Detection of a hybrid PrPfr state in the dark reversion of a bathy phytochrome indicates inter-dimer allostery

Phytochromes are photosensor proteins found in plants, fungi, and bacteria. They photoswitch between red light absorbing (Pr) and far-red light absorbing (Pfr) states. Thermal reversion in the dark, however, is an equally important factor in controlling their signaling levels. Phytochromes are generally dimeric proteins, therefore, mixed PrPfr states are possible. These states have been implicated in dark reversion studies of plant phytochromes, but not in bacterial phytochromes. Here, we investigate the dark reversion kinetics of the 'bathy' phytochrome from P. aeruginosa (PaBphP) using UV-Vis absorption spectroscopy. A single set of time-resolved spectra does not conclusively reveal the presence of a mixed PrPfr state, as both a direct Pr → Pfr model or a sequential Pr → PrPfr → Pfr model fit the spectral kinetics. However, a systematic analysis of dark reversion kinetics with varying Pr/Pfr ratios can only be satisfactorily fit by the sequential model, which indicates the presence of an intermediate PrPfr state. A newly designed monomeric variant of PaBphP provides strong support for this interpretation. Temperature-dependent kinetics revealed similarly low activation energies for the dark reversion processes of both proteins, consistent with a previously proposed keto-enol tautomerization preceding dark reversion. Interestingly, our results suggest allosteric regulation of dark reversion across the dimer, which we propose to be a contributing factor in phytochrome signaling.