A point mutation in the ATP synthase of
Rhodobacter capsulatus
results in differential contributions of DpH and Du in driving
the ATP synthesis reaction
Paola Turina and B. Andrea Melandri
Department of Biology, Laboratory of Biochemistry and Biophysics, University of Bologna, Italy
The interface between the c-subunit oligomer and the
a subunit i n the F
0
sector of the ATP synthase is believed to
form the core o f the rotating motor powered by the protonic
flow. Besides the essential cAsp61 and aArg210 residues
(Escherichia coli numbering), a few other residues at this
interface, although nonessential, show a h igh degree of
conservation, among these aGlu219. The homologous resi-
due aGlu210 in the ATP s ynthase o f t he photosynthetic
bacterium Rhodobacter capsulatus has been substituted by a
lysine. Inner membranes prepared from the mutant strain
showed approximately half of the ATP synthesis activity
when driven both by light and by a cid-base transitions. As
estimated with the ACMA assay, proton pumping rates in
the i nner membranes were also reduced to a similar extent in
the mutant. The most striking impairment of ATP synthesis
in the mutant, a decrease as low as 12 times as compared to
the wild-type, w as observed in the absence of a transmem-
brane e lectrical m embrane potential (Du)atlowtrans-
membrane pH difference ( DpH). Therefore, the mutation
seems t o affect both the mechanism responsible for coupling
F
1
with proton translocation by F

1
consists of five types of subunits in stoichiometry a
3
b
3
cde
and F
0
consists of three types of subunits in stoichiometry
ab
2
c
9)12
.Thec subunit monomers span the membrane as a
hairpin of two a helices [6] and are arranged in a oligomer in
the form of a ring (see, for example, the crystallographic
evidence in [7]). Subunit a most likely consists of five
transmembrane helices [8–10], the fourth of which has been
shown by extensive cross-linking analysis to pack against
the second transmembrane segment of subunit c [11]. The
fourth and fifth transmembrane helices, residues 206–271,
house the most conserved regions of the subunit.
In view of the ATP-driven rotation of the c-and
e-subunit shaft within the a
3
b
3
subunit barrel in F
1
,itis

cantly higher than that of either of the single mutation
strains [25], and their close position in the proposed
topological models [8–10]. Although t hese residues were
shown to be nonessential by extensive mutagenic analysis
Correspondence to B. A. Melandri, Laboratory of Biochemistry and
Biophysics, Department of Biology, University of Bologna, Via
Irnerio, 42, I-40126 Bologna, Italy. Fax: + 39 051 242576,
Tel.: + 39 051 2091293, E-mail:
Abbreviations: GTA, gene transfer agent; Bchl, bacteriochlorophyll;
ACMA, 9-amino-6-chloro-2-methoxyacridine; RC, photosynthetic
reaction center; Á
~
ll
H
þ
, transmembrane difference of electrochemical
potential of protons; Du, bulk-to-bulk transmembrane electrical
potential difference; Dw, surface electrical potential difference.
Enzyme: ATP synthase (EC 3.6.3.14).
Note: a website is available at />(Received 12 November 2001, revised 21 February 2002, accepted 21
February 2002)
Eur. J. Biochem. 269, 1984–1992 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02843.x
[25], their impo rtant functional role is indicated both by
their high degree o f conservation a nd by the d eleterious
effects of their mutations on the E. coli ATP syn thase.
In this work, the photosynthetic bacterium Rhodobacter
capsulatus has been used as a convenient system for
genetic manipulation and for investigating catalytic prop-
erties of the ATP synthase, as a variety of functional
studies can be carried out in its well-coupled inner

containing malate as a carbon source [30]; kanamycin and
tetracycline were added at 25 and 2 lgÆmL
)1
, respectively.
Cultures were illuminated by two opposite panels e ach
carrying nine 100-W i ncandescent light bulbs; excessive
warming was prevented by water cooling. The mutant and
wild-type strain we re grown in p arallel and cells were
harvested at D
600
¼ 1.2–1.4. Intra-cytoplasmic mem-
branes (chromatophores) were prepared according to the
method described previously [30], r esuspended in 50 m
M
glycyl-glycine/NaOH, 5 m
M
MgCl
2
, pH 7.5, rapidly
frozen as small d roplets in liquid n itrogen, and stored a t
)80 °C.
Subunit a mutagenesis
The wild-type copy of subunit a was c arried by the pFo16
plasmid which contained the whole atp2 operon of
Rb. c apsulatus cloned into the pTZ19U plasmid [27]. The
aE210 fi K mutation was introduced into this plasmid by
using the QuickChange Site-Directed Mutagenesis K it
(Stratagene), based on linear PCR, using the following
mutagenic oligonucleotides: 5¢-CGCGATGTATGCGC
TC

