Unexpectedly, in the presence of D~lHþ, IF1 was foundto promote the conversion of active F0F1–ATPase to a latent form which remains inactive in ATP hydrolysis even after IF1 release.. Cu
Trang 1Functional transitions of F0F1-ATPase mediated by the inhibitory
peptide IF1 in yeast coupled submitochondrial particles
Mikhail Galkin1,*, Rene´e Venard1, Jacques Vaillier2, Jean Velours2and Francis Haraux1
1
Service de Bioe´nerge´tique & CNRS-URA 2096, Gif-sur-Yvette, France;2Institut de Biochimie et Ge´ne´tique Cellulaires du CNRS, Bordeaux, France
The mechanism of inhibition of yeast F0F1-ATPase by its
naturally occurring protein inhibitor (IF1) was
investi-gated in submitochondrial particles by studying the
IF1-mediatedATPase inhibition in the presence andabsence
of a protonmotive force In the presence of protonmotive
force, IF1 added during net NTP hydrolysis almost
completely inhibitedNTPase 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-inhibitedF0F1-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 F0F1-ATPase back to the active form The rela-tionship between the different steps of the catalytic cycle, the mechanism of inhibition by IF1 andthe 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 carriedout by membrane-boundF0F1-ATPase
complex or ATP synthase [1] The extrinsic F1subcomplex
is composedof five types of subunits in the stoichiometry
a3b3cde Three fast-exchangeable nucleotide-binding sites,
presumably catalytic, andthree 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 F0subcomplex promotes proton
translocation, energetically coupledto ATP synthesis/
hydrolysis via a stalk connecting F0 andF1 F1 can be
biochemically separatedfrom F0andremains competent for
uncoupled ATP hydrolysis Numerous data, including
direct observations of the rotation of the central axis of
F0F1 relative to a3b3 crown during ATP hydrolysis [5,6],
indicate that F0F1is a rotary motor, with an asymmetrical
rotor composed, in mitochondria, of c, d and e subunits,
anchoredto a membranous decameric ring of c subunits
thought to compose a proton-driven turbine [7]
Mitochondrial F0F1is regulatedby a naturally occurring
inhibitor protein calledIF1 [8] IF1 is a small acid- and
heat-stable protein, which stoichiometrically binds to the F1 sector of ATP synthase andinhibits ATP hydrolysis [8–19] D~lHþ is believedto 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 MF1–IF1 complex drawn from radiocristallographic data showed IF1 bound
at the a/b interface, andinteracting weakly with c [24] The F0F1–IF1 interaction is modulated by a number of factors such as pH, ionic strength, D~lHþ andnucleotides [8–16,25–31] Interaction of F0F1–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~lHþ, nucleotide occupancy and enzyme turnover in the IF1-relatedregulatory processes However, it is well establishedthat ATPase turnover is necessary for IF1 binding [12], and a complex relationship was foundbetween ATP concentration andrate of IF1 binding to isolated bovine MF1subcomplex [31]
In this work, we have investigatedthe influence of D~lHþ
on IF1-mediatedinhibition, using yeast IF1 andcoupled submitochondrial particles (SMP) with ATP and GTP as substrates Unexpectedly, in the presence of D~lHþ, IF1 was foundto promote the conversion of active F0F1–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: francis.haraux@cea.fr
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.
