F0F1-ATP synthase is absent from about half of chromatophore

15 Pages·2001·0.57 MB·English

Biochimica et Biophysica Acta 1506 (2001) 189^203
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Coupling of proton £ow to ATP synthesis in Rhodobacter capsulatus:
F F -ATP synthase is absent from about half of chromatophores
0 1
Boris A. Feniouk a;b, Dmitry A. Cherepanov a;c, Wolfgang Junge a,
Armen Y. Mulkidjanian a;b;*
a Division of Biophysics, Faculty of Biology/Chemistry, University of Osnabru«ck, D-49069 Osnabru«ck, Germany
b A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia
c Institute of Electrochemistry, Russian Academy of Sciences, Leninskii prosp. 31, 117071 Moscow, Russia
Received 8 May 2001; received in revised form 10 August 2001; accepted 13 August 2001
Abstract
F F -ATPsynthase(H(cid:135)-ATPsynthase,F F )utilizesthetransmembraneprotonmotiveforcetocatalyzetheformationof
0 1 0 1
ATP from ADP and inorganic phosphate (P). Structurally the enzyme consists of a membrane-embedded proton-
i
translocatingF portionandaprotrudinghydrophilicF partthatcatalyzesthesynthesisofATP.Inphotosyntheticpurple
0 1
bacteria a single turnover of the photosynthetic reaction centers (driven by a short saturating flash of light) generates
protonmotive force that is sufficiently large to drive ATP synthesis. Using isolated chromatophore vesicles of Rhodobacter
capsulatus, we monitored the flash induced ATP synthesis (by chemoluminescence of luciferin/luciferase) in parallel to the
transmembranechargetransferthroughF F (byfollowingthedecayofelectrochromicbandshiftsofintrinsiccarotenoids).
0 1
WiththehelpofspecificinhibitorsofF (efrapeptin)andofF (venturicidin),wedecomposedthekineticsofthetotalproton
1 0
flowthroughF F into(i)thosecoupledtotheATPsynthesisand(ii)thede-coupledprotonescapethroughF .Takingthe
0 1 0
coupled proton flow, we calculated the H(cid:135)/ATP ratio; it was found to be 3.3(cid:254)0.6 at a large driving force (after one
saturating flash of light) but to increase up to 5.1(cid:254)0.9 at a smaller driving force (after a half-saturating flash). From the
results obtained, we conclude that our routine chromatophore preparations contained three subsets of chromatophore
vesicles. Chromatophores with coupled F F dominated in fresh material. Freezing/thawing or pre-illumination in the
0 1
absence of ADP and P led to an increase in the fraction of chromatophores with at least one de-coupled F (F ). The
i 0 1
disclosed fraction of chromatophores that lacked proton-conducting F (F ) (approx. 40% of the total amount) remained
0 1
constant upon these treatments. (cid:223) 2001 Elsevier Science B.V. All rights reserved.
Keywords: Proton transfer; ATP synthesis; Electrochromism; Membrane potential,; Rhodobacter capsulatus
Abbreviations: DCCD, N,NP-dicyclohexylcarbodiimide; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; P(cid:135), oxidized
stateoftheprimaryelectrondonorinthephotosyntheticreactioncenter;FWHM,full-widthhalf-maximum;RC,photosyntheticreaction
center of purple phototrophic bacteria; DMF, dimethylferrocene; BSA, bovine serum albumin; P, inorganic phosphate
i
* Corresponding author. Fax: [phone].
E-mail address: [email protected] (A.Y. Mulkidjanian).
0005-2728/01/$ ^ see front matter (cid:223) 2001 Elsevier Science B.V. All rights reserved.
PII: S0005-2728(01)00213-4
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190 B.A. Feniouk et al./Biochimica et Biophysica Acta 1506 (2001) 189^203
1. Introduction the electrochromism of membrane carotenoids
[15,21,22] and by pH-indicators [23^25]. The £ash-
F F -ATP synthase (H(cid:135)-ATP synthase, F F ) is induced ATP yield was measured by luciferin-lucifer-
0 1 0 1
the key enzyme in energy transduction. It either syn- ase [17,26]. In agreement with previous observations
thesizes ATP using the transmembrane electrochem- for Rhodobacter sphaeroides [24], we discriminated
icalpotentialdi¡erenceoftheproton(vW~ ),orvice two components in the proton £ow related to
H(cid:135)
versa, it generates vW~ by ATP hydrolysis (see F F : one component was induced by simultaneous
H(cid:135) 0 1
[1^3] for recent reviews). Structurally the enzyme addition of ADP and inorganic phosphate (P) and
i
consists of two multisubunit portions. The mem- was inhibited by efrapeptin, a potent F inhibitor.
