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 beta-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 DeltapH at constant Q = [ATP]/([ADP][P(i)]). From the shift of the equilibrium DeltapH as a function of Q the standard Gibbs free energy for phosphorylation, DeltaG(p)(0)'; and the H(+)/ATP ratio were determined. It resulted DeltaG(p)(0)' = 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.
Steigmiller
Stefan
S
Institut für Physikalische Chemie, Universität Freiburg, Albertstrasse 23a, D-79104 Freiburg, Germany.
Turina
Paola
P
Gräber
Peter
P
D000255:Adenosine Triphosphate / Q000096:biosynthesis / Q000378:metabolism*; D002736:Chloroplasts / Q000201:enzymology*; D004926:Escherichia coli / Q000201:enzymology*; D006868:Hydrolysis; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D018892:Proton-Motive Force; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011522:Protons; D013816:Thermodynamics
D008081:Liposomes; D011522:Protons; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases