time), and k
app
is the apparent deactivation rate constant.
k
app
did not depend on NTP concentrations from 0.5 m
M
to

2m
M
Nonlinear least-square minimization was carried out
using the solver of Microsoft
EXCEL
software.
Reagents and peptides
ADP, ATP and GDP were from Roche-Boehringer
Mannheim. ATP contained 0.65% (w/w) ADP, and GTP
contained 2.4% (w/w) GDP and no detectable adenine
nucleotides (by HPLC analysis). Oligomycin, gramicidin
and FCCP were from Sigma. They were prepared as stock
solutions in methanol. All other chemicals were of analytical
grade from Sigma or Merck. Synthetic IF1 from Neosystem
(Strasbourg, France) as well as IF1 purified from yeast were
used, and gave identical results [39].
Results
Effect of protonmotive force on interaction
of IF
1
with ATPase
The effect of D
~
l
H
þ
on the IF
1
–enzyme interaction was
studied using SMPs coupled with substoichiometric amount

of oligomycin. Fig. 1 shows hydrolysis of ATP in the
presence of D
~
l
H
þ
generated by succinate oxidation (a
respiratory pathway which does not generate or consume
scalar protons). As expected, FCCP addition stimulates
Fig. 1. Time-course of ATP hydrolysis in coupled SMPs. The reaction
was monitored by scalar H
+
release as described in Materials and
methods. The reaction mixture contained 0.9 m
M
ATP (curves a–c), or
0.9 m
M
ATP and 2 m
M
malonate (curve d). Arrows indicate different
additions: SMP 9 lg (a–c) or 20 lg (d), FCCP 5 l
M
(a–d) and IF
1
0.85 l
M
(b–d). Labels near the curves give rates of hydrolysis in
lmolÆmin
)1

Æmg protein
)1
. Ordinate scales, indicated by vertical arrows
and given in l
M
H
+
, are different for a–b, c and d.
1964 M. Galkin et al. (Eur. J. Biochem. 271) Ó FEBS 2004
ATP hydrolysis (curve a), and IF1 addition inhibits FCCP-
uncoupled ATP hydrolysis (curve b). Curve c shows the
time-course of ATP hydrolysis in the presence of succinate-
dependent D
~
l
H
þ
, before and after addition of IF1 at the
same concentration as in curve b. Upon IF1 addition, fast
ATPase inhibition occurred. The most remarkable obser-
vation was made after addition of FCCP to these IF1-
inhibited SMP (third part of curve c). Quite unexpectedly,
FCCP addition resulted in a transient recovery of ATPase
activity, reaching 70–90% of the control activity, which is
given by the activity in the presence of FCCP in curves a–b.
This recovered ATPase activity decayed afterwards, at
approximately the same rate as after addition of IF1 to
SMP previously uncoupled with FCCP (compare terminal
parts of curves b and c).
In the experiment shown in Fig. 1 curve d, inhibition by

IF1 was studied using SMP in the presence of malonate,
which fully inhibited the respiratory chain (control not
shown). Addition of IF1 to SMP during ATP hydrolysis
resulted in fast inhibition of ATPase activity, as previously.
However, in contrast with respiring SMP, addition of
FCCP to IF1-inhibited material resulted in only poor
transient ATPase reactivation. This shows that the extent of
recovery of ATPase activity following uncoupler addition
strongly depends on the magnitude of the D
~
l
H
þ
,whichwas
generated here only by ATP hydrolysis and which presum-
ably became dramatically low after IF1 addition.
The fact that an almost complete inhibition of ATPase by
IF
1
in the presence of D
~
l
H
þ
is followed by an almost full
recovery of ATPase activity just after D
~
l
H
þ

collapse, cannot
be readily interpreted. It suggests that a special enzyme
state, formed in the presence of D
~
l
H
þ
from the IF1-inhibited
state and remaining ATPase-inactive as long as D
~
l
H
þ
is
maintained, immediately becomes active upon uncoupling.
After uncoupling, the reactivated form of the enzyme
deactivates again. The rate of deactivation is apparently the
same as that observed after adding IF1 to SMP previously
treated with FCCP (compare curves b and c, Fig. 1),
suggesting that it is also controlled by IF1 binding. To check
this hypothesis, we studied IF1-inhibition of coupled
ATPase and recovery of its activity using different IF1
concentrations.
Relationship between IF1 concentration and
uncoupler-induced ATPase reactivation
Fig. 2A shows the time course of ATP hydrolysis in coupled
SMP, with successive additions of IF1 (0.5 l
M
)andFCCP
separated by 2 min. Fig. 2B shows the time course of ATP