1
brane F portion (a complex of a b c subunits The second component was present even in the ab-
0 1 2 10(cid:255)14
in bacteria) translocates protons. The hydrophilic F sence of ADP and P, and was insensitive to efrapep-
1 i
portion (a complex of K L Q N O subunits) protrudes tin, but was inhibited by speci¢c inhibitors of F
3 3 1 1 1 0
about 100 A(cid:238) from the membrane and catalyzes the (venturicidin, DCCD or oligomycin) [20].
synthesis/hydrolysis of ATP. These portions are con- It is noteworthy that the apparent kinetics of pro-
nected by two ‘stalks’ that are formed by the cen- ton escape through F F might be in£uenced by the
0 1
trally located QO-complex and by the eccentric b non-uniformity of the sample that, in the particular
2
dimer (for the latest structural reviews see [4^7]). case of chromatophore vesicles, could have a dual
ATPase is a rotary machine: evidence for the ATP origin. First, the coupling of proton transfer to the
hydrolysis-coupled movement of the Q O c com- ATP synthesis can vary from a tight one to a loose
1 1 10(cid:255)14
plex (the rotor) inside the K L ring (that together one leading to a di¡erence in the kinetics of proton
3 3
with a b N forms the stator) were obtained by using transfer (see [27] for the respective studies on chlo-
1 2 1
cleavable crosslinks [8,9], by polarized photochemis- roplast thylakoids). The second type of possible in-
try [10], and by microscopic observations of the ro- homogeneity is speci¢c to membrane vesicles that are
tation of a £uorescent actin ¢lament attached to the as smallas chromatophores. Their typical diameter is
Q subunit of F [11]. Mechanism for torque genera- only about 60 nm [28], much smaller than those of
1
tion by proton £ow and for the coupling of proton submitochondrial particles or chloroplast thylakoids,
transfer to ATP synthesis has been proposed, e.g., in so that each chromatophore might contain just a
[2,12,13]. few, if any, ATP synthases. Then, at low numbers
Both elements, F and F , are likely rotary step- of ATP synthases per chromatophore, the kinetics of
0 1
pers. The mismatch between the 3-fold pseudosym- proton escape through F F might essentially vary
0 1
metry in F and 10^14-fold symmetry in the c-oligo- from vesicle to vesicle and cause a kinetic inhomoge-
1
mer ring implies some kind of elasticity inherent to neity of the type that is absent in larger membrane
the enzyme coupling machinery, as has been sug- vesicles (see [29] for a general discussion of the small-
gested in [2,14]. ness-due e¡ects in chromatophores).
In photosynthetic systems an excitation by a short To determine the impact of the chromatophore
£ash of light leads to the prompt generation of mem- non-uniformity on the coupling, we monitored here
brane protonmotive force. Thereby the membrane the proton £ow through F F in parallel to the ATP
0 1
vW~ -dependent enzymes, including the ATP syn- synthesis in variously treated samples. Thereby we
H(cid:135)
thase, can be studied in a light-triggered mode [15^ determined the properties and kinetic features of
19]. Recently we applied this approach to native the coupled, ‘useful’ proton £ow, leading to the syn-
membrane vesicles (chromatophores) isolated from thesis of ATP, as well as of the de-coupled, ‘futile’
a purple phototrophic bacterium Rhodobacter capsu- proton transfer. For the coupled proton transfer, we
latus [20]. Thereby we combined the technique of the determined that H(cid:135)/ATP ratio was about 3.3(cid:254)0.6 at
complete tracking of proton £ow developed for chlo- large driving force but increased up to 5.1(cid:254)0.9 at
roplast ATP synthase [19], with the quantitative smaller driving force. We also found out that about
monitoring of the £ash-induced ATP release. The 40% of chromatophores in our preparations did not
proton transfer across the membrane was traced by contain proton-conducting F (F ) at all.
0 1
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B.A. Feniouk et al./Biochimica et Biophysica Acta 1506 (2001) 189^203 191
2. Materials and methods concentration of 0.3^0.7 mM and used on the same
day or after storage at +4‡C overnight.
