(Received for publication, February 4, 1997, and in revised form, March 25, 1997)
From the Institut für Biochemie der Pflanzen,
Heinrich-Heine-Universität Düsseldorf,
Universitätsstraße 1, D-40225 Düsseldorf, Federal Republic
of Germany and § Laboratoire de Biochimie
végétale, Université de Neuchâtel,
CH-2007 Neuchâtel, Switzerland
The kinetics of thiol modulation of the
chloroplast H+-ATPase
(CF0CF1) in membrana were analyzed
by employing thioredoxins that were kept reduced by 0.1 mM
dithiothreitol. The kinetics of thiol modulation depend on the extent
of the proton gradient. The process is an exponential function of the
thioredoxin concentration and reaction time and can be described by an
irreversible second order reaction. The results indicate that the
formation of the complex between thioredoxin and
CF0CF1 is slow compared with the subsequent
reduction step. Furthermore we have compared the efficiencies of the
Escherichia coli thioredoxin Trx and the two chloroplast thioredoxins Tr-m and Tr-f. The second order rate constants are 0.057 (Tr-f), 0.024 (Trx), and 0.010 s1
µM
1 (Tr-m) suggesting that Tr-f rather than
Tr-m is the physiological reductant for the chloroplast ATPase. The
often employed artificial reductant dithiothreitol exhibits a second
order rate constant in thiol modulation of 1.02·10
6
s
1 µM
1.
The H+-translocating ATPase of chloroplasts (CF0CF1,1 chloroplast ATP synthase) is a latent enzyme. Its physiological activation requires a transmembrane electrochemical proton potential difference (1-4). Hence, the proton gradient in addition to its role as the driving force of phosphorylation is a factor that controls CF0CF1 activity. The obvious physiological meaning of this control mechanism is the suppression of unproductive ATP hydrolysis under conditions that would energetically allow this reaction, i.e. at low proton gradients (low light or dark) and high phosphate potentials.
A superimposed regulatory device is the so-called thiol modulation of
CF0CF1. The structural basis for thiol
modulation is a sequence motif of nine amino acids comprising two
cysteines in the subunit of CF1 (5). This segment is
present in higher plants (6) and green algae (7) but not in
cyanobacteria (8-10) or in diatoms (11) suggesting that thiol
modulation is an acquisition of the chlorophyll a + b plants only. In the demodulated (oxidized) state the two
cysteines form a disulfide bond whereas the modulated state is obtained
by reduction of this disulfide bridge. In vitro reduction
can be achieved by dithiothreitol or other dithiols, but the natural
reductant is a reduced thioredoxin. In chloroplasts at least two
different thioredoxins occur, thioredoxin-m (Tr-m) and thioredoxin-f
(Tr-f) (12). The former is thought to be involved in light/dark
regulation of the chloroplast NADP-specific malate dehydrogenase, and
the latter is responsible for the light/dark regulation of fructose
bisphosphatase and other Calvin cycle enzymes (13, 14). The
thioredoxins are reduced via ferredoxin and ferredoxin-thioredoxin
reductase (15) by electrons from the photosynthetic electron transport
chain. In most of the experiments carried out so far, however, thiol
modulation of CF0CF1 was conducted with the
artificial reductant dithiothreitol, and in a few studies Escherichia coli thioredoxin (Trx) was used (16, 17). Little information is known about the action of the naturally occurring chloroplast thioredoxins on CF0CF1
(18-20).
Thiol modulation requires illumination of the chloroplasts to allow
reduction of the disulfide bridge. Apparently, the regulatory segment of the subunit, which is hidden in the dark, becomes accessible as a consequence of
µH+-induced
CF0CF1 activation (16, 21). Decay of the proton
gradient in the dark leads to deactivation of the ATP synthase. The
most significant difference between the reduced and oxidized active states concerns the velocity of deactivation. While the oxidized form
is immediately deactivated upon relaxation of the gradient, deactivation of thiol-modulated CF0CF1 takes
several minutes. For this reason only chloroplasts with thiol-modulated
CF0CF1 are capable of hydrolyzing added ATP
after transition from light to dark (22, 23).
