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

Spectroscopic and Thermodynamic Comparisons of Escherichia coli

Escherichia coli DNA photolyase and cryptochrome 1 isolated from Vibrio cholerae, a member of the CRY-DASH family, are directly compared using a variety of experimental methods including UV-vis and Raman spectroscopy, reduction potential measurements, and isothermal titration calorimetry. The semiquinone form of the cryptochrome has an absorption spectrum that is red-shifted from that of the photolyase, but the Raman spectrum indicates that the FAD binding pocket is similar to that of photolyase. The FADH - /FADH· reduction potential of the cryptochrome is significantly higher than that of the photolyase at 164 mV vs NHE, but it also increases upon substrate binding (to 195 mV vs NHE), an increase similar to what is observed in photolyase. The FADH - /FADH· reduction potential for both proteins was found to be insensitive to ATP binding. Isothermal titration calorimetry found that photolyase binds tighter to substrate (K A ∼ 10 5 M -1 for photolyase and ∼10 4 M -1 for cryptochrome 1), and the binding constants for both proteins were slightly sensitive to oxidation state. Based upon this work, it appears that this cryptochrome has significant spectroscopic and electrochemical similarities to CPD photolyase. The thermodynamic cycle of the enzymatic repair in the context of this work is discussed

Photoexcitation of the Blue Light Using FAD Photoreceptor AppA Results in Ultrafast Changes to the Protein Matrix

Photoexcitation of the flavin chromophore in the BLUF photosensor AppA results in a conformational change that leads to photosensor activation. This conformational change is mediated by a hydrogen-bonding network that surrounds the flavin, and photoexcitation is known to result in changes in the network that include a strengthening of hydrogen bonding to the flavin C4-O carbonyl group. Q63 is a key residue in the hydrogen-bonding network, and replacement of this residue with a glutamate results in a photoinactive mutant. While the ultrafast time-resolved infrared (TRIR) spectrum of Q63E AppABLUF is characterized by flavin carbonyl modes at 1680 and 1650 cm–1, which are similar in frequency to the analogous modes from the light activated state of the wild-type protein, a band is also observed in the TRIR spectrum at 1724 cm–1 that is unambiguously assigned to the Q63E carboxylic acid based on U-13C labeling of the protein. Light absorption instantaneously