Spectroscopic Characterization of Flavin Mononucleotide Bound to the LOV1 Domain of Phot1 from Chlamydomonas reinhardtii¶

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Abstract
The absorption and emission behavior of flavin mononucleotide (FMN) in the light‐, oxygen‐ and voltage‐sensitive (LOV) domain LOV1 of the photoreceptor Phot1 from the green alga Chlamydomonas reinhardtii was studied. The results from the wild‐type (LOV1‐WT) were compared with those from a mutant in which cysteine 57 was replaced by serine (LOV1‐C57S), and with free FMN in aqueous solution. A fluorescence quantum yield of φF= 0.30 and a fluorescence lifetime of τF= 4.6 ns were determined for FMN in the mutant LOV1‐C57S, whereas these quantities are reduced to about φF= 0.17 and τF= 2.9 ns for LOV1‐WT, indicating an enhanced intersystem crossing in LOV1‐WT because of the adjacent sulfur of C57. A single‐exponential fluorescence decay was observed in picosecond laser time‐resolved fluorescence measurements for both LOV1‐WT and LOV1‐C57S as expected for excited singlet state relaxation by intersystem crossing and internal conversion. An excitation intensity dependent fluorescence signal saturation was observed in steady‐state fluorescence measurements for LOV1‐WT, which is thought to be because of the formation of a long‐lived intermediate flavin‐C(4a)–cysteinyl adduct in the triplet state (few microseconds triplet lifetime, adduct lifetime around 150 s). No photobleaching was observed for LOV1‐C57S, because no thiol group is present in the vicinity of FMN for an adduct formation.