Deactivation of the modulated enzyme may proceed with or without reoxidation of the dithiol group (16). Most likely the natural actual oxidant is the oxidized form of thioredoxin. Hence at least four CF0CF1 forms may be discerned: the oxidized inactive (Eiox) and active (Eaox) enzyme, and the reduced inactive (Eired) and active (Eared) enzyme. Due to the lower deactivation rate, the equilibrium of active to inactive enzyme is shifted toward lower proton gradients when CF0CF1 is in the reduced form (24). Fast deactivation of the modulated enzyme at light-dark transition is achieved by micromolar concentrations of ADP (25, 26) accompanied with tight binding of the nucleotide molecule to one of the three catalytic sites (27).
In the present paper the interaction of CF0CF1 with thioredoxin is analyzed kinetically, and the thioredoxin specificity for thiol modulation is investigated. The efficiencies can be expressed by rate constants for the binding of the different thioredoxins.
Chloroplast thylakoids were isolated from spinach leaves as described in Ref. 28. The reaction medium contained 25 mM Tricine buffer, pH 8.0, 50 mM KCl, 5 mM MgCl2, 50 µM phenazine methosulfate, 2 mM phosphoenolpyruvate, 20 units/ml pyruvate kinase, 50 nM valinomycin (to cancel the electrical potential difference); the total volume was 2.5 ml. Further additions (dithiothreitol and thioredoxin) are indicated in the legends. The concentration of thylakoids was equivalent to 25 µg of chlorophyll/ml.
Recombinant spinach chloroplast thioredoxins were produced as described
in Ref. 29. All experiments were conducted at 20 °C with clamped
pH (30), which was measured by the 9-aminoacridine fluorescence
quench (31) and monitored as described in Ref. 32. The concentration of
9-aminoacridine was 5 µM. The calibration of the
fluorescence signal was done as in Refs. 33 and 34. The activity of
reduced CF0CF1 was measured by the initial
ATP-hydrolyzing activity at
pH 0 following illumination (35). Since
the portion of oxidized active enzyme is immediately discharged at the
time of transition to
pH 0 but the reduced enzyme retains activity, the resulting ATP-hydrolyzing activity is a measure of the reduced active form only.
Usually the thylakoids were preilluminated for 30 s to
achieve a steady pH. Then thioredoxin + 0.1 mM
dithiothreitol was added while illumination was continued. It was
ascertained that 0.1 mM dithiothreitol was sufficient to
reduce the thioredoxins completely at all concentrations employed. The
insignificant ATPase activity induced by this low concentration of
dithiothreitol was always subtracted. After different times of
illumination, the light was switched off and a mix consisting of 1 mM [
-32P]ATP and 0.5 µM
nigericin (final concentrations) was injected simultaneously. The
initial rate of ATP-hydrolyzing activity was ascertained from the
contents of released [32P]Pi in samples taken
after 3, 6, 9, and 12 s that were deproteinized by 0.5 M HClO4 and analyzed as described in Ref. 36.
Rates have been corrected for isotope dilution (less than 5%) that was
caused by the ATP-regenerating pyruvate kinase system.
The combined reversible activation/thiol modulation process may be described by Scheme 1.
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The kinetic measurements of thiol modulation are based on the
suppositions that follow. 1) The ATP-hydrolyzing activity at pH 0 reflects the amount of active and reduced
CF0CF1 since only an active enzyme is able to
catalyze ATP hydrolysis and only the reduced enzyme retains its
activity after transition to
pH 0. 2) Thiol modulation requires the
enzyme to be in its activated oxidized state.
The reduction step is practically irreversible under the employed
conditions; all enzyme molecules should become reduced at infinite
reaction time independent of the extent of the proton gradient and the
thioredoxin concentration. On the other hand, the enzyme activity
should depend on the pH to indicate the equilibrium between the
reduced active and the reduced inactive form. For a general kinetic
analysis we used commercially available Trx from E. coli. In
Fig. 1, time courses of thiol modulation are shown at 3 µM Trx and three different extents of membrane
energization. The final enzyme activities reflecting the equilibrium
Eared
Eired increase with
increasing light intensity. At
pH 2.7 about 50% of the maximal
activity is obtained. By acid-base transition Junesch and Gräber
(24) found half-maximal activity of the reduced enzyme at
pH 2.2. At
pH 2.7 they found about 50% of the maximal phosphorylation rate and
concluded that in case the enzyme is in the thiol-modulated form,
phosphorylation is limited by the catalytic reaction whereas the
activity of the ATP synthase is the limiting factor for phosphorylation
when the enzyme is oxidized. More recent results, however, have shown
that the "activating protons" cannot be discerned kinetically and
that the activating protons show the same cooperativity as the
"catalytic protons" (37). Hence activation may be a step of the
catalytic cycle, and the two processes should have the same
pH
profile. The apparent difference of the profiles may be due to the fact
that the two processes, which have been measured under rather different
experimental conditions, are affected differently by factors like ADP
or phosphate concentrations (33).