deletion) either on the chromosomes or
on the pKFo102 plasmid. Restriction analysis of the
plasmid isolated from such colonies allowed the selection
of those carrying the F
0
deletion on the chromosomes. Two
mutated s trains were selected, w hich carried mutated
plasmids originated from two different PCR runs. A
pseudo-wild-type strain was constructed in parallel, which
carried the wild-type F
0
operon on the plasmid and the
deletion of the chromosomal F
0
operon. The cells used for
chromatophores preparations were routinely checked for
the presence of the mutation by XhoII restriction analysis of
the resident plasmid.
Western blot
The amount of ATP synthase in the membrane was
evaluated by quantitative Western blot on SD S/PAGE
isolated chromatophores protein, using a yeast anti-(b
subunit) antiserum (kindly provided by J. Velours, Bor-
deaux), the luminol assay for detection, and a purified ATP
synthase (isolated from Rb. capsulatus as described previ-
ously [34]) as a standard. The amounts of chromatophores
and standard protein in the different lanes of a single gel
were kept in the linear range of the luminol assay response.
Light-induced ATP synthesis
Light-driven ATP synthesis was carried out at 30 °Cin

Fig. 1, the cuvette contained 2 0 l
M
ADP, 6 0 l
M
lucif-
erine and 2–10 lgÆmL
)1
purified luciferase (32–160 ·
10
3
light units ÆmL
)1
) from Sigma (L9009) in the reaction
mixture described above, and the luminescence was
detected in real-time a t room temperature essentially as
described previously [35]. The assay mixture was illumi-
nated by a halogen lamp (160 WÆm
)2
light intensity,
filtered through 1 cm water and two layers of 8 8 A
Wratten fi lters) and different illumination times were
determined by an electronic shutter controlled by a
Uniblitz T132 Driver . The photomultiplier was shielded
against actinic light by a copper sulfate solution. The
amount of sy nthetized ATP w as evaluated by a dding
10–25 n
M
standard ATP.
ATP synthesis induced by acid-base transitions
Acid-base driven ATP synthesis was carried out similarly

/valinomycin
diffusion potential (monitored by the carotenoid shift
signal) i nduced by the initial K
+
gradient [36]. The
chromatophores were then mixed with the acidic solution
[30 m
M
succinic acid/NaOH, pH 4.6–6.5, 2 m
M
MgCl
2
,
5m
M
P
i
,either1m
M
(+Du) or 100 m
M
KCl (–Du), 1 m
M
AMP, 5 l
M
valinomycin] and incubated at room tempera-
ture at variable t imes between 2 and 30 min, depending on
the pH of t he suspending buffer, prior t o injection of
100 lL i nto the basic solution. This latter contained
850 lL o f basic solution so that the final concentrations

light units ÆmL
)1
). The final Bchl c oncentration
varied between 1 and 8 l
M
. The ATP concentration was
evaluated by adding 100–200 n
M
standard ATP in each
cuvette. The basic solution was thermostated so that the
ATP synthesis reaction took place a t 13 °C. The p H
measured after mixing the chroma tophores with the acidic
solution was taken as the internal pH, the pH measured
after mixing the acidified chromatophores with the basic
solution (8.55 ± 0.05) was taken as the external pH. Their
difference is the indicated DpH. F or the lowest DpH
differences the Ôacidic Õ solution contained 20 m
M
Tricine
instead of succinic acid. Assuming complete equilibration
of the K
+
during the 1 h preincubation (see above), the
value of t he K
+
/valinomycin diffusion potential deter-
mined by the K
+
transmembrane concentration difference
during the acid-base transition can be approximated, ( on

