title: On the reaction mechanism of adduct formation in LOV domains of the plant blue-light receptor phototropin.
abstract: The blue-light sensitive photoreceptor, phototropin, is a flavoprotein which regulates the phototropism response of higher plants. The photoinduced triplet state and the photoreactivity of the flavin-mononucleotide (FMN) cofactor in two LOV domains of Avena sativa, Adiantum capillus-veneris, and Chlamydomonas reinhardtii phototropin have been studied by time-resolved electron paramagnetic resonance (EPR) and UV-vis spectroscopy at low temperatures (T < or = 80 K). Differences in the electronic structure of the FMN as reflected by altered zero-field splitting parameters of the triplet state could be correlated with changes in the amino acid composition of the binding pocket in wild-type LOV1 and LOV2 as well as in mutant LOV domains. Even at cryogenic temperatures, time-resolved EPR experiments indicate photoreactivity of the wild-type LOV domains, which was further characterized by UV-vis spectroscopy. Wild-type LOV1 and LOV2 were found to form an adduct between the FMN cofactor and the functional cysteine with a yield of 22% and 68%, respectively. The absorption maximum of the low-temperature photoproduct of wild-type LOV2 is red-shifted by about 15 nm as compared with the FMN C(4a)-cysteinyl adduct formed at room temperature. In light of these observations, we discuss a radical-pair reaction mechanism for the primary photoreaction in LOV domains.
authors.affiliation: Contribution from the Institut für Experimentalphysik, Freie Universität Berlin, Arnimallee 14, 14195 Berlin, Germany.
authors.forename: Erik
authors.initials: E
authors.lastname: Schleicher
authors.forename: Radoslaw M
authors.initials: RM
authors.lastname: Kowalczyk
authors.forename: Christopher W M
authors.initials: CW
authors.lastname: Kay
authors.forename: Peter
authors.initials: P
authors.lastname: Hegemann
authors.forename: Adelbert
authors.initials: A
authors.lastname: Bacher
authors.forename: Markus
authors.initials: M
authors.lastname: Fischer
authors.forename: Robert
authors.initials: R
authors.lastname: Bittl
authors.forename: Gerald
authors.initials: G
authors.lastname: Richter
authors.forename: Stefan
authors.initials: S
authors.lastname: Weber
mesh_terms: D035681:Adiantum / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D018554:Avena / Q000737:chemistry; D016825:Chlamydomonas reinhardtii / Q000737:chemistry; D056931:Cryptochromes; D004578:Electron Spin Resonance Spectroscopy; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism; D005420:Flavoproteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D013056:Spectrophotometry, Ultraviolet
chemical_list: D056931:Cryptochromes; D005420:Flavoproteins; D005486:Flavin Mononucleotide