Peter Gräber

Proton-powered subunit rotation in single membrane-bound F0F1-ATP synthase.

Synthesis of ATP from ADP and phosphate, catalyzed by F0F1-ATP synthases, is the most abundant physiological reaction in almost any cell. F0F1-ATP synthases are membrane-bound enzymes that use the energy derived from an electrochemical proton gradient for ATP formation. We incorporated double-labeled F0F1-ATP synthases from Escherichia coli into liposomes and measured single-molecule fluorescence resonance energy transfer (FRET) during ATP synthesis and hydrolysis. The γ subunit rotates stepwise during proton transport–powered ATP synthesis, showing three distinct distances to the b subunits in repeating sequences. The average durations of these steps correspond to catalytic turnover times upon ATP synthesis as well as ATP hydrolysis. The direction of rotation during ATP synthesis is opposite to that of ATP hydrolysis.

Conformational change of the chloroplast ATPase induced by a transmembrane electric field and its correlation to phosphorylation

Abstract The energy-dependent release of bound [14C]nucleotides trom the chloroplast coupling factor CF1, has been used to monitor conformational changes in CF1. The following results were obtained: 1. (1) Similar as in continuous light conformational changes of CF1 are observed on energetization of the thylakoid membrane by short light pulses. Under these conditions the transmembrane electric potential difference induced is about 200 mV and the pH gradient set up across the membrane is about 1.0. 2. (2) Conformational changes are observed also in the dark when external voltage pulses are used for energization. Under these conditions the transmembrane electric potential difference induced is about 200 mV whereas the pH gradient between the inner and outer thylakoid space is zero. 3. (3) Only a fraction of the total number of coupling factors change their conformation. The size of this fraction depends non-linearly on the magnitude of the electric potential difference induced by light pulses or external voltage pulses. 4. (4) In a light or a voltage pulse of 30-ms duration, the amount of ATP generated is 5–8 times larger than the amount of CF1 which have changed their conformation. This factor is independent of the magnitude of the electric potential difference. If the observed conformational changes are coupled with phosphorylation these results may be explained tentatively by the following concept. The proton flux which is used for phosphorylation is focussed only to a fraction of the total number of ATPases. This fraction varies strongly with the electric potential difference (and probably also with the pH gradient). The variation occurs in such a way that the flux via these “active” ATPases and their turnover time is nearly constant (about 5 ms).

Influence of the redox state and the activation of the chloroplast ATP synthase on proton-transport-coupled ATP synthesis/hydrolysis

Abstract The membrane-bound ATP synthase from chloroplasts can occur in different redox and activation states. In the absence of reductants the enzyme usually is oxidized and inactive, Eoxi. Illumination in the presence of dithiothreitol leads to an active, reduced enzyme, Ereda. If this form is stored in the dark in the presence of dithiothreitol an inactive, reduced enzyme Eredi is formed. The rates of ATP synthesis and ATP hydrolysis catalyzed by the different enzyme species are measured as a function of ΔpH (Δψ = 0 mV). The ΔpH was generated with an acid-base transition using a rapid-mixing quenched flow apparatus. The following results were obtained. (1) The oxidized ATP synthase catalyzes high rates of ATP synthesis, voxmax = 400 ATP per CF0F1 per s. The half-maximal rate is obtained at ΔpH = 3.4. (2) The active, reduced ATP synthase catalyzes high rates of ATP synthesis, vredmax = 400 ATP per CF0F1 per s. The half-maximal rate is obtained at ΔpH = 2.7. It catalyzes also high rates of ATP hydrolysis vredmax = −90 ATP per CF0F per s at ΔpH = 0. (3) The inactive species (both oxidized and reduced) catalyze neither ATP synthesis nor ATP hydrolysis. The activation/inactivation of the reduced enzyme is completely reversible. (4) The activation of the reduced, inactive enzyme is measured as a function of ΔpH by measuring the rate of ATP hydrolysis catalyzed by the active species. Half-maximal activation is observed at ΔpH = 2.2. (5) On the basis of these results a reaction scheme is proposed relating the redox reaction, the activation and the catalytic reaction of the chloroplast ATP synthase.