482 nm for excitation and emission, respectively) at 1 5 °C
in the following mixture: 20 m
M
Tricine/KOH, pH 8.0,
50 m
M
KCl, 0.5 m
M
MgCl
2
,0.2m
M
succinic acid , 5 l
M
Antimycin, 0.2 l
M
myxothiazol, 2 l
M
valinomycin, 1.5 l
M
ACMA, 20 l
M
Bchl, 400 l
M
ATP. The response of ACMA
to DpH was empirically calibrated using artificially induced
protonic g radients, established by HCl and NaOH a ddi-
tions, under similar temperature and buffer c onditions,
except for the presence of 20 m
M

ation times, the rat io function was truncated for times £ 150 ms.
1986 P. Turina and B. A. Melandri (Eur. J. Biochem. 269) Ó FEBS 2002
ATP hydrolysis assays
ATP hydrolysis was measured routinely at 30 °Cinthe
following buffer: 20 m
M
Tricine/KOH, pH 8.0, 50 m
M
KCl, 2 m
M
MgCl
2
,0.2m
M
succinic acid, 20 l
M
Bchl. Th e
reaction was started by adding 1 m
M
ATP. After stopping
the reaction at different time s with 5% trichloroacetic acid,
the P
i
concentration was measured by molybdate colori-
metric assay as described previously [38]. For more sensitive
measurements, the re leased P
i
was measu red w ith t he
EnzCheck Phosphate Assay Kit (Molecular Probes) or with
the malachite green assay [39], both methods giving similar

plasmid introduced by conjugation into Rb. capsulatus
wild-type cells. Finally, a GTA transfer was allowe d to
take place , wh ich generated the deletion o f the chromoso-
mal atp2 operon by substitution with a kanamycin
resistance cassette. Therefore, the resulting strain carried
the deletion of the chromosomal atp2 operon and several
copies of a plasmid carrying the mutated atp2 operon. As a
control, a parallel at p2-deleted strain w as created, in which
the r esident p lasmid carried the w ild-type operon. This
pseudo-wild-type strain is referred to as wild-type i n the
following procedures.
Characterization of mutant chromatophores
Phototrophic g rowth of the aE210K mutant cells was
slower than the wild-type cells. Accordingly, the light-
induced ATP synthesis r ate catalyzed by the mutant
chromatophores was about 40% lower on a Bchl basis
than the rate c atalyzed by wild-type c hromatophores
(Table 1). T he same reduction res ulted also when the
ADP concentration was varied between 20 and 500 l
M
,
indicating that the mutation does not affect the apparent K
m
for ADP. In contrast, no significant difference could be
observed in t he ATP hydrolysis r ate. The concentration of
ATP synthase was estimated on a Bchl basis by quantitative
Western blot analysis and was found to be the same within
experimental error for both mutant and wild-type chro-
matophores. The specific activity of ATP s ynth esis was
8±2and13±3ATP/(F

very similar in both strains. The most striking difference was
found in the protein content, which w as approximately
1.6-fold lower in the mutant chromatophores on a B chl
basis. It is possible that this difference affects the adsorption
of ACMA to the m embrane and therefore t he probe
response to DpH (see below).
Table 1. Catalytic activities and composition of chromatophores from
wild-type and mutant cells. All values reported are from a s ingle
chromatophores preparation but are representative of several different
preparations.
Wild-type Mutant aE210 fi K
Photophosphorylation rate
a
74 ± 4 44 ± 3
(mM ATP/M Bchl/s)
ATP Hydrolysis Rate
b
13 ± 3 11 ± 2
(mM P
i
/M Bchl/s)
Bchl/ATP Synthase Ratio
c
178 ± 35 180 ± 31
(moles/mole)
Protein/Bchl 48.4 29.7
(mg/lmole)
Bchl/RC 71 89
(moles/mole)
phospholipids/Bchl 13 ± 1.8 12 ± 2.0