hydrolysis by coupled SMP to which IF1 was previously
added, as above, with focusing on the last step of reactivation
by FCCP. Curves 1 and 2 show FCCP-induced reactivation
of ATPase previously inhibited by IF1 2 l
M
and 0.5 l
M
,
respectively. The higher the IF1 concentration, the lower
FCCP-induced ATPase activity. However, it is difficult to
discriminate between the effects of IF1 concentration on the
initial ATPase activity and on the subsequent rate of decay.
To make the picture clearer, we have made new
experiments using GTP instead of ATP for two reasons:
(a) in contrast to ADP, GDP does not produce hysteretic
inhibition [33]; (b) more generally, GDP can accumulate to
significant amounts without slowing down the rate of
hydrolysis. Accordingly, coupled GTPase activity was per-
fectly stable for at least 5 min, unlike ATPase activity, and
subsequent addition of uncoupler caused twofold stimula-
tion of the activity which remained constant for at least
5 min; furthermore, the rate of IF1-dependent inhibition of
uncoupled GTP hydrolysis was not sensitive to GDP
accumulation (data not shown).
Figure 3A shows typical kinetics of GTP hydrolysis by
coupled SMP in the presence of succinate. IF1 at various
concentrations was added during GTP hydrolysis (curves 1–
2) and FCCP was added 2.5 min later (control experiments
revealed that the rate of coupled ATP hydrolysis no longer
varied between 2.5 and 4 min after IF1 addition). Curve 3

shows what happened when IF1 (at the same concentration
as in curve 2) was added after FCCP, and comparison of
curves 2 and 3 shows that at a low IF1 concentration, the
extent of GTPase recovery after FCCP addition is almost
100%. As in Fig. 2, the recovery decreases with IF1
concentration. This is more visible on Fig. 3B which focuses
on a short time range before and after FCCP addition and
shows how rates of GTP hydrolysis are computed. Rates
just before and after FCCP additions are plotted vs. IF1
concentration in Fig. 4A. These two rates obviously follow
different patterns: half-inhibition of the coupled activity is
reached at about 0.2 l
M
IF1 (curve 1), and half-inhibition
of the uncoupler-recovered activity at about 1 l
M
IF1
(curve 2). Curve 3 (dashed) shows what GTP hydrolysis rate
after FCCP addition should be if it obeyed the same pattern
as coupled GTP hydrolysis. Curve 4 (dashed) shows what
Fig. 2. IF1-dependent inhibition and FCCP-induced recovery of ATPase
activity in SMPs. The reaction was monitored as in Fig. 1. The reac-
tion mixture contained 1.4 m
M
ATP. Additions (SMP 60 lg, IF1,
FCCP 5 l
M
) are indicated by arrows. (A) ATPase inhibition by IF1
0.5 l
M

in the presence of D
~
l
H
þ
and after FCCP-induced recovery.
(B) ATPase inhibition after FCCP addition to SMP previously
inhibited with IF1 2 l
M
(curve 1) or 0.5 l
M
(curve 2), as in (A). Only
the final stage is shown.
Ó FEBS 2004 IF1-mediated transitions of F1-ATPase in yeast SMP (Eur. J. Biochem. 271) 1965
GTP hydrolysis after FCCP addition should be if it
was only due to a subpopulation of tightly coupled,
IF1-insensitive SMP (see Discussion). Kinetics of GTP
hydrolysis after FCCP addition were fitted to a mono-
exponential decay [39], and the resulting rate constants of
deactivation k
app
were plotted in Fig. 4B. k
app
is propor-
tional to IF1 concentration [39], which confirms that the
final decay of GTPase activity is due to IF1 rebinding.
Kinetics of ATPase reactivation in IF1-pretreated SMPs
To study ATPase reactivation further we used SMP
preincubated with MgATP in succinate-free medium, with
or without highly concentrated IF1 (50 l

M
). SMP were then
diluted 100-fold in the reaction medium containing succi-
nate and checked for ATP hydrolysis under different
conditions. This allowed the experiment to be started with
ATPase fully inhibited by IF1 in a reactivation medium
containing a limited concentration of IF1 (0.5 l
M
). Fig. 5A
indeed shows that in FCCP-containing medium, IF1-
pretreated SMP were initially fully inactive (trace 2),
compared to SMP preincubated without IF1 (trace 1).
When IF1-treated SMP were diluted in FCCP-free medium
containing succinate, ATPase activity was initially negligible
and was recovered mainly after FCCP addition (Fig. 5A,
trace 3). Fig. 5B (black squares) shows the extent of
Fig. 3. IF1-dependent inhibition and FCCP-induced recovery of
GTPase activity in SMPs. The reaction was monitored as in Figs 1
and 2. The reaction mixture contained 2 m
M
GTP. (A) GTP hydrolysis
initiated by addition of SMP, inhibited by IF1 at two different con-
centrations, and further reactivated with FCCP. Additions (SMP
18 lg, IF1, FCCP 5 l
M
) are indicated by arrows. Curves 1 and 2:
addition of IF1 1.25 l
M
and 0.125 l
M

