Chemical mechanism of allosteric and asymmetric dark reversion in a bacterial phytochrome uncovered by cryo-EM.

Phytochromes are light-sensitive proteins found in plants, fungi, and bacteria. They switch between two functional states, Pr and Pfr, distinguished by E/Z isomers of their bilin chromophore. The chromophore can photoswitch between these states, but also thermally react in darkness. Given the high activation energy of the isomerization reaction in solution, it remains unclear how this reaction can proceed in the dark within the phytochrome. Here, we present time-resolved single-particle cryo-EM structures of the Pseudomonas aeruginosa bacteriophytochrome (PaBphP) captured at multiple time points during dark reversion from Pr to Pfr. We identify structural asymmetries in the precursor Pr state stretching from the homodimer interface to a conserved histidine (H277) next to the bilin and find that these lead to a strong imbalance of the dark reversion reaction rate of the two protomers. Supported by molecular modelling, we conclude that small, protomer-dependent changes of the conserved histidine control the hydrogen-bonding network around the chromophore, thereby exerting control over the activation energy of the isomerization reaction. This mechanism explains how phytochromes can thermally dark revert and how allosteric control is asserted; and it provides a structural framework for tuning phytochrome signaling lifetimes in optogenetic applications.