Molecular insights into cytochrome b6f driven PMF tuning

在光合作用的电子传递过程中，细胞色素b6f复合体(B6f)参与PMF组分的产生，包括增量pH和增量psi。这一过程由Q循环驱动，并以两种不同的模式工作-通过线性电子流(LEF)期间的典型Q循环和循环电子转移(CEF)期间的交替Q循环，将铁还蛋白-质醌还原酶活性归因于b6f。本项目旨在提供对b6f中腔QO部位和间质QO部位的结构-功能调节的机械性见解(另见图形摘要P1)。QO-位负责PQH2的氧化和电子分叉，以及与pH相关的电子传递减慢和通过未知机制启动的“光合作用控制”。由于Q周期从正则模到交替模的转换，b6f中的Qi位对于接受来自CEF的叠层电子是重要的。在这方面，我们的目标是(I)确定STT7，一种与类囊体体相关的丝氨酸/苏氨酸蛋白激酶，和/或b6f亚基-IV和Peto亚基的磷酸化(S)在结构-功能调节中的作用。因此，我们的目标是(Ii)通过定点突变研究b6f复合体中的候选残基，揭示有助于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络合物进行比较的低温EM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复合体的结合。在这条线上，我们也将研究来自wt和b6f突变体的天然类囊体膜，在存在和不存在AA的情况下，通过冷冻-ET。我们还将利用便携式柱塞将光谱分析和质谱分析与低温分析直接进行比较。此外，我们的目标是(Iv)设计b6f配合物作为叶绿体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。

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.