Phytochromes are light-sensitive proteins that are found in plants, fungi, and bacteria. They exist in two functional states, Pr and Pfr, distinguished by Z/E isomers of their bilin chromophore. The chromophore can photoswitch between these states but also thermally converts in darkness. Despite the importance of the latter reaction, it is unknown how it is controlled by the phytochrome's structure. Here, we present single-particle cryo-EM measurements on the Pseudomonas aeruginosa bacteriophytochrome (PaBphP) carried out at multiple time points during dark reversion from Pr to Pfr. These experiments resolved the structure of a PrPfr hybrid state as a transient intermediate. Surprisingly, we find that only protomer B converts back to Pfr in the hybrid, while protomer A remains in Pr. We identify structural asymmetries in the precursor Pr state, which extend from the homodimer interface to a conserved histidine (H277). The hydrogen-bonding network around the chromophore is modulated, explaining how a phytochrome exerts control over the isomerization reaction. These findings establish that dark reversion is governed by conformational selection between two substates, whereby one is "dark-reversion ready" and the other blocks the reaction. Moreover, we explain how the equilibrium of the states is allosterically controlled across the dimer. Together, these findings provide a structural framework for tuning phytochrome signaling lifetimes in optogenetic applications.
Bódizs et al. (2025) studied this question.