H+/ATP ratio of proton transport-coupled ATP synthesis and hydrolysis catalysed by CF0F1–liposomes

The H+/ATP ratio and the standard Gibbs free energy of ATP synthesis were determined with a new method using a chemiosmotic model system. The purified H+‐translocating ATP synthase from chloroplasts was reconstituted into phosphatidylcholine/phosphatidic acid liposomes. During reconstitution, the internal phase was equilibrated with the reconstitution medium, and thereby the pH of the internal liposomal phase, pHin, could be measured with a conventional glass electrode. The rates of ATP synthesis and hydrolysis were measured with the luciferin/luciferase assay after an acid—base transition at different [ATP]/([ADP][Pi]) ratios as a function of ΔpH, analysing the range from the ATP synthesis to the ATP hydrolysis direction and the ΔpH at equilibrium, ΔpH (eq) (zero net rate), was determined. The analysis of the [ATP]/([ADP][Pi]) ratio as a function of ΔpH (eq) and of the transmembrane electrochemical potential difference, Δμ̃H+ (eq), resulted in H+/ATP ratios of 3.9 ± 0.2 at pH 8.45 and 4.0 ± 0.3 at pH 8.05. The standard Gibbs free energies of ATP synthesis were determined to be 37 ± 2 kJ/mol at pH 8.45 and 36 ± 3 kJ/mol at pH 8.05.

Stepwise rotation of the γ-subunit of EF0F1-ATP synthase observed by intramolecular single-molecule fluorescence resonance energy transfer1

The EF0F1‐ATP synthase mutants bQ64C and γT106C were labelled selectively with the fluorophores tetramethylrhodamine (TMR) at the b‐subunit and with a cyanine (Cy5) at the γ‐subunit. After reconstitution into liposomes, these double‐labelled enzymes catalyzed ATP synthesis at a rate of 33 s−1. Fluorescence of TMR and Cy5 was measured with a confocal set‐up for single‐molecule detection. Photon bursts were detected, when liposomes containing one enzyme traversed the confocal volume. Three states with different fluorescence resonance energy transfer (FRET) efficiencies were observed. In the presence of ATP, repeating sequences of those three FRET‐states were identified, indicating stepwise rotation of the γ‐subunit of EF0F1.

Membrane-bound ATP synthesis generated by an external electrical field☆

In the primary act of photosynthesis an electric potential difference, A~, is generated across the energy coupling membrane by a light-induced vectorial electron transfer [1,2]. In a consecutive step protolytic reactions with the charges at the outer and the inner membrane surface lead to the formation of a pH gradient, ApH [3]. Through measurements of the relaxation of A~ simultaneously with the formation of ATP quantitative relationships were obtained between both events in respect to the extent, rate and functional unit [4]. This indicates that phosphorylation is coupled with the discharging of the electrically energized membrane. A coupling of ATP formation with the relaxation of ApH was first demonstrated by Jagendorf and Uribe [5]. Regarding the cooperation of A~ and ApH quantitative relations were elaborated in respect to the kinetics of ATP synthesis [6]. In respect to the energetics there is accumulating evidence that the free energy, AG, stored in A~ ~ 100 mV [7] and ApH ~ 3 [8,9] is with H÷/ATP ~ 2.5 [6,10] in agreement with data of AG [ 11 ] necessary for ATP synthesis. These and other results support the electrochemical hypothesis of Mitchell [12]. Under natural conditions electron transfer, field generation and ApH formation are always coupled with each other. Therefore, with respect to the mechanism of

Movements of the ε-subunit during catalysis and activation in single membrane-bound H+-ATP synthase

