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 Synechocystis reconstituted with phycocyanobilin were investigated by light-induced optical and Fourier-transform infrared (FT-IR) difference spectroscopy at low temperatures for the first time. The photochemistry was found to be deferred below -100 degrees C for the transformation of red-absorbing form of phytochrome (Pr)-->far-red-absorbing form of phytochrome (Pfr), and no formation of an intermediate similar to the photoproduct of phytochrome A obtained at -140 degrees C (lumi-R) was observed. Two intermediates could be stabilized below -40 degrees C and between -40 and -20 degrees C, and were denoted as meta-Ra and meta-Rc, respectively. Above -20 degrees C Pfr was obtained. In the reverse reaction two intermediates could be stabilized below -60 degrees C (lumi-F) and between -60 and -40 degrees C (meta-F). The FT-IR difference spectra of the late Pr-->Pfr photoreaction show great similarities to the spectra obtained from oat phytochrome A suggesting similar conformation of the chromophore and interactions with its protein environment, whereas deviations in the spectra of meta-Ra were observed. A large band around 1700 cm-1 in the difference spectra between the intermediates and Pr which is tentatively assigned to the C19=O group of the prosthetic group indicates the Z,E isomerization around the C15=C16-methine bridge of the chromophore during the formation of meta-Ra. In the difference spectra 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 Pfr. Since the FT-IR difference spectra between lumi-F and Pfr show great similarities to the spectra of the parent states, it is assumed that during the formation of lumi-F the chromophore largely returns into the primary Pr conformation. The FT-IR spectra recorded in a medium of 2H2O generally show a downshift of the significant bands due to the isotope effect. The appearance of a characteristic band around 935 cm-1 in all 2H2O spectra suggests an assignment to an N-2H bending vibration of the chromophore.
Foerstendorf
H
H
Institut für Biophysik und Strahlenbiologie, Albert-Ludwigs Universität, Freiburg, Germany.
Lamparter
T
T
Hughes
J
J
Gärtner
W
W
Siebert
F
F
D003080:Cold Temperature; D000458:Cyanobacteria / Q000737:chemistry*; D010777:Photochemistry; D010834:Phytochrome / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods
D011994:Recombinant Proteins; D010834:Phytochrome