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

Resonance Raman (RR) scattering from type A large phytochrome of pea was measured at cryogenic as well as ambient temperatures to determine an intermediate in which deprotonation of the chromophore takes place. The RR bands of the red-absorbing (Pr) and far-red-absorbing forms (Pfr) 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 RR spectrum of large phytochrome excited at 364 nm at -120 degrees C, where Pr and a photointermediate, I700 (= lumi-R), are trapped, showed a strong band at 1625 cm-1 with a shoulder at 1648 cm-1 in the C C=C stretching region. The shoulder disappeared, and a new band appeared at 1597 cm-1 upon raising the temperature to -80 degrees C, where transformation from I700 to meta-Ra proceeds. The RR spectra remained unchanged until -10 degrees C, indicating that the RR spectra of meta-Rb and meta-Rc are close to that of meta-Ra, and we call them comprehensively the bleached intermediate, Ibl. At ambient temperatures where photo-steady-states among a few species are attained, strong RR bands were observed at 1625 and 1599 cm-1 upon excitation at 364 nm under simultaneous far-red illumination, and the 1599-cm-1 band was appreciably intensified under simultaneous red- instead of far-red illumination. By comparison of these spectra with those at low temperatures, the 1625- and 1599-cm-1 bands were reasonably assigned to Pr and Ibl, respectively. A chemically prepared model of the bleached form, Pbl, also gave a prominent band at 1599 cm-1.(ABSTRACT TRUNCATED AT 250 WORDS)