Ultrafast excited-state isomerization in phytochrome revealed by femtosecond stimulated Raman spectroscopy

Photochemical interconversion between the red-absorbing (P_r) and the far-red-absorbing (P_fr) forms of the photosensory protein phytochrome initiates signal transduction in bacteria and higher plants. The P_r-to-P_fr transition commences with a rapid Z-to-E photoisomerization at the C₁₅ = C₁₆ methine bridge of the bilin prosthetic group. Here, we use femtosecond stimulated Raman spectroscopy to probe the structural changes of the phycocyanobilin chromophore within phytochrome Cph1 on the ultrafast time scale. The enhanced intensity of the C₁₅-H hydrogen out-of-plane (HOOP) mode, together with the appearance of red-shifted CAC stretch and N - H in-plane rocking modes within 500 fs, reveal that initial distortion of the C₁₅ = C₁₆ bond occurs in the electronically excited I* intermediate. From I*, 85% of the excited population relaxes back to Pr in 3 ps, whereas the rest goes on to the Lumi-R photoproduct consistent with the 15% photochemical quantum yield. The C₁₅-H HOOP and skeletal modes evolve to a Lumi-R-like pattern after 3 ps, thereby indicating that the C₁₅ = C₁₆ Z-to-E isomerization occurs on the excited-state surface.