Molecular insights into cytochrome b6f driven PMF tuning

在光合电子传递中，细胞色素b6 f复合物（b6 f）有助于产生PMF组分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循环和通过循环电子转移（CEF）期间的替代Q循环，将铁氧还蛋白-质体醌还原酶活性归因于b6 f。 该项目旨在提供b6 f中内腔Q-位点和基质Qi-位点的结构-功能调节的机制见解（参见图形摘要P1）。的Q-网站是负责PQH 2氧化和电子分叉，以及pH依赖性的电子转移和发病的“光合控制”通过一个未知的机制放缓。B6 f中的Qi位点对于接受来自CEF的基质电子以及由于Q循环从正则模式切换到替代模式是重要的。在这条路线中，我们的目标是（i）确定STT 7（一种类囊体相关的Ser/Thr蛋白激酶）和/或b6 f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）揭示有助于光合作用控制在Qo网站通过调查的候选残基B6 F复合物的定点诱变的分子机制。第三，我们的目标（iii）进行比较cryo-EM结构分析，包括化学交联和质谱，分离的WT和突变体b6 f复合物。我们还旨在从结构上阐明抗霉素-A（AA）结合的b6 f复合物。在这条线中，我们还将研究天然类囊体膜WT和b6 f突变体在AA的存在和不存在下，通过冷冻ET。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（iv）工程b6 f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第1、2、3、4和5届部长级会议的总体目标。

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.