Resonance Raman study on intact pea phytochrome and its model compounds: evidence for proton migration during the phototransformation

Abstract

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 (Pr) and far-red-light-absorbing form (Pfr) under laser illumination were estimated from the absorption spectra. The most prominent RR band of Pr obtained by 364-nm excitation under 740-nm pumping exhibited a frequency shift between H2O and D2O solutions, but that of Pfr 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 Pr and Pfr, this observation indicates migration of a proton from the chromophore of Pr to the protein moiety of Pfr. As model compounds, octaethylbiliverdin (OEBV-h3), its deuterated and 15N derivatives, and their protonated forms were also studied with both RR and 1H and 15N 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 Pr, suggesting a similar protonated structure for the pyrrolic rings of Pr. The RR spectral difference between OEBV-h3 and OEBV-d3 and that between H2O and D2O solutions of Pfr 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.

Resonance Raman spectra of the intermediates in phototransformation of large phytochrome: Deprotonation of the chromophore in the bleached intermediate

“…The major RR band in the C=C stretching region of P, (1625 and 1626 cm-' for large and intact pea phytochromes, respectively) showed a deuteration shift, while the corresponding bands of Pf, (1631 and 1591 cm-I for large and 1633 and 1591 cm-I for intact pea phytochromes) did not. It was inferred that this difference arose from protonation of the C-ring nitrogen of the tetrapyrrole chromophore in P, (Fodor et al, 1988;Mizutani et al, 1991) and its deprotonation in Pf, (Mizutani et al, 1991). The present results confirmed this proposal and furthermore suggest different protonated structures of the chromophore between P, and Ibl, since the 1599-cm-' band of Ibl did not show a deuteration shift while the 1 625-cm-1 band of P, showed it.…”

“…The R R bands of the red-absorbing (PI) and far-red-absorbing forms (Pf,) of large pea phytochromes at ambient temperatures are almost the same in their frequencies as those of the intact form reported previously (Mizutani et al, 1991). The R R spectrum of large phytochrome excited at 364 nm at -120 OC, where P, and a photointermediate, 1700 (=lumi-R), are trapped, showed a strong band at 1625 cm-l with a shoulder at 1648 cm-l in the C=C stretching region.…”

“…However, application of this technique to phytochrome has long been unsuccessful due to its intense fluorescence. Recently, RR spectra of phytochrome have been measured by a few groups: RR spectra of oat phytochrome with far-red excitation (Fodor et al, 1988(Fodor et al, ,1990Hildebrandt et al, 1992), ofoatphytochrome with surface-enhanced Raman effects upon blue excitation Rospendowski et al, 1989), and of pea phytochrome by using the two-color excitation technique Mizutani et al, 1991). In the last one, we used deep blue excitation for probing Raman scattering to avoid the interference by inherent fluorescence, and simultaneously illuminated the sample by red or far-red light to increse the population of either Pfr or P, in the photosteady-state.…”

Highly Conserved Residues Asp-197 and His-250 in Agp1 Phytochrome Control the Proton Affinity of the Chromophore and Pfr Formation

“…The respective Hill coefficients of 0.85, 1.02, and 0.81 suggest that one proton is involved. Similar spectrophotometric titration experiments with the cyanobacterial phytochrome Cph1 led to a pK a value of 9.5 (12), which was attributed to the deprotonation of the PCB chromophore on the basis of a comparison with the pH-dependent spectra of the model compound octaethylbiliverdin (33). Here we show by RR spectroscopy that in Agp1 the underlying acid-base reaction is also linked to the deprotonation of the chromophore (Fig.…”

Events in the Phytochrome Molecule After Irradiation

“…The assumption of a protonated chromophore not only in the Pr form, but also in the Pfr form as deduced from FT-IR spectra (Siebert et al, 1990), would support such a view of lack of a proton transfer during the photoconversion. This interpretation has, however, been questioned (Tokutomi et al, 1990;Mizutani et al, 1991). If the proposed assignments are correct, the resonance Raman spectra would indicate instead a protonated Pr chromophore and a deprotonated Pfr chromophore.…”