Primary intermediate in the photocycle of a blue-light sensory BLUF FAD-protein, Tll0078, of Thermosynechococcus elongatus BP-1.
Proteins with a BLUF (sensor of blue light using flavin adenine dinucleotide) domain represent a newly recognized class of photoreceptors that is widely distributed in the genomes of photosynthetic bacteria, cyanobacteria, and Euglena. Recently, Okajima et al. [Okajima, K., Yoshihara, S., Geng, X., Katayama, M. and Ikeuchi, M. (2003) Plant Cell Physiol. 44 (Suppl), 162] purified BLUF protein Tll0078 encoded in the genome of thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 by expressing the protein in Escherichia coli. We investigated the photocycle of Tll0078 by measuring the picosecond fluorescence kinetics, transient absorption changes, and the UV-visible absorption spectra at 10 to 330 K. The absorption spectrum of the FAD moiety of Tll0078 showed a 10-nm red shift upon illumination at 278-330 K. The quantum efficiency of the formation of the red-shifted form was 29%. Illumination at 10 K, on the other hand, caused only a 5-nm red shift in about one-half of the protein population. The 5-nm-shifted form was stable at 10 K. The 5-nm red-shifted form was converted into the 10-nm red-shifted form at 50-240 K upon warming in the dark. At room temperature, the 10-nm red-shifted final product appeared within 10 ns after laser flash excitation. The lifetime of the fluorescence of FAD was found to be 120 ps at room temperature. These results reveal a fast and efficient photoconversion process from the singlet-excited state to the final product at room temperature. A photocycle of BLUF protein is proposed that includes the 5-nm red-shifted intermediate form as the precursor for the 10-nm red-shifted final product. The temperature dependence of each step of the photocycle is also discussed.
Fukushima
Yoshimasa
Y
Division of Material Science, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan.
Okajima
Koji
K
Shibata
Yutaka
Y
Ikeuchi
Masahiko
M
Itoh
Shigeru
S
D001483:Base Sequence; D000458:Cyanobacteria / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects*; D004269:DNA, Bacterial / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D016680:Genome, Bacterial; D010777:Photochemistry; D020283:Photoreceptors, Microbial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013816:Thermodynamics
D004269:DNA, Bacterial; D020283:Photoreceptors, Microbial; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide