Primary Reactions of the LOV2 Domain of Phototropin, a Plant Blue-Light Photoreceptor

Publisher Website
Google Scholar
Abstract
The phototropins constitute an important class of plant photoreceptor kinases that control a range of physiological responses, including phototropism, light-directed chloroplast movement, and light-induced stomatal opening. The LOV2 domain of phototropin binds a molecule of flavin mononucleotide (FMN) and undergoes a photocycle involving light-driven covalent adduct formation between a conserved cysteine residue and the C(4a) atom of FMN. This product state promotes C-terminal kinase activation and downstream signal transduction. Here, we report the primary photophysics and photochemistry of LOV2 domains of phototropin 1 of Avena sativa (oat) and of the phy3 photoreceptor of Adiantum capillus-veneris (maidenhair fern). In agreement with earlier reports [Swartz, T. E., et al. (2001) J. Biol. Chem. 276, 36493-36500], we find that the FMN triplet state is the reactive species from which the photoreaction occurs. We demonstrate that the triplet state is the primary photoproduct in the LOV2 photocycle, generated at 60% efficiency. No spectroscopically distinguishable intermediates precede the FMN triplet on the femtosecond to nanosecond time scale, indicating that it is formed directly via intersystem crossing (ISC) from the singlet state. Our results indicate that the majority of the FMN triplets in the LOV2 domain exist in the protonated form. We propose a reaction mechanism that involves excited-state proton transfer, on the nanosecond time scale or faster, from the sulfhydryl group of the conserved cysteine to the N5 atom of FMN. This event promotes adduct formation by increasing the electrophilicity of C(4a) and subsequent nucleophilic attack by the cysteine's thiolate anion. Comparison to free FMN in solution shows that the protein environment of LOV2 increases the ISC rate of FMN by a factor of 2.4, thus improving the yield of the cysteinyl-flavin adduct and the efficiency of phototropin-mediated signaling processes.