Magdalena Gauden

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

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 3–8. 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.

The photochemistry of novel flavin-binding photoreceptors

Blue-light sensing using FAD (BLUF) domains are a distinct family of flavin-binding photoreceptors that show no significant relationship to other sensor proteins in sequence or structure. AppA is a two-component protein that can sense blue light at its N-terminal domain, and oxygen tension at its cysteine-rich C-terminal domain, and is shown to control photosynthesis gene expression in response to high-intensity blue-light irradiation and variation of the oxygen tension. This chapter uses femtosecond transient absorption spectroscopy to investigate the photochemical events that lead to the formation of the redshifted signaling state in the AppA and Synechocystis Slr1694 BLUF domains. The time-resolved data are globally analyzed in terms of a kinetic scheme with sequentially interconverting species. The global analysis procedure indicates that six components are required for an adequate description of the time-resolved data.