The photoconversion of phytochrome (phytochrome A from Avena satina) from the inactive (Pr) to the physiologically active form (Pfr) was studied by near-infrared Fourier transform resonance Raman spectroscopy at cryogenic temperatures, which allow us to trap the intermediate states. Nondeuterated and deuterated buffer solutions were used to determine the effect of H/D exchange on the resonance Raman spectra. For the first time, reliable spectra of the "bleached" intermediates meta-R(A) and meta-R(C) were obtained. The vibrational bands in the region 1300-1700 cm(-)(1), which is particularly indicative of structural changes in tetrapyrroles, were assigned on the basis of recent calculations of the Raman spectra of the chromophore in C-phycocyanin and model compounds [Kneip, C., Hildebrandt, P., Németh, K., Mark, F., Schaffner, K. (1999) Chem. Phys. Lett. 311, 479-485]. The experimental resonance Raman spectra Pr are compatible with the Raman spectra calculated for the protonated ZZZasa configuration, which hence is suggested to be the chromophore structure in this parent state of phytochrome. Furthermore, marker bands could be identified that are of high diagnostic value for monitoring structural changes in individual parts of the chromophore. Specifically, it could be shown that not only in the parent states Pr and Pfr but also in all intermediates the chromophore is protonated at the pyrroleninic nitrogen. The spectral changes observed for lumi-R confirm the view that the photoreaction of Pr is a Z --> E isomerization of the CD methine bridge. The subsequent thermal decay reaction to meta-R(A) includes relaxations of the CD methine bridge double bond, whereas the formation of meta-R(C) is accompanied by structural adaptations of the pyrrole rings B and C in the protein pocket. The far-reaching similarities between the chromophores of meta-R(A) and Pfr suggest that in the step meta-R(A) --> Pfr the ultimate structural changes of the protein matrix occur.

The parent states of the 124-kDa phytochrome (phy A from Avena sativa) and intermediates of its photocycle were studied by low-temperature Fourier-transform resonance Raman spectroscopy. Spectra of the primary photoproducts I700 and lumi-F and of the thermal intermediate meta-F have been obtained for the first time. The spectra of the stable photochromic forms of photochrome, Pr and Pfr, presented in this work are significantly better in signal-to-noise ratio and resolution than previously published spectra, demonstrating the distinct advantages of our experimental approach. The high spectral quality allows for the identification of subtle details of the vibrational band pattern so that the resonance Raman spectra, which have been measured from samples in H2O and D2O, constitute a solid basis for the structural analysis of the various forms of phytochrome. Notwithstanding the current uncertainty in the vibrational assignment of many resonance Raman bands, the spectral changes of the tetrapyrrole chromophore can plausibly be interpreted in terms of conformational changes at two different methine bridges, i.e., torsions around two single bonds and the E/Z isomerization of a double bond. Within the framework of this interpretation, which is based on a vibrational analysis of biliverdin dimethyl ester (Smith, K. Matysik, J., Hlldebrandt, P., & Mark, F. (1993) J. Phys. Chem. 97, 11887-11900), a consistent model is proposed to describe the molecular events in the chromophore during the photocycle. The involvement of a proton transfer in the primary photoprocess of Pr can safely be ruled out. However, previous conclusions concerning the chromophore protonation in the individual states appear premature at the present state of the vibrational assignment. In particular, the attribution of a broad band at 1100 cm-1 to the N-H out-of-plane bending of the protonated pyrrolenin nitrogen (Hildebrandt, P., Hoffmann, A., Lindemann, P., Heibel, G., Braslavsky, S. E., Schaffner, K., & Schrader, B. (1992) Biochemistry 32, 7957-7962) has now been found to be erroneous.