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 chromophore structures in the parent states Pr and Pfr as well as in the photocycle intermediate Lumi-R of oat phytochrome phyA are determined by comparison of the experimental resonance Raman spectra with calculated Raman spectra that have been obtained by density functional theory calculations (B3LYP) using scaled force fields. The spectra were calculated for various tetrapyrrole geometries including more than twenty different methine bridge isomers. For the parent states Pr and Pfr the best agreement in terms of vibrational frequencies, isotopic shifts, and Raman intensities was achieved with the ZZZasa and ZZEssa geometry, respectively. For the first intermediate Lumi-R, the chromophore geometry is concluded to be the ZZEasa configuration. These finding imply that the primary step of the photoactivation of phytochrome is the Z/E isomerization of the C-D methine bridge double bond, whereas the single bond remains in the anti conformation. The subsequent transition to the physiologically active state Pfr includes a (partial) single bond rotation of the A-B methine bridge.

A phytochrome-encoding cDNA from the cyanobacterium Synechocystis has been heterologously expressed in Escherichia coli and reconstituted into functional chromoproteins by incubation with either phycocyanobilin (PCB) or phytochromobilin (PPhiB). These materials were studied by Raman spectroscopy and nanosecond flash photolysis. The Raman spectra suggest far-reaching similarities in chromophore configuration and conformation between the Pfr forms of Synechocystis phytochrome and the plant phytochromes (e.g. phyA from oat), but some differences, such as torsions around methine bridges and in hydrogen bonding interactions, in the Pr state. Synechocystis phytochrome (PCB) undergoes a multistep photoconversion reminiscent of the phyA Pr --> Pfr transformation but with different kinetics. The first process resolved is the decay of an intermediate with red-shifted absorption (relative to parent state) and a 25-micros lifetime. The next observable intermediate grows in with 300 (+/-25) micros and decays with 6-8 ms. The final state (Pfr) is formed biexponentially (450 ms, 1 s). When reconstituted with PPhiB, the first decay of this Synechocystis phytochrome is biexponential (5 and 25 micros). The growth of the second intermediate is slower (750 micros) than that in the PCB adduct whereas the decays of both species are similar. The formation of the Pfr form required fitting with three components (350 ms, 2.5 s, and 11 s). H/D Exchange in Synechocystis phytochrome (PCB) delays, by an isotope effect of 2.7, both growth (300 micros) and decay rates (6-8 ms) of the second intermediate. This effect is larger than values determined for phyA (ca. 1.2) and is characteristic of a rate-limiting proton transfer. The formation of the Pfr state of the PCB adduct of Synechocystis phytochrome shows a deuterium effect similar as phyA (ca. 1.2). Activation energies of the second intermediate in the range 0-18 degrees C are 44 (in H2O/buffer) and 48 kJ mol-1 (D2O), with essentially identical pre-exponential factors.