Petra Mészáros: Structural basis of photoactivation in reverse phytochromes

Abstract

Phytochromes are red/far-red light–sensing proteins that regulate diverse biological processes by interconverting between two photochromic states in a cyclic fashion, termed Pr and Pfr. While the forward reaction (Pr-to-Pfr) has been extensively studied, the molecular mechanism of the reverse photocycle (Pfr-to-Pr) remains poorly understood. This thesis elucidates the structural and kinetic basis of the reverse photoreaction in the bathy phytochrome from Pseudomonas aeruginosa (PaBphP). Using a combination of time-resolved serial crystallography, cryo-electron microscopy, ultrafast spectroscopy, and biochemical assays, we characterize PaBphP across multiple functional states and timescales. Femtosecond-to-millisecond structural snapshots of the photosensory core module reveal that photoexcitation initially induces collective atomic displacements within the chromophore-binding pocket, which subsequently resolve into discrete states. As the C-D bridge of the biliverdin chromophore undergoes ultrafast rotation, the hydrogen bonds to nearby amino acids weaken, which enables the formation of the primary photoproduct via an unexpected one-bond-flip isomerization. Cryo-EM structures of the full-length protein show that the Pr state represents the catalytically active conformation, mediating trans-autophosphorylation between protomers, whereas the open structure of the Pfr state exhibits pronounced structural flexibility consistent with catalytic inactivity. Lastly, studying the dark reversion kinetics of the reverse photocycle reveals that, similarly to plant phytochromes, the two protomers in bacteriophytochromes do not revert from the PrPr light-activated state to the PfrPfr ground state simultaneously, but rather through a hybrid PrPfr intermediate state. Together, these findings establish a fundamental mechanism governing photoactivation through chromophore isomerization, signal propagation, and the importance of dimer formation in signal transduction, and clarify the relationship between bathy and canonical members of the phytochrome family.