2.1. Cell growth and chromatophore preparation
2.3. Spectrophotometric measurements
Cells of Rb. capsulatus (wild-type, strain B10) were
grown photoheterotrophically on malate as a carbon The kinetic £ash-spectrophotometer was con-
source at +30‡C [30]. The light intensity was 18 structed according to [36]. Monitoring light was pro-
W/m2. Cells were harvested at the logarithmic vided by a 200-W halogen lamp, and was heat-¢l-
growth phase, washed twice with 30 mM HEPES^ tered (KG 2 ¢lter, Schott, Mainz). The desired
KOH (pH 7.4), 5 mM MgCl , 0.5 mM DTT, 50 mM wavelengths were cut out by appropriate interference
2
KCl, 10% sucrose and resuspended in the same pH- ¢lters (Schott). A shutter was placed in front of the
bu¡er. A few £akes of DNase were added. The cells cuvette to eliminate the actinic e¡ect of the monitor-
were disrupted by sonication (Branson Soni¢er B15, ing beam between the measurements. The delay time
4^5 exposures for 15 s with 1 min incubation on ice between the opening of the shutter and the actinic
inbetween), andcentrifuged(20000Ug,20min,4‡C) £ash was 400 ms. Changes in transmitted light inten-
to remove large cell fragments. The pellet was sus- sity(vI) weremonitoredby aphotomultiplier(Thorn
pended in the same pH-bu¡er and re-sonicated as EMI, 9801B) shielded with two blue ¢lters (BG 39,
above; the re-sonicated suspension was centrifuged Schott) from the actinic £ash. The DC-output from
again (20000Ug, 20 min, 4‡C). Supernatants were the photomultiplier was pre-set to 1 V (load resistor:
collected and centrifuged (180000Ug, 90 min, 10 k6) by varying the current on the photomultiplier
+4‡C). The pellet was resuspended in 30 mM power supply. The photomultiplier output was con-
HEPES^KOH (pH 7.4), 5 mM MgCl , 0.5 mM nected to the positive input of a di¡erence ampli¢er
2
DTT, 50 mM KCl, 20% sucrose. It contained chro- (TektronixAM502).Beforean actinic£ashwas¢red,
matophores at bacteriochlorophyll concentration of the signal was sampled and held by a home-made
0.3^0.7 mM. Chromatophores were stored at 380‡C ampli¢er (designed and constructed by N. Spreckel-
until use. In some experiments ‘freshly prepared’ meier) connected to the negative input of the di¡er-
chromatophores were used immediately after the ence ampli¢er. The di¡erence signal was ampli¢ed
preparation or, otherwise, were kept at +4‡C over- 100-fold, digitized and stored by an averaging oscil-
night and used the next morning. The concentration loscope (Nicolet, Pro 10). The analogue bandshift
of bacteriochlorophyll in the samples was determined width was 3 kHz and the digital time-per-address
spectrophotometrically in the acetone/methanol ex- was 200 Ws. The optical path was 1 cm, both for
tract at 772 nm according to [31]. The amount of the exciting and for the measuring beam. The ¢nal
functionally active RCs was estimated from the ex- concentration of bacteriochlorophyll in the cuvette
tent of £ash-induced absorption changes at 603 nm was 8^12 WM.
as in [32]. Repetitive signals measured at 0.08 Hz were aver-
aged. The dark adaptation time of 12 s between
2.2. Preparation of chromatophores stripped of F £ashes was chosen to allow the decay of transmem-
1
brane electrical potential di¡erence (vi). This time
Chromatophores were depleted of F by EDTA- was still short enough to prevent the de-activation
1
treatment [33^35]. Chromatophores from frozen of the F F -ATP synthase (the expected lifetime of
0 1
stock were thawed and diluted 25-fold with 1 mM the vi-activated state of the F F -ATP synthase
0 1
EDTA (pH 8) in daylight. The suspension was soni- under conditions of our experiments was 30^70 s
cated5timesfor20swith1-mininterval(onice)and [37]).
centrifuged at 180000Ug, 90 min, +4‡C. The pellet Electrochromic carotenoid bandshifts at 522 nm
was resuspended in a medium containing 20 mM were used to monitor vi[21,22,38,39]. Notably, up
HEPES^KOH (pH 7.4), 5 mM MgCl , 0.5 mM to 15% of the £ash-induced response was due not to
2
DTT, 50 mM KCl to yield a bacteriochlorophyll the delocalized vi, but to local electrochromic band-
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192 B.A. Feniouk et al./Biochimica et Biophysica Acta 1506 (2001) 189^203
shift (presumably in response to the P(cid:135) formation) 3. Results
that was insensitive to uncouplers.
Saturating actinic £ashes were provided by a xe- 3.1. Two types of proton conduction via
non £ash lamp (FWHM approx. 10 Ws, the £ash F F -ATP synthase
0 1
energy density on the cuvette was 12 mJ/cm2). A
red optical ¢lter (RG 780, Schott) covered the £ash. Fig. 1A shows typical absorption transients at 522
Excitation by the xenon lamp £ash gave 8^11% nm in a suspension of Rb. capsulatus chromato-
greater signals than a laser pulse (duration 6100 phores in response to a saturating actinic £ash.
ns), indicating double turnover in about 10% of Chromatophores were stored frozen at 380‡C in a
RCs, in agreement with the recent report on the medium containing 50% glycerol, and were thawed
presence of a 3-Ws component in the kinetics of immediately before the measurement. The transients
the Q reduction in chromatophores of Rb. sphae- at 522 nm were due to the electrochromic bandshift
B
roides and Rb. capsulatus [40]. Because the deviation of the membrane carotenoids and re£ected the
from single turnover conditions was not pro- changes in the transmembrane electrical potential
nounced, we used the more convenient xenon £ash di¡erence (vi). A minor, here unspeci¢ed contribu-
in our experiments, correcting the calculations by tion from local electrochromic bandshifts (up to 15%
10%. of the total response, see Materials and Methods)
was also present at this wavelength.
