Transient IR spectroscopy identifies key interactions and unravels new intermediates in the photocycle of a bacterial phytochrome.

Phytochromes are photosensory proteins in plants, fungi, and bacteria, which detect red- and far-red light. They undergo a transition between the resting (Pr) and photoactivated (Pfr) states. In bacterial phytochromes, the Pr-to-Pfr transition is facilitated by two intermediate states, called Lumi-R and Meta-R. The molecular structures of the protein in these states are not known and the molecular mechanism of photoconversion is not understood. Here, we apply transient infrared absorption spectroscopy to study the photocycle of the wild-type and Y263F mutant of the phytochrome from Deinococcus radiodurans (DrBphP) from nanoto milliseconds. We identify two sequentially forming Lumi-R states which differ in the local structure surrounding the carbonyl group of the biliverdin D-ring. We also find that the tyrosine at position 263 alters local structure and dynamics around the D-ring and causes an increased rate of Pfr formation. The results shed new light on the mechanism of light-signalling in phytochrome proteins.