(Received3 February 2003, revised18 March 2004,
accepted25 March 2004)
Trang 2specific microscopic states andcatalytic steps play an
explicit role in IF1 locking andin the interconversion
between active andlatent 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] anddiluted
to 5–8 mgÆmL)1in 10 mMTris/HCl, 100 lMEDTA pH 8.1
The suspension was saturatedwith argon for 2 min, then
sonicatedon ice using a W-225R tip sonicator (Ultrasonics
Inc.) Two 20 s cycles (output control 2; 60% duty cycle)
separatedby 30 s interval were applied Then the suspension
was centrifugedat 23 000 g for 20 min at 4°C The
supernatant containing SMP was centrifugedat 100 000 g
for 40 min at 4°C The pellet of SMP was suspended in
0.65M mannitol ATPase andATP synthase activities by
these SMP were stable for at least 1–2 days when stored at
room temperature SMP couldalso be rapidly frozen in
liquidnitrogen for future use Protein content was
deter-minedaccording 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) in a
medium containing (final concentrations) 0.65Mmannitol,
10 mMHepes, 5 mMpotassium phosphate pH 8.0, 100 lM
EDTA, 2 mMmalonate, anda substoichiometric amount
of oligomycin (0.1 lgÆmg)1protein) The suspension was
incubatedfor 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 hadthe following activities
(lmolÆmin)1Æmg protein)1): coupledATP hydrolysis, 1–1.5;
uncoupled ATP hydrolysis, 2–3; succinate-mediated ATP
synthesis, 0.3–0.5; coupledsuccinate oxidation, 0.3; coupled
NADH oxidation, 0.37; uncoupled NADH oxidation, 1.5
(respiratory control with NADH, 4)
ATP hydrolysis measurements
The reaction was monitoredcontinuously at 25°C in a
stirredcuvette as H+release [42], with phenol redas pH
indicator (A557) in 1 or 2 mL of the standard mixture
containing 0.65M mannitol, 10 mM phosphate pH 8.0,
10 mMsuccinate, 100 lMEDTA (potassium salts), 17 mM
KCl, 5 mMMgCl2, 60 lMPhenol red, and 1 mgÆmL)1BSA
(Mg)ATP and (Mg)GTP were added as indicated in the
figures NTP concentrations > 0.8 mM were practically
saturating Carbonyl-cyanide
p-(trifluoromethoxy)phenyl-hydrazone (FCCP; 5 lM) or 2.5 lgÆmL)1gramicidin D was
usedas an uncoupler The same results were obtainedwith
either uncoupler The different additions (nucleotides, IF1,
uncouplers) hadnegligible effects on the pH of the medium
containing no SMP or SMP treatedwith excess of
oligomycin First-order kinetics of inhibition by IF1 [39]
was fittedto the equation:
yðtÞ ¼ V1tþ ½ðV0 V1Þ=kapp ð1 ek t
appÞþ y0 Eqnð1Þ where y0and y(t) are A557at zero time andt time after IF1 addition, respectively, V0is the initial rate of A557change (at zero time), V1is the final rate of A557change (at infinite time), and kappis the apparent deactivation rate constant
kappdid not depend on NTP concentrations from 0.5 mMto
2 mMNonlinear least-square minimization was carriedout using the solver of MicrosoftEXCELsoftware
Reagents and peptides ADP, ATP andGDP were from Roche-Boehringer Mannheim ATP contained0.65% (w/w) ADP, andGTP contained2.4% (w/w) GDP andno detectable adenine nucleotides (by HPLC analysis) Oligomycin, gramicidin andFCCP were from Sigma They were preparedas 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 purifiedfrom yeast were used, and gave identical results [39]
Results Effect of protonmotive force on interaction
of IF1with ATPase The effect of D~lHþ on the IF1–enzyme interaction was studiedusing SMPs coupledwith substoichiometric amount
of oligomycin Fig 1 shows hydrolysis of ATP in the presence of D~lHþ generatedby 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 monitoredby 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 and2 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)1Æmg protein)1 Ordinate scales, indicated by vertical arrows andgiven in l H+, are different for a–b, c and d.
Trang 3ATP hydrolysis (curve a), and IF1 addition inhibits
FCCP-uncoupledATP hydrolysis (curve b) Curve c shows the
time-course of ATP hydrolysis in the presence of
succinate-dependent D~lHþ, 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-inhibitedSMP (thirdpart 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 recoveredATPase activity decayedafterwards, at
approximately the same rate as after addition of IF1 to
SMP previously uncoupledwith FCCP (compare terminal
parts of curves b andc)
In the experiment shown in Fig 1 curve d, inhibition by
IF1 was studied using SMP in the presence of malonate,
which fully inhibitedthe respiratory chain (control not
shown) Addition of IF1 to SMP during ATP hydrolysis
resultedin fast inhibition of ATPase activity, as previously
However, in contrast with respiring SMP, addition of
FCCP to IF1-inhibitedmaterial resultedin 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~lHþ, which was
generatedhere only by ATP hydrolysis andwhich
presum-ably became dramatically low after IF1 addition
The fact that an almost complete inhibition of ATPase by
IF1in the presence of D~lHþ is followedby an almost full
recovery of ATPase activity just after D~lHþcollapse, cannot
be readily interpreted It suggests that a special enzyme
state, formedin the presence of D~lHþfrom the IF1-inhibited
state andremaining ATPase-inactive as long as D~lHþ is
maintained, immediately becomes active upon uncoupling
After uncoupling, the reactivatedform of the enzyme
deactivates again The rate of deactivation is apparently the
same as that observed after adding IF1 to SMP previously
treatedwith FCCP (compare curves b andc, Fig 1),
suggesting that it is also controlledby IF1 binding To check
this hypothesis, we studied IF1-inhibition of coupled
ATPase andrecovery 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 lM) and FCCP
separatedby 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 and2 show FCCP-inducedreactivation
of ATPase previously inhibitedby IF1 2 lM and0.5 lM,
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 andon the subsequent rate of decay
To make the picture clearer, we have made new
experiments using GTP insteadof 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 remainedconstant for at least
5 min; furthermore, the rate of IF1-dependent inhibition of uncoupledGTP hydrolysis was not sensitive to GDP accumulation (data not shown)
Figure 3A shows typical kinetics of GTP hydrolysis by coupledSMP 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 revealedthat the rate of coupledATP hydrolysis no longer variedbetween 2.5 and4 min after IF1 addition) Curve 3 shows what happenedwhen IF1 (at the same concentration
as in curve 2) was added after FCCP, and comparison of curves 2 and3 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 reachedat about 0.2 lMIF1 (curve 1), andhalf-inhibition
of the uncoupler-recoveredactivity at about 1 lM 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 monitoredas in Fig 1 The reac-tion mixture contained1.4 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~ lHþ andafter FCCP-inducedrecovery (B) ATPase inhibition after FCCP addition to SMP previously inhibitedwith IF1 2 l M (curve 1) or 0.5 l M (curve 2), as in (A) Only the final stage is shown.