2.4. ATP synthesis A £ash of light caused a fast biphasic increase in
vidue to the sequential operation of the photosyn-
ATP synthesis was monitored by chemolumines- thetic reaction center (RC) and the cytochrome-bc
1
cence of luciferin^luciferase [26] in the same sample complex (ubiquinol:cytochrome c oxidoreductase).
2
that was used for measurements of absorption Electrogenic reactions in the RC included (i) electron
changes. The photomultiplier was shielded against transfer from the light-excited bacteriochlorophyll
actinic light by a stack of three blue ¢lters (BG 39, dimer P to the ubiquinone electron acceptors on
Schott). Aliquots of 0.5 mM freshly prepared ATP the other side of the membrane (V200 ps); (ii) the
solution were added to up to 2 WM ¢nal concentra- reduction of the oxidized P(cid:135) by water soluble cyto-
tion for calibration; linear calibration curves were chrome c (3^150 Ws); and (iii) the trapping of one
obtained in the range from 0.05 to 2 WM ATP. The proton, on average, by the secondary quinone Q
B
traces were corrected for linear drift due to the ATP (100 Ws under our experimental conditions). Corre-
hydrolysis (2^120 nM/min). spondingly, the fast, here unresolved component of
vigeneration re£ected the transfer of one electron
2.5. Incubation medium per RC across the whole chromatophore membrane.
The slower component of the viincrease at milli-
The samples were suspended in a medium which second time scale re£ected the electrogenic oxidation
contained 50 mM KCl, 0.3% bovine serum albumin of ubiquinol to ubiquinone by the cytochrome-bc
1
(BSA,wasusedasapH-bu¡er),5mMMgCl ,2mM complex (see [18,41] for reviews on electrogenic reac-
2
succinate/2 mM fumarate (as a redox bu¡er), 5 or 10 tions in chromatophores).
WM DMF (as a redox mediator) and 2 mM KCN (to The following decay of the light-induced voltage
prevent the oxidation of the redox-bu¡er by the ter- transientsat 522 nm was markedly accelerated by the
minal oxidase). In some experiments 2 mM addition of ADP and inorganic phosphate (P, see
i
K Fe[CN] was present instead of succinate^fuma- Fig. 1A, trace 2), in agreement with previous obser-
4 6
rate redox bu¡ering system. When ATP synthesis vations with chromatophores of Rb. sphaeroides
was measured, 8 Wg/ml luciferase and 0.2 mM luci- [15,16]. The e¡ect of ADP+P was reversed by efra-
i
ferin were present in the medium. peptin, a peptide inhibitor that binds between sub-
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B.A. Feniouk et al./Biochimica et Biophysica Acta 1506 (2001) 189^203 193
Fig. 1. E¡ect of freezing and storage on the components of proton transport through ATP synthase. Changes in absorption at 522
nm re£ect changes in vi, which, in turn, are linearly proportional to the net transmembrane charge transfer. Measuring medium con-
tained 2 mM K HPO , 2 mM K [Fe(CN) ], 5 WM DMF, 2 mM KCN, 5 mM MgCl , 50 mM KCl, 0.3% BSA, pH 7.9. Arrows mark
2 4 4 6 2
actinic £ashes. Same trace numeration is used in all panels: 1, no additions; 2, ADP to 400 WM; 3, ADP to 400 WM, efrapeptin to
200 nM; 4, ADP to 400 WM, efrapeptin to 200 nM, venturicidin to 200 nM. Di¡erence trace +/3 efrapeptin and +/3 venturicidin are
marked 3-2 and 4-3, respectively. (A) Chromatophores stored 1 week at 380‡C in a pH-bu¡er containing 50% glycerol. (B) Freshly
prepared chromatophores. (C) Di¡erence traces (+/3 efrapeptin) for A and B samples scaled to compare the kinetics (scaling factor:
2.4). (D) Chromatophores as in B but stored in the measuring medium overnight at +4‡C. (E) EDTA-treated, F -stripped chromato-
1
phores. (F) Di¡erence traces (+/3 venturicidin) for A, D and E samples scaled to compare the kinetics (scaling factors: 3.23 (A), 1.48
(D)).
