RESONANCE RAMAN-STUDY ON INTACT PEA PHYTOCHROME AND ITS MODEL COMPOUNDS - EVIDENCE FOR PROTON MIGRATION DURING THE PHOTOTRANSFORMATION

Resonance Raman (RR) scattering from intact pea phytochrome was observed in resonance with the blue band at ambient temperature. The relative populations of the red-light-absorbing form (P(r)) and far-red-light-absorbing form (P(fr)) under laser illumination were estimated from the absorption spectra. The most prominent RR band of P(r) obtained by 364-nm excitation under 740-nm pumping exhibited a frequency shift between H2O and D2O solutions, but that of P(fr) obtained by 407-nm excitation under 633-nm pumping did not, indicating a distinct difference in a protonation state of their chromophores. Since the protonation level of a whole molecule of intact phytochrome remains unchanged between P(r) and P(fr), this observation indicates migration of a proton from the chromophore of P(r) to the protein moiety of P(fr). As model compounds, octaethylbiliverdin (OEBV-h3), its deuterated and N-15 derivatives, and their protonated forms were also studied with both RR and H-1 and N-15 NMR spectroscopies. The RR spectrum of the protonated form, for which the protonation site was determined to be C-ring pyrrole nitrogen by NMR, displayed a deuteration shift corresponding to that of P(r) suggesting a similar protonated structure for the pyrrolic rings of P(r). The RR spectral difference between OEBV-h3 and OEBV-d3 and that between H2O and D2O solutions of P(fr) suggested that the N-H protons of the A-, B-, and D-rings of intact phytochrome are replaced with deuterons in D2O. A role of the 7-kDa segment of phytochrome is discussed on the basis of RR spectral differences between the intact and large phytochromes.