The initial rates of thiol modulation likewise depend on the employed
pH (Fig. 1). According to the reaction scheme the rate should be
dependent on the concentration of active oxidized enzyme molecules
present at the beginning of thiol modulation, i.e. on the
equilibrium Eiox
Eaox established by
preillumination. Accordingly, while the steady-state activities in Fig.
1 represent the activation equilibrium of the reduced form of
CF0CF1, the initial rates of thiol modulation represent the activation equilibrium of the oxidized form. Compared with the reduced CF0CF1 the activation profile
of the oxidized enzyme is shifted toward higher
pH values (24). The
initial rates and the final levels of thiol modulation likewise depend differently on
pH in the expected manner (Table
I).
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According to the results of Junesch and Gräber (24) both
activation equilibria should be completely on the side of the active
forms at pH
4. To create clear-cut conditions for the action
of thioredoxin, we employed illumination at saturating light intensity
to achieve a saturating proton gradient. Saturation was ascertained by
the fact that the activity of the oxidized enzyme could not be
increased by a further increase of
pH. At light saturation the
extent of the gradient is well above 4
pH units, but in this range
where the 9-aminoacridine calibration curve shows a progressive
deflection from linearity, the
pH could not be precisely
determined.
Fig. 2 shows at light saturation time courses of thiol
modulation with 0.5 and 3 µM Trx. As expected, the curves
show different initial slopes depending on the Trx concentration.
Whereas the initial rates are proportional to the concentration of
thioredoxin both curves level off at the same final activity. This
result permits the conclusions that at saturating pH 1) thiol
modulation (at least up to 3 µM Trx) is limited by the
velocity of Trx binding, 2) the reduction of
CF0CF1 is practically unidirectional, and 3)
the equilibrium Eared
Eired is far on the left
side. This context may be described by Scheme 2.
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(Eq. 1) |
Equation 1 predicts that the degree of thiol modulation is an
exponential function of both the reaction time and the
thioredoxin concentration. In an experiment shown in Fig.
3 the degree of thiol modulation as measured by the
ATPase activity is plotted as a function of the thioredoxin
concentration at three different reaction times. The curves were again
fitted according to Equation 1 and yielded the rate constants 0.026 s1 µM
1 (at 10 s), 0.021 s
1 µM
1 (at 30 s), and
0.020 s
1 µM
1 (at 120 s
reaction time), respectively. The mean value including standard
deviation of k for Trx from all experiments was 0.024 ± 0.003 s
1 µM
1.
Futhermore the efficiencies of the chloroplast thioredoxins Tr-m and
Tr-f in thiol modulation are compared with Trx and dithiothreitol. Concentration dependences at 2 min modulation time indicated that under
all conditions the same final activity is attained (not shown). In Fig.
4 ATPase activities are shown after a reaction time of
10 s in the presence of different concentrations of Tr-f, Trx, and
Tr-m. The rate constants calculated from the best fits are summarized
in Table II. The results indicate that among the three
thioredoxins Tr-f is most efficient followed by Trx and Tr-m. The rate
constant for dithiothreitol is at least 4 orders of magnitude lower
(Table II).
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Thiol modulation of CF0CF1 in
membrana requires illumination of the thylakoids and a dithiol
reductant to reduce the disulfide bond in subunit . Originally this
complex interplay was expressed by the term "light-triggered
ATPase" (22, 39). Now it is clear that light is necessary for
CF0CF1 activation and that it is the transmembrane proton gradient that is essential (1). As a consequence of energization a series of conformational changes occur in
CF0CF1. Changes in the region of the nucleotide
binding sites located in the
-
subunits lead to the release of
tightly bound adenine nucleotides (1, 2, 28),
subunit is available
for an antibody (40), and Lys-109 of the
subunit becomes accessible to chemical modification by pyridoxal 5
-phosphate (41). Likewise the
so-called "light site" of the
subunit, a cysteine residue in
position 89, becomes accessible to modification by
o-phenylenedimaleimide (42). Similarly the target sequence
for thiol modulation, the disulfide bridge formed between Cys-199 and
Cys-205 in the
subunit, is exposed. This domain, which is hidden in
the inactive enzyme, becomes available to tryptic cleavage upon
activation (43) and accessible to reduction by dithiol reductants. At
full CF0CF1 activation the pure kinetics of
reduction can be measured, and a second order rate constant can be
determined. The rate constant depends of course on the nature of the
reductant and is much higher for the thioredoxins than for
dithiothreitol.
