Resonance Raman (RR) scattering from large pea phytochrome was observed at ambient temperature for the first time by using a micro-spinning cell and the two color excitation technique. The relative population of the red-absorbing form (Pr), the far red-absorbing form (Pfr), and the bleached intermediate (Ibl) under laser illumination was estimated from the absorption spectra. The RR spectrum of Pr obtained by the 363.8 nm excitation under the 740.0 nm pumping exhibited a frequency shift between the H2O and D2O solutions, but those of Pfr and Ibl obtained by the 406.7 nm excitation under the 632.8 nm pumping did not, indicating a distinct difference in their protonation levels, presumably in the protonation of ring C.