, respectively, 2.5 min after
FCCP. Curve 3, control with IF1 0.125 l
M
added after FCCP. Only
one sample of the first part of the curves is shown, because it does not
change with subsequent additions. (B) Time course of GTP hydrolysis
just before and just after FCCP addition (extended scale), for three
differentIF1concentrations.Curves1,2,3:IF10.125 l
M
,0.25 l
M
and
1 l
M
, respectively. Straight lines were obtained from linear regression
using all the displayed data before FCCP additions, and the data of the
first 30 s, 20 s and 15 s, respectively, after FCCP addition. Their slopes
are proportional to GTPase activities.
Fig. 4. Rate of GTP hydrolysis and GTPase inhibition as a function of
IF1 concentration. Conditions as in Fig. 3. (A) Rates of GTP hydro-
lysis just before (s, curve 1) and just after (d, curve 2) FCCP addition
to coupled SMP, as a function of IF1 concentration. Rates were cal-
culated as shown in Fig. 3B. Curve 3 (dashed) was obtained by mul-
tiplying ordinates of curve 1 by the ratio between uncoupled and
coupled GTPase rates in the absence of IF1. It indicates expected
uncoupled activity if catalysed by the same enzyme form as coupled
activity. Curve 4 (dashed) was obtained by subtracting curve 1 to the
uncoupled activity without IF1. It gives expected activity due to a
hypothetical tight-coupled SMP subpopulation resistant to IF1 before
FCCP addition. Neither curve 3, nor curve 4 correctly fits the data (see

text for details). (B) Apparent rate constant of inhibition after FCCP
addition, vs. IF1 concentration.
1966 M. Galkin et al. (Eur. J. Biochem. 271) Ó FEBS 2004
recovery of ATPase activity as a function of the time
separating dilution of pretreated SMP and FCCP addition.
It is also shown that some recovery of ATPase activity was
actually observed before FCCP addition (white squares),
but it was weak, even though one multiplies its value by a
factor two (Fig. 5B, dashed curve) to take into account the
back pressure effect of D
~
l
H
þ
, which is about 50%. This
comparison confirms that ATPase activities measured
before and after FCCP are different by nature. Lastly,
black triangles in Fig. 5B show the ATPase activity
recovered in the presence of FCCP, present from the
beginning. It is about twice the activity recovered without
FCCP, as expected if the only difference between these two
modes of slow activation is the back-pressure effect exerted
by D
~
l
H
þ
. Anyway, in both cases, this background reacti-
vation, due to the high pH of the reaction medium [39] and
not related to membrane energization, remains well below

that triggered by D
~
l
H
þ
and further revealed by FCCP
addition.
Discussion
It is thought that unidirectional IF
1
binding to ATP
synthase is independent of the presence of D
~
l
H
þ
[17],
whereas its release depends on D
~
l
H
þ
[10–17]. Here, inhibi-
tion of coupled NTP hydrolysis by IF
1
, and the unexpected
reactivation of IF
1
-inhibited enzyme on uncoupler addition,
were measured in the same assay, in conditions where

consumption of NTP had little effect (ATP case) or no effect
(GTP case) in itself. This uncoupler-induced recovery of
ATPase activity is hardly compatible with the assumption of
only two states of the enzyme (active, without IF1 and
inactive, with IF
1
bound), which suggests that the proton-
motive force converts the IF1-inhibited ATPase into a form
which has a lower affinity for IF1, but which remains
inactive even after IF1 release, unless D
~
l
H
þ
is collapsed.
Before developing this idea, it is necessary to examine other
explanations. A possible interpretation of our data could be
that SMP preparation would be a heterogeneous mixture of
energized vesicles, always insensitive to IF1 unless FCCP is
added, and permanently deenergized vesicles, sensitive to
IF1. The recovery of ATPase activity after FCCP addition
should only be due to well-coupled vesicles. This does not
seem realistic for quantitative reasons. In Fig. 1 indeed,
the maximal uncoupled ATPase activity, representing in
the context of heterogeneity the uncoupled activity of the
whole preparation, is 3 lmolÆmin
)1
Æmg protein
)1
(curve a),

