Protonation state and structural changes of the tetrapyrrole chromophore during the P-r -> P-fr phototransformation of phytochrome: A resonance Ramanspectroscopic study

The photoconversion of phytochrome (phytochrome A from Avena satina) from t he inactive (P-r) to the physiologically active form (P-fr) was studied by near-infrared Fourier transform resonance Raman spectroscopy at cryogenic t emperatures, which allow us to trap the intermediate states. Nondeuterated and deuterated buffer solutions were used to determine the effect of H/D ex change 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 v ibrational bands in the region 1300-1700 cm(-1), which is particularly indi cative of structural changes in tetrapyrroles, were assigned on the basis o f recent calculations of the Raman spectra of the chromophore in C-phycocya nin and model compounds [Kneip, C., Hildebrandt, P., Nemeth, K., Mark, F., Schaffner, K. (1999) Chem. Phys. Lett. 311, 479-485]. The experimental reso nance Raman spectra P, are compatible with the Raman spectra calculated for the protonated ZZZasa configuration, which hence is suggested to be the ch romophore structure in this parent state of phytochrome. Furthermore, marke r bands could be identified that are of high diagnostic value for monitorin g structural changes in individual parts of the chromophore. Specifically, it could be shown that not only in the parent states P, and P-fr but also i n all intermediates the chromophore is protonated at the pyrroleninic nitro gen. The spectral changes observed for lumi-R confirm the view that the pho toreaction of P-r is a Z --> E isomerization of the CD methine bridge. The subsequent thermal decay reaction to meta-RA includes relaxations of the CD methine bridge double bond, whereas the formation of meta-R-C is accompani ed by structural adaptations of the pyrrole rings B and C in the protein po cket. The far-reaching similarities between the chromophores of meta-R-A, a nd P-fr suggest that in the step meta-R-A --> P-fr the ultimate structural changes of the protein matrix occur.