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Photochemical and mutational analysis of the FMN-binding domains of the plant blue light receptor, phototropin.
- Source :
-
Biochemistry [Biochemistry] 2000 Aug 08; Vol. 39 (31), pp. 9401-10. - Publication Year :
- 2000
-
Abstract
- 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.
- Subjects :
- Amino Acid Sequence
Amino Acid Substitution drug effects
Amino Acid Substitution genetics
Avena metabolism
Binding Sites drug effects
Binding Sites genetics
Circular Dichroism
Computer Simulation
Cryptochromes
Cysteine genetics
Flavin Mononucleotide metabolism
Flavoproteins metabolism
Light
Maleimides pharmacology
Models, Molecular
Molecular Sequence Data
Oxygen
Phosphoproteins chemistry
Phosphoproteins genetics
Phosphoproteins metabolism
Photochemistry
Protein Serine-Threonine Kinases
Protein Structure, Tertiary drug effects
Protein Structure, Tertiary genetics
Receptors, G-Protein-Coupled
Spectrometry, Fluorescence
Spectrophotometry, Ultraviolet
Arabidopsis Proteins
Avena chemistry
Avena genetics
Drosophila Proteins
Eye Proteins
Flavin Mononucleotide chemistry
Flavin Mononucleotide genetics
Flavoproteins chemistry
Flavoproteins genetics
Mutagenesis, Site-Directed
Photoreceptor Cells, Invertebrate
Subjects
Details
- Language :
- English
- ISSN :
- 0006-2960
- Volume :
- 39
- Issue :
- 31
- Database :
- MEDLINE
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 10924135
- Full Text :
- https://doi.org/10.1021/bi000585+