unitQandtheK L hexamerinF [42](Fig.1A,trace bition by the F -inhibitors was observed at the fol-
3 3 1 0
3). The inhibitor decreased the decay rate of the lowing incubation times: from 20 to 90 min for
light-induced voltage transients approximately to DCCD (depending on pH), 15 min for oligomycin,
the level that was observed in the absence of ADP and 1^2 min for venturicidin. Because of its e⁄-
and P. A further slowing of the voltage decay was ciency, we mostly used venturicidin throughout this
i
achieved when the inhibitors of proton conduction work.
through the F -portion of ATP synthase, namely Previously, by combining hydrophilic and amphi-
0
venturicidin, oligomycin, or DCCD [43,44], were philic pH-indicators, we showed that the membrane
added over efrapeptin (Fig. 1A, trace 4 compared discharge is due to the vW~ -driven proton escape
H(cid:135)
totrace3).Theformerinhibitorsdecreasedthedecay from the chromatophore lumen through F F [20].
0 1
rate also when they were added in the absence of TheADP+P-inducedvoltagedischargethatwassen-
i
ADP or phosphate (see e.g. [20]). The maximal inhi- sitive to efrapeptin could be then attributed provi-
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194 B.A. Feniouk et al./Biochimica et Biophysica Acta 1506 (2001) 189^203
sionally to the ‘useful’ proton transfer through the and to efrapeptin, but was sensitive to venturicidin
F F -ATP synthase, coupled to the ATP synthesis, (Fig. 1E).
0 1
whereas the surplus venturicidin-sensitive discharge The traces in Fig. 1F represent the scaled di¡er-
to some kind of a non-coupled, ‘futile’ proton £ow. encetracesbetweenthevoltagetransientsobtainedin
We found out that the relative extent of the the presence of venturicidin and those obtained in
ADP+P-induced component of proton £ow de- the presence of efrapeptin, with frozen/thawed,
i
pended on the choice of cryoprotector for the chro- aged, and EDTA-stripped chromatophores. In the
matophore storage. When chromatophores were rou- case of freshly prepared chromatophores this di¡er-
tinely frozen in a pH-bu¡er containing 50% (v/v) ence was negligible. In all the preparations the ‘futile’
glycerol, the ADP+P-induced and the ‘futile’ com- proton transfer kinetics had the same time constant
i
ponents of proton transfer were approximately equal of approximately 10 ms.
in magnitude (Fig. 1A). Among other cryoprotectors Thepatternoftheoverallvidecayinthepresence
tested (DMSO, ethylene glycol, ¢coll, sucrose, and of venturicidin was more or less the same in all ex-
glycerol) sucrose (25%) was the most e⁄cient in periments and was independent of the presence of
maintaining the ADP+P-induced proton £ow. Nota- ADP, phosphate, efrapeptin or chromatophore stor-
i
bly, if the chromatophores were not frozen at all, the age conditions (see Fig. 1A,B,D,E).
‘futile’ proton transfer was very small if not negli-
gible (Fig. 1B). 3.2. ATP synthesis in various chromatophore
The time course of the ADP+P-induced proton preparations
i
discharge was obtained by subtraction of kinetic
traces measured in the presence of ADP+P from We determined the amount of ATP that was syn-
i
those recorded in the presence of efrapeptin. The thesized after a £ash excitation in di¡erently treated
respective di¡erence traces obtained both from Fig. chromatophore preparations. The extent of lumines-
1A (frozen/thawed sample) and Fig. 1B (fresh sam- cence transients of luciferin^luciferase was propor-
ple) are plotted in Fig. 1C after scaling their ampli- tional to the yield of ATP per £ash, but its time
tudes. Although the absolute extents di¡ered by a course did not truly re£ect the kinetics of ATP syn-
factor of two (see the ¢gure legend), the discharge thesis, because of a V100-ms lag in the luminescence
characteristic times were equal, of about 20 ms (com- increase [45]. The respective ATP-yields are given in
pare the two data sets in Fig. 1C). the boxes in Fig. 1A,B. The amount of ATP synthe-
Cold storage of chromatophores at +4‡C over- sized was maximal in freshly prepared, non-frozen
night, eventually, abolished the sensitivity of the chromatophores (Fig. 1B). It was less in chromato-
voltage discharge both to ADP+P and to efrapeptin. phores that were frozen and kept at 380‡C after the
i
The sensitivity to venturicidin, however, remained preparation (Fig. 1A). In chromatophores that were
(see Fig. 1D). Apparently, upon freezing/thawing stored overnight at 4‡C (Fig. 1D) or in the F -de-
1
or ageing of chromatophore preparation, the extent pleted chromatophores (Fig. 1E), the ATP yield after
of the ‘futile’ component of proton transport a single £ash, if any, was below the resolution of our
through ATP synthase increased at the expense of luminometer. In a series of similar experiments we
the ADP+P-inducible, efrapeptin-sensitive compo- found out that the relative yield of ATP was propor-
i
nent. tional to the relative extent of the ADP+P-induced
i
We compared the kinetics of proton transfer and efrapeptin-sensitive, ‘useful’ charge transfer.
