Blue light perception in plants. Detection and characterization of a light-induced neutral flavin radical in a C450A mutant of phototropin.
The LOV2 domain of Avena sativa phototropin and its C450A mutant were expressed as recombinant fusion proteins and were examined by optical spectroscopy, electron paramagnetic resonance, and electron-nuclear double resonance. Upon irradiation (420-480 nm), the LOV2 C450A mutant protein gave an optical absorption spectrum characteristic of a flavin radical even in the absence of exogenous electron donors, thus demonstrating that the flavin mononucleotide (FMN) cofactor in its photogenerated triplet state is a potent oxidant for redox-active amino acid residues within the LOV2 domain. The FMN radical in the LOV2 C450A mutant is N(5)-protonated, suggesting that the local pH close to the FMN is acidic enough so that the cysteine residue in the wild-type protein is likely to be also protonated. An electron paramagnetic resonance analysis of the photogenerated FMN radical gave information on the geometrical and electronic structure and the environment of the FMN cofactor. The experimentally determined hyperfine couplings of the FMN radical point to a highly restricted delocalization of the unpaired electron spin in the isoalloxazine moiety. In the light of these results a possible radical-pair mechanism for the formation of the FMN-C(4a)-cysteinyl adduct in LOV domains is discussed.
Kay
Christopher W M
CW
Institut für Experimentalphysik, Freie Universität Berlin, Arnimallee 14, 14195 Berlin, Germany.
Schleicher
Erik
E
Kuppig
Andreas
A
Hofner
Heidi
H
Rüdiger
Wolfhart
W
Schleicher
Michael
M
Fischer
Markus
M
Bacher
Adelbert
A
Weber
Stefan
S
Richter
Gerald
G
D018554:Avena / Q000502:physiology*; D056931:Cryptochromes; D029721:Drosophila Proteins; D004578:Electron Spin Resonance Spectroscopy; D005136:Eye Proteins; D005415:Flavins / Q000096:biosynthesis* / Q000737:chemistry; D005420:Flavoproteins / Q000737:chemistry* / Q000235:genetics; D008027:Light; D008969:Molecular Sequence Data; D017956:Photoreceptor Cells, Invertebrate; D010957:Plasmids; D043562:Receptors, G-Protein-Coupled; D013056:Spectrophotometry, Ultraviolet
D056931:Cryptochromes; D029721:Drosophila Proteins; D005136:Eye Proteins; D005415:Flavins; D005420:Flavoproteins; D043562:Receptors, G-Protein-Coupled; C119828:cry protein, Drosophila