The Primary Photophysics of the Avena sativa Phototropin 1 LOV2 Domain Observed with Time‐resolved Emission Spectroscopy^†

Abstract

The phototropins are blue-light receptors that base their light-dependent action on the reversible formation of a covalent bond between a flavin mononucleotide (FMN) cofactor and a conserved cysteine in light, oxygen or voltage (LOV) domains. The primary reactions of the Avena sativa phototropin 1 LOV2 domain were investigated by means of time-resolved and low-temperature fluorescence spectroscopy. Synchroscan streak camera experiments revealed a fluorescence lifetime of 2.2 ns in LOV2. A weak long-lived component with emission intensity from 600 to 650 nm was assigned to phosphorescence from the reactive FMN triplet state. This observation allowed determination of the LOV2 triplet state energy level at physiological temperature at 16600 cm−1. FMN dissolved in aqueous solution showed pH-dependent fluorescence lifetimes of 2.7 ns at pH 2 and 3.9–4.1 ns at pH 3 to 8. Here, too, a weak phosphorescence band was observed. The fluorescence quantum yield of LOV2 increased from 0.13 to 0.41 upon cooling the sample from 293 to 77 K. A pronounced phosphorescence emission around 600 nm was observed in the LOV2 domain between 77 and 120 K in the steady-state emission.

Bright blue-shifted fluorescent proteins with Cys in the GAF domain engineered from bacterial phytochromes: fluorescence mechanisms and excited-state dynamics

“…Time-resolved fluorescence measurements were carried out with a streak camera setup, as described previously373839 at room temperature (~20 °C). Femtosecond laser pulses from an integrated Ti:Sapphire oscillator (Vitesse, Coherent, 800 nm, 80 MHz, 100 fs) was incorporated into a Ti:Sapphire regenerative amplifier (RegA, Coherent, 800 nm, 50 kHz, 100 fs).…”

Structure and Function of the ZTL/FKF1/LKP2 Group Proteins in Arabidopsis

“…In crystal structures of LOV proteins, typically two orientations of the active site Cys residue are observed with only the conformation placing the cysteine thiol moiety above the C4a position competent for photoactivation [55,57]. Following excitation by blue-light, the flavin is promoted to an excited singlet state that rapidly converts to a triplet species [53,54,58,59]. The flavin triplet then causes an electron transfer event between the flavin and active site Cys to generate a flavin-semiquinone and a Cys-radical [37,58].…”

Quantum Mechanics/Molecular Mechanics Study on the Photoreactions of Dark‐ and Light‐Adapted States of a Blue‐Light YtvA LOV Photoreceptor

“…Thel ight-adapted signaling state of light-activated LOVp hotoreceptors is generated through the formation of af lavin-(C4a)-cysteinyl covalent bond with ac onserved cysteine residue in the active site of LOVd omains (Figure 1). [5,6] In the past decade,t he excited-state dynamics of the dark-adapted state of LOVp hotoreceptors has been thoroughly investigated experimentally [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] while there have been less such studies on the light-adapted state.…”