Trang 4GTP 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 kapp were plottedin Fig 4B kapp 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
preincubatedwith MgATP in succinate-free medium, with
or without highly concentratedIF1 (50 lM) SMP were then diluted 100-fold in the reaction medium containing succi-nate and checked for ATP hydrolysis under different conditions This allowedthe experiment to be startedwith ATPase fully inhibitedby IF1 in a reactivation medium containing a limitedconcentration of IF1 (0.5 lM) Fig 5A indeed shows that in FCCP-containing medium, IF1-pretreatedSMP were initially fully inactive (trace 2), comparedto SMP preincubatedwithout IF1 (trace 1) When IF1-treatedSMP were dilutedin 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 monitoredas in Figs 1
and2 The reaction mixture contained2 m M GTP (A) GTP hydrolysis
initiatedby addition of SMP, inhibitedby IF1 at two different
con-centrations, andfurther reactivatedwith FCCP Ad d itions (SMP
18 lg, IF1, FCCP 5 l M ) are indicatedby arrows Curves 1 and2:
addition of IF1 1.25 l M and0.125 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
d ifferent IF1 concentrations Curves 1, 2, 3: IF1 0.125 l M , 0.25 l M and
1 l M , respectively Straight lines were obtainedfrom linear regression
using all the displayed data before FCCP additions, and the data of the
first 30 s, 20 s and15 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) andjust after (d, curve 2) FCCP addition
to coupledSMP, as a function of IF1 concentration Rates were cal-culatedas shown in Fig 3B Curve 3 (dashed) was obtainedby mul-tiplying ordinates of curve 1 by the ratio between uncoupled and coupledGTPase rates in the absence of IF1 It ind icates expected uncoupledactivity if catalysedby the same enzyme form as coupled activity Curve 4 (dashed) was obtained by subtracting curve 1 to the uncoupledactivity without IF1 It gives expectedactivity due to a hypothetical tight-coupledSMP 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.
Trang 5recovery of ATPase activity as a function of the time
separating dilution of pretreatedSMP andFCCP 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~lHþ, which is about 50% This
comparison confirms that ATPase activities measured
before andafter FCCP are different by nature Lastly,
black triangles in Fig 5B show the ATPase activity
recoveredin the presence of FCCP, present from the
beginning It is about twice the activity recoveredwithout
FCCP, as expectedif the only difference between these two
modes of slow activation is the back-pressure effect exerted
by D~lHþ Anyway, in both cases, this
backgroundreacti-vation, due to the high pH of the reaction medium [39] and
not relatedto membrane energization, remains well below
that triggeredby D~lHþ andfurther revealedby FCCP
addition
Discussion
It is thought that unidirectional IF1 binding to ATP
synthase is independent of the presence of D~lHþ [17],
whereas its release depends on D~lHþ [10–17] Here,
inhibi-tion of coupledNTP hydrolysis by IF1, andthe unexpected
reactivation of IF1-inhibited enzyme on uncoupler addition,
were measuredin the same assay, in conditions where
consumption of NTP hadlittle 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 bound), which suggests that the
proton-motive force converts the IF1-inhibitedATPase into a form which has a lower affinity for IF1, but which remains inactive even after IF1 release, unless D~lHþ is collapsed Before developing this idea, it is necessary to examine other explanations A possible interpretation of our data could be that SMP preparation wouldbe a heterogeneous mixture of energizedvesicles, always insensitive to IF1 unless FCCP is added, and permanently deenergized vesicles, sensitive to IF1 The recovery of ATPase activity after FCCP addition shouldonly be due to well-coupledvesicles This does not seem realistic for quantitative reasons In Fig 1 indeed, the maximal uncoupledATPase activity, representing in the context of heterogeneity the uncoupledactivity of the whole preparation, is 3 lmolÆmin)1Æmg protein)1(curve a), whereas the FCCP-induced ATPase recovery, believed
to be only due to well-coupled vesicles, is 2.2 lmolÆmin)1Æmg 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)1Æmg protein)1) wouldbe the activity of permanently uncoupledvesicles, the only ones to be inhibited by IF1 added before the uncoupler This activity shouldbe subtractedfrom the apparent coupledactivity (1.4 lmolÆmin)1Æmg protein)1, curve c) to obtain the true coupledATPase activity of the subpopulation of energized vesicles The resulting activity is expectedto be resistant to IF1, so ATPase activity after IF1 and before FCCP addition shouldremain as high as 0.6 lmolÆmin)1Æmg protein)1 This