through the ATP synthase in chromatophores that
were stored overnight at 4‡C (Fig. 1D) with those 3.3. The e¡ect of pre-illumination in the absence of
in chromatophores that were depleted in F by the ADP and P on proton transfer and
1 i
EDTA treatment plus sonication (Fig. 1E). Accord- ATP synthesis
ing to this well established technique (see [33^35]),
such a treatment yields approximately 85% F deple- The pre-illumination of chromatophores in the ab-
1
tion. In the chromatophores treated in such way the sence of ADP led to an increase in the proton £ux
proton£owwas insensitivebothtoADP+P addition through F F as it followed from the acceleration of
i 0 1
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B.A. Feniouk et al./Biochimica et Biophysica Acta 1506 (2001) 189^203 195
videcay (Fig. 2A). Applying speci¢c inhibitors of more pronounced when the post-illumination incu-
F and F (efrapeptin and venturicidin, respectively) bation was prolonged for 30 min: the ATP synthetic
1 0
to such pre-illuminated chromatophores, we revealed activity was restored up to 70% of the control level
that the videcay acceleration was due to the in- (Fig. 2B).
crease in the ‘futile’ component of proton transfer
at the expense of the coupled, i.e., ADP+P-induced 3.4. Dependence of the ATP synthesis on the
i
and efrapeptin-sensitive, component. Pre-illumina- £ash intensity
tion by red light for 20 s was su⁄cient to almost
totally abolish the ADP+P-induced component (see We studied how the coupled, ADP+P-sensitive
i i
traces 1 and 2 in Fig. 2A). proton transfer and the ATP yield depended on the
When ADP was added to the pre-illuminated sam- transmembrane voltage. To vary the latter, we de-
ple, a gradual recovery of the ADP+P-induced, efra- creased the intensity of the actinic £ash by mounting
i
peptin-sensitive component was observed. When neutral density optical ¢lters on it. Fig. 3 shows that
efrapeptin was added 30 min after ADP, the decrease when the actinic £ash was attenuated so that vi
in the rate of videcay was pronounced, although it generation dropped about 2-fold, the ‘useful’ proton
had not reached the level of the initial control trace transfer through ATP synthase decreased by a factor
(compare traces 3, 4 and 1 in Fig. 2A). However, if of V2.4, whereas the drop in ATP yield was, on
efrapeptin was added immediately after the trace average, more than 4-fold. Table 1 summarizes the
with ADP was recorded, no signi¢cant slowing of data from several experiments.
the videcay was observed, so that the pattern re-
sembled those in Fig. 1D (not documented). 3.5. Proton transfer via F after two subsequent
0
ATP synthesis, as measured by luciferin^luciferase £ashes of light
luminescence, was also reduced to about 35% of the
control, when the sample was pre-illuminated by red As could be seen in Fig. 1C, the kinetics of proton
light before ADP was added (Fig. 2B). In this case, discharge sensitive to ADP+P and to efrapeptin had
i
however, there was always a dead time of about a time constant of about 20 ms [20]. Then one would
5 min after ADP addition that was necessary for expectthatvidecayedalmosttozeroinapprox.100
the sample equilibration before the measurement. ms. Still viremained signi¢cant even 200 ms after
We believe that the partial recovery of the enzyme the £ash in the presence of ADP and phosphate, i.e.,
during the 5-min incubation in the presence of under ATP synthesis conditions (Fig. 4A). By anal-
ADP+P accounted for the notable ATP yield in ogy with plant thylakoids [46], we had previously
i
the pre-illuminated sample. The recovery was even attributed this phenomenon to the vithreshold of
Table 1
Yield of ATP and the amount of protons transferred through ATP synthase in dependence on £ash intensity
Expt. Fullflashvi Fullflashvi FullflashvC R
RC R (cid:136) Efra R (cid:136) ATP ATP
no. Weakflashvi protons Weakflashvi ATP WeakflashvC R
RC Efra ATP protons
1 2.44 2.94 4.80 1.63
2 2.35 2.72 3.72 1.37
3 2.27 2.42 3.43 1.42
4 2.32 2.70 4.69 1.74
5 2.59 3.71 4.32 1.36
6 2.18 2.38 4.96 2.08
Average2.36(cid:254)0.14 2.81(cid:254)0.49 4.32(cid:254)0.62 1.60(cid:254)0.28
Full £ash vi and Weak £ash vi denote the extent of the fast vigeneration component attributed to charge separation in the
RC RC
reaction centers after a saturating and an attenuated £ash, respectively. Full £ash vi and Weak £ash vi denote the extent of
Efra Efra
the charge transfer through ATP synthase that is sensitive to efrapeptin; their ratio R re£ects a relative decrease in this transfer
protons
after £ash attenuation. Full £ash vC and Weak £ash vC denote the increase in ATP concentration; their ratio R re£ects
ATP ATP ATP
relative decrease in ATP yield after £ash attenuation.
