Faculty of Physics, Moscow State University, Moscow 119991, Russia.
Faculty of Physics, Moscow State University, Moscow 119991, Russia.
Biochim Biophys Acta Biomembr. 2017 Mar;1859(3):388-401. doi: 10.1016/j.bbamem.2016.11.016. Epub 2016 Dec 2.
This work is devoted to critical analysis and reexamination of the problem of lateral heterogeneity of the trans-thylakoid pH difference (ΔpH=pH-pH) in thylakoid membranes of chloroplasts. Correct measurements of ΔpH may be complicated by nonuniform partitioning of the protons pumped into the lumen of granal (stacked) and stroma-exposed thylakoids. We have compared results of ΔpH estimations in isolated bean chloroplasts by two different methods. One of the methods is based on the use of pH-sensitive spin-probes. Another approach relies on the analysis of pH-dependent post-illumination reduction of P - oxidized reaction center of photosystem I (PSI). Both methods lead to virtually the same values of ΔpH, as measured in the state of photosynthetic control when the ATP synthase complexes are inactive (ΔpH2.3-2.5 at pH8). Under the photophosphorylation conditions (metabolic state 3), ΔpH decreases due to the proton drain from the lumen to stroma via active ATP synthases (ΔpH1-2 at pH8). In this state, ΔpH values derived from kinetic data are smaller than ΔpH measured with the pH-probing amines. Such a discrepancy can be explained by the coexistence of thylakoids with different pH established in granal and stromal thylakoids. The kinetic method is equivalent to the use of a "local pH-meter", which is sensitive to less significant decrease in pH inside the stroma-exposed thylakoids. Otherwise, pH-indicating probes give information on pH values averaged out at both granal and stromal thylakoids. Experimental results have been analyzed within the framework of our mathematical model developed for simulation of electron and proton transport processes in laterally heterogeneous thylakoids. The model provides a reasonable description of experimental data, supporting the notion that the long-range diffusion of protons within the lumen and obstructed diffusion of mobile electron carriers (PQH and Pc) influence the lateral profiles of pH along the thylakoid membranes. The model predicts significant alkalization of the inter-thylakoid gap and the establishment of nonuniform lateral profiles of ΔpH under the photophosphorylation conditions. These results are discussed in the context of the problem of energy coupling in laterally heterogeneous lamellar system of chloroplasts.
这项工作致力于对叶绿体类囊体膜中跨类囊体 pH 差（ΔpH=pH-pH）的横向异质性问题进行批判性分析和再检验。正确测量 ΔpH 可能会受到质子不均匀分配的影响，这些质子被泵入粒状（堆叠）和基质暴露的类囊体的腔室中。我们比较了两种不同方法在分离的豆叶绿体中 ΔpH 估计的结果。其中一种方法基于使用 pH 敏感的自旋探针。另一种方法依赖于分析光系统 I（PSI）的 P 氧化反应中心在光照后的 pH 依赖性还原。当 ATP 合酶复合物不活跃时（在 pH8 时，ΔpH2.3-2.5），两种方法都导致光合作用控制状态下的 ΔpH 几乎相同，此时测量值。在光合磷酸化条件下（代谢状态 3），由于质子通过活跃的 ATP 合酶从腔室排到基质中，ΔpH 减小（在 pH8 时，ΔpH1-2）。在这种状态下，从动力学数据得出的 ΔpH 值小于使用 pH 探针胺测量的值。这种差异可以通过在粒状和基质类囊体中建立不同 pH 的类囊体共存来解释。动力学方法相当于使用“局部 pH 计”，它对基质暴露的类囊体中 pH 较小的下降敏感。否则，指示 pH 的探针给出了在粒状和基质类囊体上平均的 pH 值的信息。实验结果在我们为模拟横向异质类囊体中的电子和质子传输过程而开发的数学模型的框架内进行了分析。该模型为实验数据提供了合理的描述，支持质子在腔室内的长程扩散和可移动电子载体（PQH 和 Pc）的受阻扩散影响跨类囊体膜 pH 横向分布的观点。该模型预测在光合磷酸化条件下，在类囊体间隙中明显碱化，并建立非均匀的 ΔpH 横向分布。这些结果在叶绿体横向异质层状系统中的能量偶联问题的背景下进行了讨论。