is not the case, the measuredactivity (curve c) is less than 0.05 lmolÆmin)1Æmg 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 preincubatedin standardsuccinate-free mixture in presence of 50 l M IF 1 , 2.3 m M ATP and8.3 m M MgCl 2 (SMP IF1 ) Additions in a 1-mL spectrophotometric cuvette (1.4 m M ATP, 5 l M FCCP,
9 lg SMP, 100 l M ADP) are indicatedby arrows (A) Time courses of ATPase activity of pretreatedSMP after 100-folddilution 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 and3 give rates of hydrolysis in lmolÆmin)1Æmg 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), CoupledATPase 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 coupledATPase activity, representing the expectedactivity 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).
Trang 6permanently deenergizedvesicles, andthen 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, noncoupledandtightly coupled, where
ATP-ases are, respectively, inhibitedand100% resistant It
shouldcontain of course partially coupledvesicles, 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 locatedbetween curves 3 and4, still
more remote from the experimental data Therefore the
results cannot be explainedsimply by functional
heterogen-eity of SMP Of course, this does not mean that SMP are
fully homogeneous, andfunctional homogeneity of SMP is
actually impossible to check But heterogeneity is generally
associatedwith poor reproducibility, andthe different SMP
preparations usedin this work hada 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 andin the presence of D~lHþ Only in the presence
of D~lHþ, does the E*IF1 complex undergo
energy-dependent conversion to a latent form of the enzyme still
inactive in ATP hydrolysis (E¼ IF1) but presenting a
lower affinity for IF1 Upon uncoupling (indicated by a
vertical arrow) the latent ATPase which is free of IF1
(E¼) 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 secondinhibition
cannot be reversedanymore
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 (E¼ in the
above scheme) of the ATPase for IF1:Kd is 1 lM This
value is much higher than the Kd for the IF1–ATPase
interaction in the absence of D~lHþ, which is 40 nM at
pH 8 under conditions similar to the present ones [39] and
is consistent with the classical energy-dependent release
of IF1
A plausible mechanism for the IF1-transition to the
ATPase latent form can be proposedby 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 andopen [45] D~lHþ tends to reverse the closed to half-closedtransition (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 closedstate is low When IF1 is bound, it is assumed to block, or to slow down, the transition from half-closedto 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~lHþ, the final state is a dead-end complex where IF1, initially loosely bound, is now locked [31] When IF1 is boundandD~lHþ present, the ADP-loaded catalytic site remains essentially closedandis then convertedinto its latent form, not representedin the above scheme As in the latent form the catalytic reaction is stoppedupstream of ADP release, IF1 remains loosely boundandcan be releasedif its concentration is low, which gives the IF1-free latent form
This outline of mechanism can explain the synergetic effect of IF1 and D~lHþ on the formation of the latent form in the presence of MgATP These three effectors must be simultaneously present to stabilize the closedstate leading to the latent form The proposed mechanism can also explain the recovery of the active form after D~lHþ
collapse The latent form indeed can be considered so sensitive to the D~lHþ back-pressure that ADP stays on the catalytic site andthe rate of ATP hydrolysis is practically zero However, when D~lHþ 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~lHþ-induced activation of mitochondrial ATPase involves two functional forms: an inactive andan 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~lHþ, IF1 couldact 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 andIF1 shouldnow take into account
Trang 7this possible additional form of ATP synthase This
IF1-dependent conversion of active ATPase into latent ATPase
resembles that induced by MgADP and D~lHþ in bovine
heart SMP [32,34] andin Pseudomonas denitrificans [46]
Whether the presently described latent state of ATPase
could synthesize ATP under appropriate conditions or not
has not been establishedso 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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