Molecular insights into cytochrome b6f driven PMF tuning

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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.