Protonation State and Structural Changes of the Tetrapyrrole Chromophore during the P_r→ P_frPhototransformation of Phytochrome:  A Resonance Raman Spectroscopic Study

Abstract

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.

Structure of the Biliverdin Cofactor in the Pfr State of Bathy and Prototypical Phytochromes

“…Although the first peak constitutes a well known marker band for the tetrapyrrole backbone conformation, the high intensity of the second peak is characteristic for tetrapyrrole chromophores in the Pfr state. In addition, the presence of the band at 1549 cm Ϫ1 , which is sensitive to H/D exchange, is indicative of a protonated tetrapyrrole, as already demonstrated by previous spectroscopic studies on phytochromes (58,59).…”

Structure of the Cyanobacterial Phytochrome 2 Photosensor Implies a Tryptophan Switch for Phytochrome Signaling

“…In particular, His 130 and His 160 , lying in the vicinity of the chromophore, were modeled with a hydrogen atom at the ⑀-nitrogen and ␦-nitrogen, respectively. In addition, based on spectroscopic evidence (27,28) the PCB chromophore is nitrogen-protonated, whereas the propionic side chains are deprotonated yielding a total charge of Ϫ1e.…”

Characterization of Two Thermostable Cyanobacterial Phytochromes Reveals Global Movements in the Chromophore-binding Domain during Photoconversion

“…The near-infrared excitation line was sufficiently close to the first electronic transition to generate a strong pre-resonance enhancement of the chromophoric vibrational bands such that Raman bands of the protein matrix remained very weak in the spectra of the parent states (18,43). All spectra were measured at Ϫ140°C using a liquid nitrogencooled cryostat (Linkam, Waterfield, Surrey, UK).…”