Electron transfer at Photosystem I and the Cytochrome b6f Complex: Mechanistic Insights and STT7 Kinase Feedback Regulation

In photosynthesis, electron transfer between cytochrome b6f (b6f) and photosystem I (PSI) is mediated by cytochrome c6 (Cyt c6) or plastocyanin (PC). Efficient electron transfer requires precise docking of these carriers to Cyt b6f and PSI. While we have high-resolution structures of PSI-PC complexes from Pea and Chlamydomonas 1,2, no structure is available for a PSI-Cyt c6 complex. To close this gap, we acquired a high-resolution cryo-EM structure of C. reinhardtii PSI chemically crosslinked with Cyt c6 in a native, electron transfer–competent state. We also explored the effects of PC phosphorylation on electron transfer with PSI and b6f. To do so, we generated several recombinant variants of PC and studied the kinetics of both Cytf oxidation and P700 re-reduction by measuring fast optical spectroscopy. We also conducted chemical protein crosslinking and structural proteomics to gain further insights on the interaction between PC and b6f. Our results show that the phosphorylation mode of PC alters the conformation in which they establish binding and electron transfer, and generated new models which elaborate the mechanism of this adaptation. To address the role of STT7 dependent phosphorylation of Thr4 in the N-terminal domain of b6f subunit IV, we generated site-directed mutants in the N-terminal domain of b6f subunit IV by chloroplast transformation. The phosphomimic mutation PetD T4E effectively inhibits STT7 kinase activity, as shown by phosphoproteomic analyses, resulting in the PetD T4E strain being locked in State 1. These findings reveal a novel feedback regulation mechanism that controls the phosphorylation capacity of STT7 kinase. Similarly, deletion of five N-terminal amino acids b6f subunit IV also disrupts STT7 function, retains cells in State 1, and, in contrast to the PetD T4E mutation, significantly impairs electron transfer within cyt b6f. These data reveal that the PetD N-terminus has crucial functions in b6f electron transfer and STT7 regulation.

References

Naschberger, A. et al. Algal photosystem I dimer and high-resolution model of PSI-plastocyanin complex. Nat Plants 8, 1191-1201 (2022). https://doi.org:10.1038/s41477-022-01253-4

Caspy, I. et al. Structure of plant photosystem I-plastocyanin complex reveals strong hydrophobic interactions. Biochem J 478, 2371-2384 (2021). https://doi.org:10.1042/BCJ20210267