Plant cryptochromes are photosensory receptors that regulate various Central aspects of plant growth and development. These. receptors consist of a photolyase homology :region (PHR) carrying the oxidized flavin adenine dinucleotide (FAD) cofactor, and a tryptochrome C-terminal extension (CCT), which is essential for signaling. Absorption of blue/UVA light leads to formation of the FAD neutral radical as the likely signaling state, and ultimately activates the CCT. Little is known about the Signal transfer from the flavin to the CCT. Here, we investigated the photoreaction of the PHR by time-resolved step-scan FT-IR spectroscopy complemented by UV-vis spectroscopy. The first spectrum at 500 ns shows major contributions from the FAD anion radical, which is demonstrated to then be protonated by aspartic acid, 396 to the neutral radical within: 3.5 mu s. The analysis revealed the existence of three intermediates characterized by:Changes in secondary structure. A marked loss of beta-sheet structure is observed in the second intermediate evolving with a time constant of 500 mu s. This change is accompanied by a conversion of a tyrosine residue, which is identified as the formation of a tyrosine radical in the UV-vis. The only beta-sheet it the PHR is located within the alpha/beta subdomain similar to 25 angstrom away from the flavin. This Subdomain has been previously attributed a role as a putative antenna binding site, but is now Suggested to have evolved: to a component in the signaling of plant cryptochromes by mediating the interaction with the CCT.