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

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.