The photoreactions of recombinant phytochrome from the cyanobacterium Synechocystis: A low-temperature UV-Vis and FT-IR spectroscopic study

The interconvertible photoreactions of recombinant phytochrome from Synecho cystis reconstituted with phycocyanobilin were investigated by light-induce d optical and Fourier-transform infrared (FT-IR) difference spectroscopy at low temperatures for the first time. The photochemistry was found to be de ferred below -100 degrees C for the tranformation of red-absorbing form of phytochrome (Pr) --> far-red-absorbing form of phytochrome (Pfr), and no fo rmation of an intermediate similar to the photoproduct of phytochrome A obt ained at -140 degrees C (lumi-R) was observed. Two intermediates could be s tabilized below -40 degrees C and between -40 and -20 degrees C, and were d enoted as meta-R-a and meta-R-c, respectively. Above -20 degrees C Pfr was obtained. In the reverse reaction two intermediates could be stabilized bel ow -60 degrees C (lumi-F) and between -60 and -40 degrees C (meta-F). The F T-IR difference spectra of the late Pr --> Pfr photoreaction show great sim ilarities to the spectra obtained from oat phytochrome A suggesting similar conformation of the chromophore and interactions with its protein environm ent, whereas deviations in the spectra of meta-R-a were observed, A large b and around 1700 cm(-1) in the difference spectra between the intermediates and Pr which is tentatively assigned to the C-19=O group of the prosthetic group indicates the Z,E isomerization around the C-15=C-16-methine bridge o f the chromophore during the formation of meta-R-a, In the difference spect ra of the parent states only small differences are observed in this region suggesting that the frequency of the carbonyl group is similar in Pr and Pf r, Since the FT-IR difference spectra between lumi-F and Pfr show great sim ilarities to the spectra of the parent states, it is assumed that during th e formation of lumi-F the chromophore largely returns into the primary Pr c onformation. The FT-IR spectra recorded in a medium of (H2O)-H-2 generally show a downshift of the significant bands due to the isotope ef-feet. The a ppearance of a characteristic band around 935 cm(-1) in all (H2O)-H-2 spect ra suggests an assignment to an N-H-2 bending vibration of the chromophore.