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.

Primary Reactions of the LOV2 Domain of Phototropin Studied with Ultrafast Mid-Infrared Spectroscopy and Quantum Chemistry

“…The frequencies of the bleaching bands agree with those recorded with FTIR, Raman, and fluorescence line-narrowing spectroscopy on AsLOV2 and several other LOV domains upon covalent adduct formation (5,35,41,42). The observation of a single-exponential decay of the FMN singlet excited state in AsLOV2 agrees with that observed for essentially all LOV domains (8,9,43,44), in contrast with the multiexponential excited-state decays found in the flavin adenine dinucleotide (FAD)-binding blue-light sensing using FAD (BLUF) domains (34,(45)(46)(47). The 1.5-ns time constant that was found here fairly agrees with that obtained previously with ultrafast visible spectroscopy (2.0 ns (8)).…”

“…The early steps of the LOV photocycle after photon absorption involve the intersystem crossing of the FMN singlet excited state to the FMN triplet state on a nanosecond timescale (8,9), after which, covalent adduct formation proceeds without apparent intermediates on a microsecond timescale (10)(11)(12)(13)(14)(15). In contrast to the apparent consensus on the spectroscopically distinguishable intermediates in the LOV photocycle, the mechanism by which covalent adduct formation occurs in the LOV domains is a matter of considerable debate.…”

Functional Characterization of a LOV‐Histidine Kinase Photoreceptor from Xanthomonas citri subsp. citri

“…Xcc‐LOV behaved differently to other LOV proteins studied so far by LIOAS as neither the structural volume change associated with triplet formation (Δ V T ) could be associated to a sharp contraction, nor was evident a contraction on a slower time scale associated with photoadduct formation (Δ V 390 ) . Curiously, the C76S mutation produced a quite large volume contraction upon triplet formation, opposite to free FMN and to the corresponding mutation in the instrumental cysteine of C. reinhardtii phototropin LOV1 ( Cr LOV1) . Notably, also in YtvA the corresponding C62S mutation induces a larger Δ V T than in the wild‐type protein (Carmen Mandalari, unpublished data), which follows the same trend as seen here for Xcc‐LOV.…”

“…The values of ΔV T are normally around −1 to −1.5 mL mol −1 , while Δ V 390 is ca −10 mL mol −1 . On the other hand, the C76S mutation produced a quite large volume contraction of −5 mL mol −1 upon triplet formation, in contrast to free FMN ( ca −1.4 mL mol −1 , see Table ) and to the corresponding C57S mutation in Chlamydomonas reinhardtii phototropin LOV1 ( Cr LOV1) where Δ V T = −0.9 mL mol −1 . Yet, this value is consistent with the corresponding C62S mutation in YtvA, which also produced a larger Δ V T than in the wild‐type protein (Carmen Mandalari, unpublished data, see Table ).…”

Photocatalytic interaction of aminophylline–riboflavin leads to ROS‐mediated DNA damage and cell death: A novel phototherapeutic mechanism for cancer

“…In biological systems Rf is found as a constituent of two prominent cofactors: Flavin mononucleotide and Flavin adenine dinucleotide . The photophysical and photochemical properties of Rf make it an excellent photosensitizer, thereby rendering this vitamin a promising tool of PDT . Aminophylline (Am) is a bronchodialating agent used in asthmatic condition.…”