whereas the FCCP-induced ATPase recovery, believed
to be only due to well-coupled vesicles, is
2.2 lmolÆmin
)1
Æmg protein
)1
(curve c; this value, calculated
some seconds after FCCP addition, is probably underesti-
mated). The difference between these two activities
(0.8 lmolÆmin
)1
Æmg protein
)1
) would be the activity of
permanently uncoupled vesicles, the only ones to be
inhibited by IF1 added before the uncoupler. This activity
should be subtracted from the apparent coupled activity
(1.4 lmolÆmin
)1
Æmg protein
)1
, curve c) to obtain the true
coupled ATPase activity of the subpopulation of energized
vesicles. The resulting activity is expected to be resistant to
IF1, so ATPase activity after IF1 and before FCCP addition
should remain as high as 0.6 lmolÆmin
)1
Æmg protein
)1
.This

is not the case, the measured activity (curve c) is less than
0.05 lmolÆmin
)1
Æmg protein
)1
(in fact practically zero). In
other words, if a special SMP subpopulation is responsible
for the FCCP-induced recovery of activity after IF1
treatment, one expects IF1 to inhibit only the activity of
Fig. 5. ATP hydrolysis in SMPs preincubated with concentrated IF1. SMP (0.9 mgÆmL
)1
) were preincubated in standard succinate-free mixture in
presence of 50 l
M
IF
1
,2.3m
M
ATP and 8.3 m
M
MgCl
2
(SMP
IF1
). Additions in a 1-mL spectrophotometric cuvette (1.4 m
M
ATP, 5 l
M
FCCP,
9 lgSMP,100l

M
ADP) are indicated by arrows. (A) Time courses of ATPase activity of pretreated SMP after 100-fold dilution in the standard
reaction medium containing succinate. Curve 1, control; curves 2–4, SMP preincubated with IF
1
(SMP
IF1
); curve 2, FCCP added before SMP
IF1
;
curve 3, FCCP added 2 min after SMP
IF1
. Labels near curves 1 and 3 give rates of hydrolysis in lmolÆmin
)1
Æmg protein
)1
. (B) Dependence of the
uncoupler-induced ATPase activity (j) on the time separating dilution of SMP
IF1
in succinate-containing medium and addition of uncoupler;
100% corresponds to the control SMP (A, trace 1). (h), Coupled ATPase activity (no FCCP) as a function of the time following SMP
IF1
addition,
calculated from slopes of pH recordings, under conditions similar to those of the part of curve 3 (A) preceding FCCP addition; dashed curve (- - -),
double of the coupled ATPase activity, representing the expected activity after FCCP addition; (m), ATPase activity as a function of the time
following SMP
IF1
addition with FCCP initially present, calculated from the slope of curves like curve 2 in (A).
Ó FEBS 2004 IF1-mediated transitions of F1-ATPase in yeast SMP (Eur. J. Biochem. 271) 1967
permanently deenergized vesicles, and then to cause the
same drop of ATPase activity before and after FCCP

addition. This is far from being observed in Fig. 1, as
previously discussed, and also in Fig. 4A, which shows the
dependency of GTPase activity on IF1 concentration before
and after FCCP addition and clearly indicates that the two
activities do not follow parallel curves (curves 2 and 4 are
clearly distinct). More, if we consider a heterogeneous
preparation, it cannot reasonably consist of two discrete
populations, noncoupled and tightly coupled, where ATP-
ases are, respectively, inhibited and 100% resistant. It
should contain of course partially coupled vesicles, where
some inhibition by IF1 is expected. To get ATPase
inhibition by IF1, complete collapse of the protonmotive
force is not necessary provided that ATPase works, even
slowly, in the direction of ATP (GTP) hydrolysis. As a
consequence, if it was due to functional heterogeneity,
FCCP-induced GTPase activity should actually follow not
curve 4, but a curve located between curves 3 and 4, still
more remote from the experimental data. Therefore the
results cannot be explained simply by functional heterogen-
eity of SMP. Of course, this does not mean that SMP are
fully homogeneous, and functional homogeneity of SMP is
actually impossible to check. But heterogeneity is generally
associated with poor reproducibility, and the different SMP
preparations used in this work had a reproducible ATPase
activity in the presence of succinate, the stimulation factor
of ATPase activity by FCCP being practically constant
(100 ± 10%).
Finally, our data are consistent with the existence of the
following states of ATPase (or GTPase):
where E is the only active form of ATPase. During ATP