In the basic work of Mills et al. (20) it was shown that Tr-f is active in unmasking the ATPase in thylakoids in the light. The result reported here suggests that Tr-f (rather than Tr-m) is indeed the natural reductant of CF0CF1 in the chloroplast. Tr-m is even less effective than Trx from E. coli. To our knowledge this is the first clear comparative kinetic study of thiol modulation of CF0CF1 in membrana with the natural chloroplast thioredoxins. Galmiche et al. (19) compared the effects of Tr-m, Trx, and human thioredoxin on the activation of isolated CF1 and found no differences between the former two, but the human thioredoxin was 10 times less effective.
In fact the differences in efficiency between Tr-f, Trx, and Tr-m are
apparent at short reaction times. This is due to the fact that
reduction of is virtually an irreversible reaction provided that
the thioredoxins are kept reduced by excess dithiothreitol. Theoretically the process includes two successive reactions, reversible binding of thioredoxin to CF0CF1 followed by
the reduction step itself. The formation of an intermolecular complex
between isolated CF1 and Trx was measured by Dann and
McCarty (16) by using fluorescent probes. They found dissociation
constants in the range of 1 µM. Nevertheless the total
process including reduction can be described by a single irreversible
second order process suggesting that the irreversible reduction step is
fast compared with thioredoxin association/dissociation. Accordingly
the efficiency may be expressed by the second order rate constant for
the association of thioredoxin with CF1. Mathematical
simulations of models assuming the reduction step as the rate-limiting
reaction could not well explain our experimental data.
Compared with dithiothreitol all thioredoxins are by a factor of at
least 104 more efficient suggesting that the specific
structure or orientation of the dithiol group in the thioredoxins bound
to CF0CF1 is essential for high reactivity. The
sequences of Tr-f and Tr-m on one hand and Tr-f and Trx on the other
hand contain about 30% identical amino acids, but the highest homology
is found between Tr-m and Trx (46% identical positions). Accordingly
the reactivities in thiol modulation are not well substantiated by the
overall primary structures. As the three thioredoxins have the same
sequence motif in the region of the reactive dithiol group (WCGPCK),
the reactivity might be based on specific amino acid domains located on
the protein surface. The three-dimensional structure of Trx from
E. coli shows a central twisted sheet composed of five
strands surrounded by four
helices. The domain containing the
reactive cysteines Cys-32 and Cys-35 is in helix
2.
Eklund et al. (44) proposed that the hydrophobic flat area
around the reactive cysteines may form contact with the target protein.
The differences in efficiency may be referred to as slight differences
in the conformation of this contact area. Recent crystallographic
analyses of the chloroplast thioredoxins confirm that they have the
same general architectures as the other analyzed
thioredoxins.2 However, due to their
sequences the two chloroplast proteins show quite different surface
structures. The surface area of Tr-f around the accessible active site
cysteine is not as flat and hydrophobic as that of Trx but quite
structured and surrounded by positive and negative charges. These
charges are probably instrumental for the proper orientation during the
protein-protein interaction. The corresponding surface area of Tr-m
resembles much more the one of Trx, except for one boundary area. The
fact that Tr-m is less efficient in thiol modulation than Trx may be
due to some topological difference between these on the whole rather
similar proteins. It might also be due to the replacement in the
boundary area of Tr-m of three hydrophobic residues present in Tr-f and Trx by three charged residues: V86(R)/A87(K)/A88(E) (notation of
E. coli-Trx; the corresponding amino acids of Tr-m are in
parentheses). These charges may reduce the binding of Tr-m to the
coupling factor and therefore be responsible for its lower efficiency
compared with Tr-f and Trx.