Photochemical and Mutational Analysis of the FMN-Binding Domains of the Plant Blue Light Receptor, Phototropin^,

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.

Molecular mechanism of light responses in Neurospora: from light-induced transcription to photoadaptation

“…After activation by light, phototropins undergo rapid photocycles (from seconds to minutes) and return to the dark state, so that they can be repeatedly activated by light (Christie et al 1999;Salomon et al 2000;Briggs and Christie 2002;Harper et al 2003). Since only a small portion of the WCC purified from the insect cells contained FAD, this prevented us from carrying out further photochemical analysis of the complex in vitro.…”

“…Like many other photoreceptors, phototropins undergo rapid photocycles in the dark after their light activation to return to the dark state (Salomon et al 2000), so that they can be repeatedly activated by light. Whether WC-1 has a photocycle and how its photochemical property affects its function in light responses are not known.…”

Phototropin Receptor Kinase Activation by Blue Light

“…7 In the dark, LOV domains bind FMN non-covalently forming a spectral species, LOV 447 , which absorbs maximally near 447 nm. 7,8 Irradiation of the domain induces the formation of a covalent bond between the C(4a) carbon of the FMN and the sulfur atom of a nearby, conserved cysteine residue within the domain. Cysteinyl adduct formation occurs within microseconds of illumination and produces a spectral species, LOV 390 , that absorbs maximally near 390 nm.…”

“…8 Mutation of the afore-mentioned active-site cysteine to either alanine or serine results in a loss of photochemical reactivity of the LOV domain. 7,8 Moreover, this mutation has been used successfully to probe the roles of LOV1 and LOV2 in regulating phototropin activity and function. [9][10][11][12] As reported previously (ref.…”

“…6 Bacterially expressed LOV domains are photochemically reactive as monitored by absorbance spectroscopy. 7 In the dark, LOV domains bind FMN non-covalently forming a spectral species, LOV 447 , which absorbs maximally near 447 nm. 7,8 Irradiation of the domain induces the formation of a covalent bond between the C(4a) carbon of the FMN and the sulfur atom of a nearby, conserved cysteine residue within the domain.…”