Fluorescence and photochemistry of recombinant phytochrome from the cyanobacterium Synechocystis.

Fluorescence and photochemical properties of phytochrome from the cyanobacterium Synechocystis were investigated in the temperature interval from 293 to 85 K. The apoprotein was obtained by overexpression in Escherichia coli and assembled to a holophytochrome with phycocyanobilin (PCB) and phytochromobilin (P phi B), Syn(PCB)phy and Syn(P phi B)phy, respectively. Its red-absorbing form, Pr, is characterized at 85 K by the emission and excitation maxima at 682 and 666 nm in Syn(PCB)phy and at 690 and 674 nm in Syn(P phi B)phy. At room temperature, the spectra are blue shifted by 5-10 nm. The fluorescence intensity dropped down by approximately 15-20-fold upon warming from 85 to 293 K and activation energy of the fluorescence decay was estimated to be ca 5.4 and 4.9 kJ mol-1 in Syn(PCB)phy and Syn(P phi B)phy, respectively. Phototransformation of Pr upon red illumination was observed at temperatures above 160-170 K in Syn(PCB)phy and above 140-150 K in Syn(P phi B)phy with a 2-3 nm shift of the emission spectrum to the blue and increase of the intensity of its shorter wavelength part. This was interpreted as a possible formation of the photoproduct of the meta-Ra type of the plant phytochrome. At ambient temperatures, the extent of the Pr phototransformation to the far-red-absorbing form, Pfr, was ca 0.7-0.75 and 0.85-0.9 for Syn(PCB)phy and Syn(P phi B)phy, respectively. Fluorescence of Pfr and of the photoproduct similar to lumi-R was not observed. With respect to the photochemical parameters, Syn(PCB)phy and Syn(P phi B)phy are similar to each other and also to a small fraction of phyA (phyA") and to phyB. The latter were shown to have low photochemical activity at low temperatures in contrast to the major phyA pool (phyA'), which is distinguished by the high extent (ca 50%) of Pr phototransformation at 85 K. These photochemical features are interpreted in terms of different activation barriers for the photoreaction in the Pr excited state.