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196 B.A. Feniouk et al./Biochimica et Biophysica Acta 1506 (2001) 189^203
Fig. 2. E¡ect of the far-red pre-illumination on proton transfer and ATP synthesis. Arrows indicate light £ashes. (A) Flash-induced
absorption changes at 522 nm. Traces were consequently recorded in the same cuvette. 1, control trace; 2, 10 min after illumination
of the sample for 20 s with far-red light; 3, immediately after ADP was added to 30 WM; 4, after 30 min dark incubation with ADP
followed by the addition of efrapeptin (250 nM); 5, after venturicidin (200 nM) was added. Note the y-axis break. (B) ATP synthesis
as measured 5 min and 45 min after a far-red pre-illumination followed by an addition of ADP (30 WM) as compared with a non-illu-
minated sample. Measuring medium contained 2 mM K HPO , 2 mM K [Fe(CN) ], 5 WM DMF, 2 mM KCN, 5 mM MgCl ,
2 4 4 6 2
50 mM KCl, 0.3% BSA, pH 7.9; 8 Wg/ml luciferase and 0.2 mM luciferin were present in B.
Fig. 3. Impact of the actinic £ash attenuation on the ATP synthesis and proton transfer as measured in the same sample. Measuring
medium contained 2 mM K HPO , 2 mM K [Fe(CN) ], 5 WM DMF, 2 mM KCN, 5 mM MgCl , 50 mM KCl, 0.3% BSA, 30 WM
2 4 4 6 2
ADP, 8 Wg/ml luciferase, 0.2 mM luciferin, pH 7.9. Flashes are indicated by arrows. (A) Electrochromic carotenoid bandshift measure-
ments. Solid curves (1 and 2), saturating actinic £ash, dotted curves (3 and 4), attenuated actinic £ash. 1 and 3, no inhibitors; 2 and
4, +400 nM efrapeptin. (B) ATP synthesis in response to an actinic £ash (monitored by luciferin^luciferase as indicated in Section 2.
1, saturating £ash; 2, attenuated £ash. The respective ATP yields are given in boxes.
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B.A. Feniouk et al./Biochimica et Biophysica Acta 1506 (2001) 189^203 197
Fig. 4. Assessment of the F (F )-lacking chromatophore fraction from the voltage transients generated by two saturating £ashes of
0 1
light ¢red at 240 ms interval. Arrows indicate actinic £ashes. (A) Freshly prepared, coupled chromatophores. Trace 1, ADP 30 WM,
2 mM phosphate; trace 2, same as 1, with 200 nM venturicidin added. (B) Reconstruction of kinetics of proton transfer in the F F -
0 1
containing chromatophore population. Trace 1 could be presented as a sum of contributions from the chromatophores lacking the
ATP synthase (3F F trace=trace 2 multiplied by 0.41, see details in Section 4) and from chromatophores containing the ATP syn-
0 1
thase (+F F trace=trace 1 minus 3F F trace). (C) Chromatophores stripped of F . Trace 1, no additions; trace 2, venturicidin to
0 1 0 1 1
200 nM. (D) Reconstruction of kinetics of proton transfer in the F -containing chromatophore population. Trace 1 could be presented
0
as a sum of traces for chromatophores lacking F (3F trace=trace 2 multiplied by 0.42, see details in Section 4) and for chromato-
0 0
phores containing F (+F =trace 1 minus 3F trace). Dashed horizontal lines represent the estimation of the threshold vilevel for
0 0 0
the proton transport through the coupled (B) and de-coupled (D) enzyme, respectively. Medium contained 2 mM succinate, 2 mM fu-
marate, 5 WM DMF, 2 mM KCN, 5 mM MgCl , 50 mM KCl, 2 mM K HPO , 0.3% BSA, pH 8.0.
