Photochemical and mutational analysis of the FMN-binding domains of the plant blue light receptor, phototropin.
The plant photoreceptor phototropin is an autophosphorylating serine-threonine protein kinase activated by UV-A/blue light. Two domains, LOV1 and LOV2, members of the PAS domain superfamily, mediate light sensing by phototropin. Heterologous expression studies have shown that both domains function as FMN-binding sites. Although three plant blue light photoreceptors, cry1, cry2, and phototropin, have been identified to date, the photochemical reactions underlying photoactivation of these light sensors have not been described so far. Herein, we demonstrate that the LOV domains of Avena sativa phototropin undergo a self-contained photocycle characterized by a loss of blue light absorbance in response to light and a spontaneous recovery of the blue light-absorbing form in the dark. Rate constants and quantum efficiencies for the photoreactions indicate that LOV1 exhibits a lower photosensitivity than LOV2. The spectral properties of the photoproduct produced for both LOV domains are unrelated to those found for photoreduced flavins and flavoproteins, but are consistent with those of a flavin-cysteinyl adduct. Flavin-thiol adducts are generally short-lifetime reaction intermediates formed during the flavoprotein-catalyzed reduction of protein disulfides. By site-directed mutagenesis, we have identified several amino acid residues within the putative chromophore binding site of LOV1 and LOV2 that appear to be important for FMN binding and/or the photochemical reactivity. Among those is Cys39, which plays an important role in the photochemical reaction of the LOV domains. Replacement of Cys39 with Ala abolished the photochemical reactions of both LOV domains. We therefore propose that light sensing by the phototropin LOV domains occurs via the formation of a stable adduct between the FMN chromophore and Cys39.
Botanisches Institut der Universität München, Germany.
M
M
Salomon
J M
JM
Christie
E
E
Knieb
U
U
Lempert
W R
WR
Briggs
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000187:drug effects / Q000235:genetics; D029681:Arabidopsis Proteins; D018554:Avena / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites / Q000187:drug effects / Q000235:genetics; D002942:Circular Dichroism; D003198:Computer Simulation; D056931:Cryptochromes; D003545:Cysteine / Q000235:genetics; D029721:Drosophila Proteins; D005136:Eye Proteins; D005486:Flavin Mononucleotide / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D005420:Flavoproteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008027:Light; D008301:Maleimides / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010100:Oxygen; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010777:Photochemistry; D017956:Photoreceptor Cells, Invertebrate; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary / Q000187:drug effects / Q000235:genetics; D043562:Receptors, G-Protein-Coupled; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D029681:Arabidopsis Proteins; D056931:Cryptochromes; D029721:Drosophila Proteins; D005136:Eye Proteins; D005420:Flavoproteins; D008301:Maleimides; D010750:Phosphoproteins; D043562:Receptors, G-Protein-Coupled; C119828:cry protein, Drosophila; D005486:Flavin Mononucleotide; C028525:N-phenylmaleimide; C103646:NPH1 protein, Arabidopsis; D017346:Protein Serine-Threonine Kinases; D003545:Cysteine; D010100:Oxygen