F0F1‐ATP synthases catalyze proton transport‐coupled ATP synthesis in bacteria, chloroplasts, and mitochondria. In these complexes, the ε‐subunit is involved in the catalytic reaction and the activation of the enzyme. Fluorescence‐labeled F0F1 from Escherichia coli was incorporated into liposomes. Single‐molecule fluorescence resonance energy transfer (FRET) revealed that the ε‐subunit rotates stepwise showing three distinct distances to the b‐subunits in the peripheral stalk. Rotation occurred in opposite directions during ATP synthesis and hydrolysis. Analysis of the dwell times of each FRET state revealed different reactivities of the three catalytic sites that depended on the relative orientation of ε during rotation. Proton transport through the enzyme in the absence of nucleotides led to conformational changes of ε. When the enzyme was inactive (i.e. in the absence of substrates or without membrane energization), three distances were found again, which differed from those of the active enzyme. The three states of the inactive enzyme were unequally populated. We conclude that the active–inactive transition was associated with a conformational change of ε within the central stalk.

The rate of ATP synthesis as a function of ΔpH in normal and dithiothreitol-modified chloroplasts

Abstract The rate of ATP synthesis catalyzed by normal and by dithiothreitol-modified ATPases is investigated as a function of ΔpH in spinach chloroplasts at constant pHout. The transmembrane ΔpH was generated by an acid-base transition and the reaction time was limited to 150 ms by using a rapidly mixing quenched-flow apparatus. The result was that the functional dependence of the rate on ΔpH is shifted to lower ΔpH values and that the shape of this curve is altered after dithiothreitol modification. The maximal rate (400 ATP / CF1 per s) is the same under both conditions.

The thermodynamic H+/ATP ratios of the H+-ATPsynthases from chloroplasts and Escherichia coli

The H+/ATP ratio is an important parameter for the energy balance of all cells and for the coupling mechanism between proton transport and ATP synthesis. A straightforward interpretation of rotational catalysis predicts that the H+/ATP coincides with the ratio of the c-subunits to β-subunits, implying that, for the chloroplast and Escherichia coli ATPsynthases, numbers of 4.7 and 3.3 are expected. Here, the energetics described by the chemiosmotic theory was used to determine the H+/ATP ratio for the two enzymes. The isolated complexes were reconstituted into liposomes, and parallel measurements were performed under identical conditions. The internal phase of the liposomes was equilibrated with the acidic medium during reconstitution, allowing to measure the internal pH with a glass electrode. An acid–base transition was carried out and the initial rates of ATP synthesis or ATP hydrolysis were measured with luciferin/luciferase as a function of ΔpH at constant Q = [ATP]/([ADP][Pi]). From the shift of the equilibrium ΔpH as a function of Q the standard Gibbs free energy for phosphorylation, ΔGp0′; and the H+/ATP ratio were determined. It resulted ΔGp0′ = 38 ± 3 kJ·mol−1 and H+/ATP = 4.0 ± 0.2 for the chloroplast and H+/ATP = 4.0 ± 0.3 for the E. coli enzyme, indicating that the thermodynamic H+/ATP ratio is the same for both enzymes and that it is different from the subunit stoichiometric ratio.

Proton transport through a peptide-tethered bilayer lipid membrane by the H+-ATP synthase from chloroplasts measured by impedance spectroscopy

A lipid membrane was tethered to a gold film by a peptide spacer molecule terminated by a sulfhydryl group. Membranes were formed by fusion of liposomes prepared from egg phosphatidylcholine on self assembled monolayers of the thiolipopeptide Myr-Lys(Myr)-Ser-Ser-Pro-Ala-Ser-Ser-Ala-Ala-Ser-Ala-Cys-amide mixed with mercaptoethanol as a diluent molecule or lateral spacer. These mixed films, although not representing a perfect lipid bilayer, have been shown to retain the activity of incorporated H(+)-ATP synthases from chloroplasts in contrast to films prepared from the pure thiolipopeptide. The activity of the protein was demonstrated by impedance spectroscopy. The resistance decreased due to proton transport across the lipid film, which occurs as a consequence of adenosine triphosphate (ATP) hydrolysis. Several effects previously determined from kinetic measurements of the enzyme reconstituted in liposomes such as saturation with respect to the substrate (ATP), inhibition by venturicidin, activation by a positive potential pulse and increase of the proton current as a function of increasingly negative potentials have been confirmed also for this tethered membrane system. Changes in the impedance spectra due to the addition of ATP were fully reversible.