2 2 4
ATP synthesis below which no coupled proton trans- connected system with well over 104 F F per thyla-
0 1
fer could occur [20]. koid network with total membrane area of 20^200
We have noted, however, that when two actinic Wm2 [47]). The situation is quite di¡erent in the case
£ashes at 240 ms interval were ¢red (Fig. 4A), the ofmuchsmallerchromatophoreswithaveragevesicle
vilevel was substantially greater 200 ms after the surface area of 0.011 Wm2. It was conceivable that a
second £ash than 200 ms after the ¢rst one. This fraction of chromatophores totally lacked proton-
di¡erence could be hardly described in the terms of conducting F F or F complexes. To check this pos-
0 1 0
a vithreshold, since the latter is expected to be the sibility, we gave two £ashes to the EDTA-treated,
same after the ¢rst and the second £ashes. F -depleted chromatophores (Fig. 4C,D). The vi
1
A chloroplast thylakoid forms a large electrically threshold for proton transfer through the ‘bare’ F
0
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198 B.A. Feniouk et al./Biochimica et Biophysica Acta 1506 (2001) 189^203
in the F -stripped chromatophores is expected to be The decrease in the ATP yield per £ash was propor-
1
much lower than in the case of the untreated, tional to the decrease in the relative extent of the
coupled ones [14]. Therefore no remarkable contri- ADP+P-induced, efrapeptin-sensitive component of
i
bution from the threshold vito the overall £ash- the proton £ow through F F . Correspondingly, this
0 1
induced voltage kinetics could be expected. Fig. 4C component of proton £ow could be identi¢ed as the
shows that the fast, 10-ms component of videcay, catalytic, useful proton £ow coupled to the ATP syn-
attributable to proton £ux through F , vanished thesis. The efrapeptin-insensitive but venturicidin-
0
while considerable viwas still present. This residual sensitive proton £ow was apparently not coupled
viwas again larger after the second £ash. This ¢nd- with the ATP synthesis and could be attributed to
ing could be taken as an evidence for the presence of the enzyme fraction that lost either the coupling be-
a chromatophore sub-population that totally lacked tween F and F in the ATP synthase or the F part
0 1 1
proton-conducting F . proper (as with chromatophores that were sonicated
0
From the data in Fig. 4 a rough upper estimate for in the presence of EDTA). Freezing/thawing, ageing
the relative fraction of such F (F )-lacking1 chroma- and/or pre-illumination increased the fraction of the
0 1
tophores could be obtained by comparing the elec- de-coupled chromatophores in the absence of ADP.
trochromic bandshift extents at ts200 ms in the
presence and in the absence of venturicidin. Appar- 4.2. Estimation of the H(cid:135)/ATP ratio. Dependence of
ently, the ratio between viextent at ts200 ms in the H(cid:135)/ATP ratio on the driving force
the absence of venturicidin and the corresponding
viextent in the presence of inhibitor should corre- A single chromatophore vesicle can be approxi-
spond roughly to the fraction of the F -lacking chro- mated by a spherical capacitor with a capacitance
0
matophores. It follows from Fig. 4C that up to 50% of 4.4U10317 F [28,48]. The transmembrane voltage
of the F -stripped chromatophores seemed to lack is proportional to the amount of charges transferred
1
proton-conducting F . This estimate is quite rough, across the membrane. As the calibration of caroten-
0
as the absorbance transients at 522 nm always con- oid electrochromic changes in millivolts by di¡usion
tained minor contribution from local electrochromic potentials (K(cid:135)/valinomycin) or with other methods
bandshifts, as was mentioned above. A more precise of vimeasurement, e.g., permeate ion distribution,
estimate of the F F -lacking chromatophore fraction gave ambiguous results ([21,22,38], see also above),
0 1
is given in Section 4. we made no attempt to calibrate vichanges in this
work. Instead, we assessed the amount of charges
that were translocated by F F by using the charge
0 1
4. Discussion transfer by the RCs as a reference. On average, 1.1
charges were transferred across the membrane by
4.1. Di¡erent types of proton conduction via each RC in response to our 10 Ws £ash (see Section
F F -ATP synthase 2). We assumed that the extent of the very fast, here
0 1
unresolved, increase in vi(see Fig. 3B) was equiv-
We monitored the £ash-induced proton transfer alent to the translocation of 1.1 charge per reaction
through the F F -ATP synthase of Rb. capsulatus center (1.1 eq/mol RC).
0 1
and the concomitant ATP production with variously We assumed that only the efrapeptin-sensitive
treated chromatophore preparations. The greatest charge transfer through a functional ATP synthase
ATP yield was found in freshly prepared chromato- was coupled to the ATP synthesis (see above). The
phores; it decreased by 55% in freeze-stored (380‡C) extent of the efrapeptin-sensitive component(see Fig.
material and was about zero in the aged material. 3B and the corresponding comments in the text) was
calibrated in charges transferred per reaction center
by comparison with the extent of voltage transients
corresponding to the charge separation in the RC.
1 Hereafter we de¢ne for simplicity a proton conducting ATP
Both the concentration of RCs in the sample and
synthasemoietyasF (F )independently,whetheritiscoupledor
0 1
not. the amount of the ATP synthesized could be rather
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