Molecular insights into cytochrome b6f driven PMF tuning

细胞色素 b6f 驱动 PMF 调节的分子见解

• 批准号: 524891100
• 负责人: Professor Dr. Michael Hippler
• 金额: --
• 依托单位: Institute of Plant Science and Resources
• 依托单位国家: 德国
• 项目类别: Research Units
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/524891100?language=en
• 关键词: Molecular; insights; into; cytochrome; b6f

In photosynthetic electron transfer, the cytochrome b6f complex (b6f) contributes to the generation of both PMF components, delta pH and delta psi. This process is driven by the Q-cycle and operates in two distinct modes – via the canonical Q cycle during linear electron flow (LEF) and via an alternative Q cycle during cyclic electron transfer (CEF), attributing a ferredoxin-plastoquinone reductase activity to the b6f. This project aims to provide mechanistic insights into the structure-function tuning of the lumenal Qo-site and the stromal Qi-site in b6f (see also Graphical Abstract P1). The Qo-site is responsible for PQH2 oxidation and electron bifurcation as well as for pH dependent slowdown of electron transfer and onset of “photosynthetic control” via an unknown mechanism. The Qi-site in b6f is important for acceptance of stromal electrons deriving from CEF and due to switching of the Q-cycle from the canonical to the alternative mode. In this line, we aim (i) to determine the role of STT7, a thylakoid-associated Ser/Thr protein kinase, and/or phosphorylation(s) of b6f subunit-IV and subunit PETO for structure-function tuning. Accordingly, we aim to (ii) reveal molecular mechanisms contributing to photosynthetic control at the Qo site through investigation of candidate residues in b6f complex by site directed mutagenesis. Thirdly, we aim (iii) to perform comparative cryo-EM structural analyses, including chemical crosslinking and mass spectrometry, for isolated WT and mutant b6f complexes. We also aim to structurally elucidate antimycin-A (AA) binding to the b6f complex. In this line we will also investigate native thylakoid membranes from WT and b6f mutants in the presence and absence of AA via cryo-ET. We will also take advantage of a portable plunger to directly compare spectroscopic and mass spectrometric analyses with cryo-ET analyses. Moreover, we aim (iv) to engineer b6f complexes as sensors for chloroplast PMF. Generally, our work will contribute to the overall aims of GoPMF 1, 2, 3, 4 and 5.

在光合作用电子转移中，细胞色素b6f复合物（b6f）有助于产生PMF成分，δ pH和δ psi。该过程由Q循环驱动，并以两种不同的模式运行-通过线性电子流（LEF）期间的规范Q循环和循环电子转移（CEF）期间的替代Q循环，将铁氧化还原蛋白-塑性醌还原酶活性归因于b6f。该项目旨在为b6f中管腔q -位点和基质q -位点的结构-功能调节提供机制见解（参见图形摘要P1）。qo位点负责PQH2氧化和电子分岔，以及pH依赖的电子转移减慢和通过未知机制开始的“光合控制”。b6f中的qi位对于接受来自CEF的基质电子和由于q周期从规范模式切换到替代模式是重要的。在这条线上，我们的目标是(i)确定STT7（一种类囊体相关的丝氨酸/苏氨酸蛋白激酶）和/或b6f亚基- iv和亚基PETO的磷酸化在结构-功能调节中的作用。因此，我们的目标是（ii）通过位点定向诱变研究b6f复合体的候选残基，揭示Qo位点光合作用控制的分子机制。第三，我们的目标是（iii）对分离的WT和突变的b6f复合物进行比较冷冻电镜结构分析，包括化学交联和质谱分析。我们还旨在从结构上阐明抗霉素- a （AA）与b6f复合物的结合。在这条线中，我们还将通过冷冻- et研究在存在和不存在AA的情况下WT和b6f突变体的天然类囊体膜。我们还将利用便携式柱塞直接比较光谱和质谱分析与冷冻et分析。此外，我们的目标是（iv）设计b6f复合物作为叶绿体PMF的传感器。总的来说，我们的工作将有助于实现GoPMF 1、2、3、4和5的总体目标。