Otto H, Marti T, Holz M, Mogi T, Lindau M, Khorana H G, Heyn M P
Biophysics Group, Freie Universität Berlin, Federal Republic of Germany.
Proc Natl Acad Sci U S A. 1989 Dec;86(23):9228-32. doi: 10.1073/pnas.86.23.9228.
Above pH 8 the decay of the photocycle intermediate M of bacteriorhodopsin splits into two components: the usual millisecond pH-independent component and an additional slower component with a rate constant proportional to the molar concentration of H+, [H+]. In parallel, the charge translocation signal associated with the reprotonation of the Schiff base develops a similar slow component. These observations are explained by a two-step reprotonation mechanism. An internal donor first reprotonates the Schiff base in the decay of M to N and is then reprotonated from the cytoplasm in the N----O transition. The decay rate of N is proportional to [H+]. By postulating a back reaction from N to M, the M decay splits up into two components, with the slower one having the same pH dependence as the decay of N. Photocycle, photovoltage, and pH-indicator experiments with mutants in which aspartic acid-96 is replaced by asparagine or alanine, which we call D96N and D96A, suggest that Asp-96 is the internal proton donor involved in the re-uptake pathway. In both mutants the stoichiometry of proton pumping is the same as in wild type. However, the M decay is monophasic, with the logarithm of the decay time [log (tau)] linearly dependent on pH, suggesting that the internal donor is absent and that the Schiff base is directly reprotonated from the cytoplasm. Like H+, azide increases the M decay rate in D96N. The rate constant is proportional to the azide concentration and can become greater than 100 times greater than in wild type. Thus, azide functions as a mobile proton donor directly reprotonating the Schiff base in a bimolecular reaction. Both the proton and azide effects, which are absent in wild type, indicate that the internal donor is removed and that the reprotonation pathway is different from wild type in these mutants.
在pH值高于8时，细菌视紫红质光循环中间体M的衰变分为两个部分：通常的与pH无关的毫秒级部分和一个额外的较慢部分，其速率常数与H⁺的摩尔浓度[H⁺]成正比。同时，与席夫碱再质子化相关的电荷转移信号也出现了类似的慢成分。这些观察结果可以用两步再质子化机制来解释。一个内部供体首先在M衰变到N的过程中使席夫碱再质子化，然后在N到O的转变过程中从细胞质中再质子化。N的衰变速率与[H⁺]成正比。通过假设从N到M的逆反应，M衰变分为两个部分，较慢的部分与N的衰变具有相同的pH依赖性。对天冬氨酸-96被天冬酰胺或丙氨酸取代的突变体（我们称为D96N和D96A）进行的光循环、光电压和pH指示剂实验表明，天冬氨酸-96是再摄取途径中涉及的内部质子供体。在这两个突变体中，质子泵浦的化学计量与野生型相同。然而，M衰变是单相的，衰变时间的对数[log(τ)]与pH呈线性关系，这表明内部供体不存在，席夫碱直接从细胞质中再质子化。与H⁺一样，叠氮化物增加了D96N中M的衰变速率。速率常数与叠氮化物浓度成正比，并且可以比野生型大100倍以上。因此，叠氮化物在双分子反应中作为直接使席夫碱再质子化的可移动质子供体。野生型中不存在的质子和叠氮化物效应都表明内部供体被去除，并且在这些突变体中再质子化途径与野生型不同。