hydrolysis, IF1 can bind to the ATPase both in the
absence and in the presence of D
~
l
H
þ
. Only in the presence
of D
~
l
H
þ
, does the E*IF1 complex undergo energy-
dependent conversion to a latent form of the enzyme still
inactive in ATP hydrolysis (E ¼ IF1) but presenting a
lower affinity for IF1. Upon uncoupling (indicated by a
vertical arrow) the latent ATPase which is free of IF1
(E ¼) immediately converts, within the time resolution
limit, into the active ATPase (E) (uncoupler-induced
activity), which again undergoes inhibition by IF1. Due to
the presence of the uncoupler, this second inhibition
cannot be reversed anymore.
In the case of GTP hydrolysis, the dependency of the
uncoupler-induced ATPase on IF1 concentration allows
one to determine the affinity of the latent state (E ¼ in the
above scheme) of the ATPase for IF
1
:K
d
is %1 l

M
.This
value is much higher than the K
d
for the IF
1
–ATPase
interaction in the absence of D
~
l
H
þ
,whichis% 40 n
M
at
pH 8 under conditions similar to the present ones [39] and
is consistent with the classical energy-dependent release
of IF
1
.
A plausible mechanism for the IF1-transition to the
ATPase latent form can be proposed by focusing on the
release of ADP from one catalytic site during net ATP
hydrolysis (we will not consider the other sites; a complete
description of the enzyme should take into account the
increase of nucleotide occupancy induced by IF1 [43,44]).
The statements are the following:
The ADP-loaded site is successively closed, half-closed
and open [45]. D
~

l
H
þ
tends to reverse the closed to half-
closed transition (back pressure effect). When the ADP-
loaded site is closed, the enzyme has some probability to be
converted into the latent form. Under normal conditions,
this conversion is negligible because the steady state
concentration of the closed state is low. When IF1 is
bound, it is assumed to block, or to slow down, the
transition from half-closed to open conformation. In
the latter case, it fully blocks the next step. IF1 acts on the
considered catalytic site either directly, or indirectly, by
blocking ATP hydrolysis on another site [24]. In the absence
of D
~
l
H
þ
, the final state is a dead-end complex where IF1,
initially loosely bound, is now locked [31]. When IF1 is
bound and D
~
l
H
þ
present, the ADP-loaded catalytic site
remains essentially closed and is then converted into its
latent form, not represented in the above scheme. As in the
latent form the catalytic reaction is stopped upstream of

ADP release, IF1 remains loosely bound and can be
released if its concentration is low, which gives the IF1-free
latent form.
This outline of mechanism can explain the synergetic
effect of IF1 and D
~
l
H
þ
on the formation of the latent
form in the presence of MgATP. These three effectors
must be simultaneously present to stabilize the closed state
leading to the latent form. The proposed mechanism can
also explain the recovery of the active form after D
~
l
H
þ
collapse. The latent form indeed can be considered so
sensitive to the D
~
l
H
þ
back-pressure that ADP stays on
the catalytic site and the rate of ATP hydrolysis is
practically zero. However, when D
~
l
H

þ
is collapsed, ADP
release occurs at a rate which may be low with respect to
the time scale of the catalytic cycle (milliseconds), but
which remains faster than the experimental time response
(seconds). Once ADP is released, one gets again the active
form of ATPase.
The simplest model of D
~
l
H
þ
-induced activation of
mitochondrial ATPase involves two functional forms: an
inactive and an active IF1-free form. Early studies have
suggested the existence of an additional IF1-bearing form
active in ATP synthesis but practically inactive in ATP
hydrolysis [10,19]. The present data suggest that in the
presence of D
~
l
H
þ
, IF1 could act as a catalyst inducing the
transformation of ATP synthase from a fully active enzyme
to a latent form, unable to hydrolyse ATP, even after IF1
release. Further investigations of the mechanism of ATPase
regulation by D
~
l

H
þ
and IF1 should now take into account
1968 M. Galkin et al. (Eur. J. Biochem. 271) Ó FEBS 2004
this possible additional form of ATP synthase. This IF1-
dependent conversion of active ATPase into latent ATPase
resembles that induced by MgADP and D
~
l
H
þ
in bovine
heart SMP [32,34] and in Pseudomonas denitrificans [46].
Whether the presently described latent state of ATPase
could synthesize ATP under appropriate conditions or not
has not been established so far.
Acknowledgements
We thank Gwe
´
nae
¨
lle Moal-Raisin for her excellent technical help, and
Dr Sigalat for HPLC analysis of nucleotides and helpful suggestions.
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