þ
was
obtained, which resulted in a linear increase of the ATP
yields with illumination time. In order to investigate the
activity of the mutated enzyme at lower D
~
ll
H
þ
values, shorter
illumination times were chosen (from 100 ms to 2 s ). The
assays were also supplemented with the ionophore valino-
mycin, which largely prevents the onset of the electrical
component of D
~
ll
H
þ
, thus further reducing its total extent
during short illumination times. Due to the low ATP yields
expected in these experiments, the luciferine/luciferase ATP
detection system was added into t he assay cuvette at high
concentration (32–160 · 10
3
light units ÆmL
)1
of luciferase),
so that the ATP-induced luminescence was directly detected.
Figure 1A shows the ATP yields obtained from m utant
and wild-type chromatophores as a function of illumination

ll
H
þ
values.
ATP synthesis induced by acid-base transitions
The functioning of the mutated enzyme was also studied by
using the technique of acid-base transitions. This technique
allows one to control the extent of DpH across vesicle
Fig. 2. ATP synthesis driven by acid-base transitions. Chromatophores
were preincubated in resuspending and acidic media as described in the
Experimental procedures, and injected into the basic medium as
indicated by the arrow. The ATP synthesis following chromatophores
injection was mo nitored continuously with t he luciferine/luciferase
ATP Monitoring Kit in a luminometer. The high signal-to-noise ratio
was obtained due to ad ded purifie d luciferase (80 · 10
3
light
unitsÆmL
)1
). The internal and external pH’s were 4.96 and 8.54,
respectively, and the [K
+
]
out
and [K
+
]
in
were 150 m
M

+
]
in
equal to 1 and 150 m
M
,
respectively, or 100 and 100 m
M
, respectively. The data points have
been fitted with arbitrary functions. (B) An enlarged view of (A),
showing only the data obtained in the absence of Du.(C)Theratioof
the best-fitting functions and of the data points of wild-type over
mutant are plotted for data in the presence (d)andabsence(m)ofDu.
1988 P. Turina and B. A. Melandri (Eur. J. Biochem. 269) Ó FEBS 2002
membranes and to independently superimpose an electrical
diffusion potential Du, by varying the K
+
gradient across
the vesicle membrane in the presence of valinomycin. In the
present work, the [K
+
]
in
and [K
+
]
out
for + Du was 1 and
150 m
M

We therefore conclude that in chromatophores of Rb. c ap-
sulatus, under these conditions, DpH represents the only
driving force for ATP synthesis.
When carried out at room temperature, the linear phase
of the D
~
ll
H
þ
-induced ATP synthesis decays within a few
hundred milliseconds [36] , due to the r elatively h igh
permeability o f t he chromatophores membrane and to the
high H
+
flow through the ATP synthase, thus requiring for
its measurement a quench-flow apparatus. In the present
work, the du ration of this linear phase was increased to a
few seconds by decreasing the reaction temperature to
13 °C, and the transition was carried out manually by
rapidly injecting the acidic chromatophores suspension into
the luminometer cuvette containing the b asic solution and
the luciferine/luciferase detection kit (plus additional
80 · 10
3
light units ÆmL
)1
of luciferase). This method has
already been applied at room temperature for measuring the
ATP synthesis activity of ATP synthases incorporated into
liposomes [45,46].

range tested. In the absence of a Du, the ATP synthesis rate
of the mutant was up to eightfold lower with respect to the
wild-type for DpH values r anging between 2 .4 and 3.3,
whereas a twofold factor was approached at the h ighest
DpH tested. The trace –Du in Fig. 3C can b e d irectly
compared to the +valinomycin trace in Fig. 1C, because, in
the absence of a Du,theDpH is low in the short illumination
times (200–500 ms), and it increases at longer times.
Efficiency of proton pumping as estimated
with the ACMA assay
The proton-pumping activities of the mutant and wild-type
chromatophores were compared first by measuring the
ATP-induced fluorescence quenching of ACMA. For better
comparison of the in itial rates of quenching, t he ATP
hydrolysis rates were slowed down by decreasing the
reaction temperature to 15 °C. The ACMA fluorescence
quenching as a function of time after addition of ATP is
shown i n Fig. 4A f or chromatophores of both strains. N o
significant difference between the two chromatophores
preparations could be detected. However, when the ACMA
quenching was calibrated as a function of known DpH’s
generated during acid-base transitions, as described previ-
ously [37], the r esponse turned out to be sign ifican tly
different for the mutant and wild-type c hromatophores
(Fig. 4 B). This different respon se was systematically found
in different experimental sessions and i n different chro-
matophores preparations. Therefore, after converting the
fluorescence quenching values into DpH values according to
this calibration procedure, the initial rate of DpH formation
resulted to be higher in the wild-type w ith respect to the

