Blue light induces radical formation and autophosphorylation in the light-sensitive domain of Chlamydomonas cryptochrome.
Cryptochromes are sensory blue light receptors mediating various responses in plants and animals. Studies on the mechanism of plant cryptochromes have been focused on the flowering plant Arabidopsis. In the genome of the unicellular green alga Chlamydomonas reinhardtii, a single plant cryptochrome, Chlamydomonas photolyase homologue 1 (CPH1), has been identified. The N-terminal 500 amino acids comprise the light-sensitive domain of CPH1 linked to a C-terminal extension of similar size. We have expressed the light-sensitive domain heterologously in Escherichia coli in high yield and purity. The 59-kDa protein bears exclusively flavin adenine dinucleotide in its oxidized state. Illumination with blue light induces formation of a neutral flavin radical with absorption maxima at 540 and 580 nm. The reaction proceeds aerobically even in the absence of an exogenous electron donor, which suggests that it reflects a physiological response. The process is completely reversible in the dark and exhibits a decay time constant of 200 s in the presence of oxygen. Binding of ATP strongly stabilizes the radical state after illumination and impedes the dark recovery. Thus, ATP binding has functional significance for plant cryptochromes and does not merely result from structural homology to DNA photolyase. The light-sensitive domain responds to illumination by an increase in phosphorylation. The autophosphorylation takes place although the protein is lacking its native C-terminal extension. This finding indicates that the extension is dispensable for autophosphorylation, despite the role it has been assigned in mediating signal transduction in Arabidopsis.
Immeln
Dominik
D
Institute of Neurosciences and Biophysics-2, Molecular Biophysics, Research Center Jülich, Jülich, Germany.
Schlesinger
Ramona
R
Heberle
Joachim
J
Kottke
Tilman
T
D000818:Animals; D002696:Chlamydomonas / Q000502:physiology* / Q000528:radiation effects*; D056931:Cryptochromes; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005420:Flavoproteins / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D005609:Free Radicals; D007700:Kinetics; D008027:Light; D055537:Light Signal Transduction; D010766:Phosphorylation; D015800:Protozoan Proteins / Q000378:metabolism* / Q000528:radiation effects; D011994:Recombinant Proteins / Q000378:metabolism / Q000528:radiation effects
D056931:Cryptochromes; D016054:DNA, Protozoan; D005420:Flavoproteins; D005609:Free Radicals; D015800:Protozoan Proteins; D011994:Recombinant Proteins