2044 Biophysical Journal Volume 85 September 2003 2044–2054
Charge Displacements during ATP-Hydrolysis and Synthesis of the
Na1-Transporting FoF1-ATPase of Ilyobacter tartaricus
Christiane Burzik,* Georg Kaim,y Peter Dimroth,y Ernst Bamberg,*z and Klaus Fendler*
*Max-Planck-Institut fu¨r Biophysik, D-60596 Frankfurt/Main, Germany; yEidgeno¨ssische Technische Hochschule Zu¨rich,
CH-8092 Zu¨rich, Switzerland; and zUniversita¨t Frankfurt, D-60439 Frankfurt-Main, Germany
ABSTRACT Transient electrical currents generated by the Na1-transporting FoF1-ATPase of Ilyobacter tartaricus were
observed in the hydrolytic and synthetic mode of the enzyme. Two techniques were applied: a photochemical ATP
concentration jump on a planar lipid membrane and a rapid solution exchange on a solid supported membrane. We have
identified an electrogenic reaction in the reaction cycle of the FoF1-ATPase that is related to the translocation of the cation
through the membrane bound Fo subcomplex of the ATPase. In addition, we have determined rate constants for the process:
For ATP hydrolysis this reaction has a rate constant of 15–30 s1 if H1 is transported and 30–60 s1 if Na1 is transported. For
ATP synthesis the rate constant is 50–70 s1.
INTRODUCTION
Driven by a transmembrane electrochemical proton gradient,
FoF1-ATPases catalyze ATP synthesis in bacteria, mito-
chondria, or chloroplasts. The ATP synthases of a few
anaerobic bacteria-like Propionigenium modestum (Lau-
binger and Dimroth, 1988) and Ilyobacter tartaricus
(Neumann et al., 1998) have acquired the ability to use the
energy stored in a Na1 electrochemical gradient for the
synthesis of ATP. These Na1-FoF1-ATPases are close
relatives of the H1 driven FoF1-ATPases in structure and
properties. Like the latter they consist of a water soluble F1
part (subunit stoichiometry a3b3gde) that harbors the
catalytic sites for ATP synthesis and hydrolysis and the
membrane-embedded Fo component (subunit stoichiometry
ab2c11), that is responsible for ion translocation (Stahlberg
et al., 2001).
The high-resolution crystal structure from bovine mito-
chondrial F1 (Abrahams et al., 1994) was in remarkable
agreement with the binding change mechanism (Boyer,
1997) suggesting a rotary catalytic mechanism, which was
proven experimentally (Noji et al., 1997). Subunits-g, -e,
and the c11 oligomer could be cross-linked without loss
of function (Tsunoda et al., 2001) and were shown to
represent the rotor of FoF1 complexes by direct visualiza-
tion of rotation with an attached actin filament (Pa¨nke
et al., 2000; Sambongi et al., 1999). More recently, coupled
ATP-driven rotation of the gec11 subassembly and rotation
during ATP synthesis has been demonstrated for the
first time with single FoF1 molecules (Bo¨rsch et al., 2002;
Kaim et al., 2002). The a-subunit is connected laterally
with the c11 oligomer (Birkenhager et al., 1995; Singh et al.,
1996; Takeyasu et al., 1996), where it is held in place
by the two b-subunits that form a peripheral stalk con-
necting subunit-a with an a-subunit of F1 with the help of
subunit-d (Dunn and Chandler, 1998; Rodgers and Capaldi,
1998).
Since the pioneering work of Drachev et al. (1974),
current measurements on planar lipid membranes (black
lipid membrane (BLM)) have been used to investigate
electrogenic membrane proteins. With an appropriate acti-
vation system they were applied to detect ion transport
reactions or conformational transitions in the reaction cycle
of ion translocating ATPases (for a review see Bamberg
et al. (1993)). Because in FoF1-ATPase hydrolysis and
synthesis of ATP are associated with ion translocation in
the membrane-embedded Fo part, an electrical signal is
expected after activation of the enzyme with ATP or ADP 1
Pi. This has been demonstrated previously using the
FoF1-ATPase from Rhodospirillum rubrum (Christensen
et al., 1988). Activation was accomplished with a photolytic
ATP- or ADP-concentration jump using caged ATP and
caged ADP, respectively. We have now applied the same
technique to the Na1-FoF1-ATPase from I. tartaricus. This
enzyme has the advantage of additional possibilities to
modulate its function due to its Na1 sensitivity. In contrast to
previous work we have used a short laser flash for the release
of ATP from caged ATP, which allowed millisecond time
resolution. In addition, we have complemented the measure-
ments with a method for a substrate concentration jump
using a solid supported membrane (SSM) (Pintschovius
et al., 1999). Using both techniques we have detected
Na1-dependent transmembrane charge displacements. We
propose that the charge displacements are associated with
the rotary movements of the ringlike assembled c11 oligomer
versus subunits ab2 in the membrane-imbedded Fo part of
the protein.
Submitted August 16, 2002, and accepted for publication April 3, 2003.
Address reprint requests to Klaus Fendler, Marie Curie Str. 15, Frankfurt/
Main, Germany D-60439. Tel.: 49-69-6303-2035; Fax: 49-69-6303-2002;
E-mail: klaus.fendler@mpibp-frankfurt.mpg.de.
