Steigmiller Stefan, Turina Paola, Gräber Peter
Institut für Physikalische Chemie, Universität Freiburg, Albertstrasse 23a, D-79104 Freiburg, Germany.
Proc Natl Acad Sci U S A. 2008 Mar 11;105(10):3745-50. doi: 10.1073/pnas.0708356105. Epub 2008 Mar 3.
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.
H⁺/ATP 比值是所有细胞能量平衡以及质子运输与 ATP 合成之间偶联机制的一个重要参数。对旋转催化的直接解释预测，H⁺/ATP 与 c 亚基与 β 亚基的比值一致，这意味着对于叶绿体和大肠杆菌的 ATP 合酶，预期该比值分别为 4.7 和 3.3。在此，利用化学渗透理论所描述的能量学来确定这两种酶的 H⁺/ATP 比值。将分离的复合物重组成脂质体，并在相同条件下进行平行测量。在重组过程中，脂质体的内相用酸性介质平衡，以便能用玻璃电极测量内部 pH 值。进行酸碱转变，并在恒定的 Q = [ATP]/([ADP][P(i)]) 条件下，以荧光素/荧光素酶测量 ATP 合成或 ATP 水解的初始速率作为 ΔpH 的函数。根据平衡 ΔpH 随 Q 的变化，确定磷酸化的标准吉布斯自由能 ΔG(p)(0)' 和 H⁺/ATP 比值。结果显示，叶绿体的 ΔG(p)(0)' = 38 ± 3 kJ·mol⁻¹，H⁺/ATP = 4.0 ± 0.2，大肠杆菌酶的 H⁺/ATP = 4.0 ± 0.3，这表明两种酶的热力学 H⁺/ATP 比值相同，且与亚基化学计量比不同。
