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 phi(F) = 0.30 and a fluorescence lifetime of tau(F) = 4.6 ns were determined for FMN in the mutant LOV1-C57S, whereas these quantities are reduced to about phi(F) = 0.17 and tau(F) = 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.

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“…At this λmax the ε observed is 1.45 x 10 4 M -1 cm -1 . This value is in good agreement of literature value 34,36,37 . Figure 3(a).…”

“…The fluorescence lifetime, τo, for riboflavin in bicontinuous microemulsion was determined using the Strickler-Berg relations 34 given in equation 2:…”

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“…Nevertheless, adduct formation is the overwhelmingly dominant process in wild-type LOV2; no radicals are observed in the wild type that may be irradiated (reversibly) for long periods without suffering degradation (22). Previous optical studies have determined that adduct formation occurs on the microsecond time scale (20,48). Hence, the competing electron transfer reaction observed in the mutant must be much less efficient and, hence, slower to ensure that only FMN-cysteinyl adduct formation occurs.…”

“…Hence, overall the excited-state flavin is optimized to undergo electron transfer but the electron transfer pathways leading to the surface of the LOV2 domain are inefficient. This may be necessary so that adduct formation with Cys-450 in the wild-type, which takes place on a microsecond time scale (20,48), could dominate alternative electron transfer processes. From this study we cannot directly draw conclusions about the time scale or quantum yield of the forward electron transfer observed in the LOV2 C450A domain.…”

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“…It should also be mentioned that we were unable to find a publication that actually determined the fluorescence quantum yield of FMN. Van den Berg and coworkers 58 cite a Φ F of 0.26 for FMN with reference to 59 , were FMN is not mentioned whereas Holzer and colleagues 60 refer to a Φ F of 0.26 with reference to 56 , where the Φ F of riboflavin, but not FMN was determined. In this study we measured Φ F = 0.246 ± 0.002 for FMN, which is identical to the value we measured for riboflavin (Φ F = 0.247 ± 0.007).…”