Abbreviations used: BLM, black lipid membrane; SSM, solid supported
membrane; NPE-caged ATP, P3-1-(2-nitro)phenylethyladenosine-59-tri-
phosphate); background ATP, ATP present in small amounts before and
after activation of the ATPase; l, rate constant of release of ATP from
caged ATP; h, fraction of ATP released from caged ATP; DCCD,
dicyclohexylcarbodiimide; DTT, dithiothreitole.
 2003 by the Biophysical Society
0006-3495/03/09/2044/11 $2.00

MATERIAL AND METHODS
Protein purification and preparation of
the proteoliposomes
The I. tartaricus cells (DSM 2382) were grown anaerobically at 308C and
collected as described (Neumann et al., 1998; Schink, 1984). Five grams of
cells were broken by passing a suspension through a French pressure cell.
The FoF1-ATPase was solubilized with Triton-X-100 (1%) and contami-
nating proteins were precipitated using polyethylene glycol PEG-6000. The
protein activity was determined by a coupled spectrophotometric assay
(Neumann et al., 1998). After further purification by gel chromatography
an activity of 10–20 units mg1 protein was achieved. The protein con-
centration was determined by the bicinchoninic acid test.
Proteoliposomes were prepared by the freeze-thaw-sonication procedure
as described by (Neumann et al., 1998) with a concentration of phospha-
tidylcholine (Sigma, St. Louis, MO; type IIS) of 30 mgml1 and a protein
concentration of 1–2 mgml1.
BLM measurements
The preparation of bilayers, the electrical recording instrumentation,
photolysis of caged ATP, and measurements of membrane conductivity
were performed as described (Fendler et al., 1996; Fendler et al., 1985).
Briefly, a planar lipid membrane (area  1 mm2) was formed between the
two compartments of a cuvette (volume 1.5 ml each compartment) filled
with electrolyte. A solution of 1,5% phosphatidylcholine and 0,025%
octadecylamine in n-decane was used to form the membrane. Proteolipo-
somes were added to one side of the membrane and allowed to adsorb to
the planar membrane by stirring the solution for 2 h. They are capacitively
coupled to the measuring system via the planar membrane. The FoF1-
ATPase in the proteoliposomes was activated by photolytic release of ATP
or ADP in the millisecond range (1mM MgCl2 and room temperature: 14
ms at pH 7.0 and 2.0 ms at pH 6.4) using a XeCl excimer laser at 308 nm.
The irradiance at the membrane plane was 180–300 mJ/cm2 leading
to a fraction of released ATP in solution of h ; 0.20–0.35. Transient
pump currents could be observed that represent the electrical activity of
the protein. The electrolyte contained buffer, salts, and various amounts
of NPE-caged ATP (P3-1-(2-nitro)phenylethyladenosine-59-triphosphate),
Na1 salt, or caged ADP, K1 salt, purchased from Calbiochem. For ex-
periments that required the absence of Na1 the (C2H5)3NH
1 salt of
NPE-caged ATP was kindly supplied by E. Grell, MPI fu¨r Biophysik,
Germany. In all ATP hydrolysis measurements the electroneutral H1/Na1
exchanger monensin (10 mM) was present to facilitate equilibration of Na1
between the cuvette and the internal volume of the liposomes (Fendler et al.,
1996).
SSM measurements
The SSM consisted of an alkanethiol monolayer covalently bound to a gold
surface via the sulfur atom, with a phospholipid monolayer on top of it.
For the preparation we followed the procedure described previously (Pints-
chovius et al., 1999; Seifert et al., 1993). As in the case of the BLM
proteoliposomes were allowed to adsorb to the SSM and transient currents
were measured via capacitive coupling. For the activation of the FoF1-
ATPase concentration jumps of ATP and ADP were generated via a rapid
solution exchange at the surface of the SSM (Pintschovius et al., 1999). The
cuvette was connected to the outlet of an electromagnetic valve, which
allowed fast switching between activating and nonactivating solutions. The
usual procedure for a concentration jump consisted of three steps: 1),
washing the cuvette with the nonactivating solution (1 s); 2), switching to the
activating solution (1 s); and 3), removing the activating substrate from the
cuvette with the nonactivating solution (1 s).
Determination of FoF1-ATPase activity in the
presence and absence of caged ATP
The activity of the protein was determined by the coupled spectrophoto-
metric assay (Neumann et al., 1998). The hydrolytic activity was measured
in the presence of 1 mM MgCl2, 2 mM NaCl and an ATP regenerating
system (phosphoenolpyruvate, pyruvate kinase, lactate dehydrogenase) in
addition to various amounts of ATP and NPE-caged ATP. For experimental
conditions, see Neumann et al. (1998).
RESULTS
Transient currents of the Na1-FoF1-ATPase
on the BLM
Proteoliposomes containing the FoF1-ATPase were allowed
to adsorb to the planar bilayer as described in Material and
Methods. Then caged ATP was added and the ion pump was
activated via the photolytic release of ATP (see Fig. 1).
FIGURE 1 BLM and adsorbed proteoliposomes. (A) ATPase in the
hydrolytic mode. The ATPase is activated by caged ATP. The current I(t) is
measured via the electrodes E. This circuit is omitted in B. (B) ATPase in the
synthetic mode. ‘‘Background ATP’’ generates the driving force for ATP
synthesis, which is activated by caged ADP.
Charge Displacements in FoF1-ATPase 2045
Biophysical Journal 85(3) 2044–2054