+
-
transporter valinomycin was either imposed by a [K
+
]
gradient across the membrane, or prevented by imposing
equally high K
+
concentrations in both intra- and extra-
vesicular compartments. In the latter case, the A TP
Ó FEBS 2002 The aE210K mutation in Rb. capsulatus ATP synthase (Eur. J. Biochem. 269) 1989
synthesis r ate was m uch lower (up to eightfold) in the
mutant with respect to the wild-type in the DpH range
between 2.4 and 3.3.
The fact that this higher impairment is seen only within a
limited pH
in
range strongly suggests that a step in the ATP
synthase functioning, which is rate-limiting at th e low H
+
in
concentrations, gradually ceases to b e r ate-limiting f or
increasing H
+
in
concentrations. It is this rate-limiting step,
apparently, which is strongly affected in the mutant. What
could be rate-limiting at t he lowest H
+
in

and upon the
potential drop Dw due to surface charges, i.e. k
in
a10
–pHin
exp(Dw/RT). As a Glu fi Lys substitution is expected to
change the e lectrostatic profile o f t he nearby region, a
possible explanation for the data presented is that residue
a210 (a219 in E. co li) belongs to the periplasmic half-
channel and that one of i ts roles is to create a favorable
electrostatic profile f or incoming protons. This role is
drastically altered when a lysine side-chain substitutes the
carboxylate.
Within this framework, the results obtained in the
presence of a Du indicate that different steps of the
reaction cycle b ecome rate-limiting under these conditions.
According to the cited model [21], the main effects of a Du
in speeding up the enzymatic rate are an increased rate o f
H
+
releaseatthecytoplasmicsideandadecreasedrateof
H
+
release at the periplasmic side. Particularly this last
effect can presumably compensate for the low bulk H
+
in
concentration b y effectively i ncreasing [H
+
] w ithin the

ATP/(M BchlÆs) for both wild-type and the E210K mutant.
1990 P. Turina and B. A. Melandri (Eur. J. Biochem. 269) Ó FEBS 2002
A s econd major effect of the aE210K mutation is the
generally lower ATP synthesis f ound regardless of D
~
ll
H
þ
extent and composition, which might also explain t he lower
growth rate, as found here and in E. coli strains carrying
the homologous aE219K mut ation [28,29]. This general
impairment of ATP synthesis amounted here ap proxi-
mately to a factor of 2. This is seen for light-driven ATP
synthesis, both for long and short illumination times
(Table 1 and Fig. 1A, respectively), a s well as f or ATP
synthesis driven b y acid-base transitions, both in the
presence of a Du andinitsabsenceathighH
+
in
concentrations (Fig. 3C).
Several hypotheses can be put forward t o e xplain this
impairment. One possibility is that the mutation perturbs
the electrostatic profile sensed b y t he unproto nated Asp61
during i ts r adial translocation, thereby i ncreasing t he
probability of protons leaking through F
0
without perform-
ing any work [21]. This perturbation would also reduce the
H
+

Finally, it should be considered that the ATP synthase of
Rb. c apsulatus had been shown t o undergo significant
D
~
ll
H
þ
-induced activation phenomena [49], whose possible
interplay w ith the catalytic activity is not yet c lear. Very
similar activation phenomena have recently been seen in the
E. coli enzyme [50]. S trikingly, in both cases, the Du
component was driving this activation significantly more
than the DpH, suggesting t he presence o f an allosteric Du
sensor in the transmembrane portion of F
0
. It cannot be
ruled out that the aGlu210 is part of this activation switch,
whose perturbation could lead both to a decrease in the
functional efficiency of the enzyme population and to a
different sensitivity towards DpH and Du, respectively, as
observed in the present work.
ACKNOWLEDGEMENTS
This work has been supported by t he grant PRIN/01, Processi
Ossidoriduttivi e Trasduzione di Energia in Membrane Procariotiche ed
Eucariotiche, from the Italian M inistery for E ducation of University
and Research (MIUR). We are grateful to G. Venturoli for many
discussions and to F. Federici and D. Giovannini for excellent help in
the experiments.
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