Absorption and emission spectroscopic characterisation of the LOV2-His domain of phot from Chlamydomonas reinhardtii

The absorption and emission behaviour of flavin mononucleotide (FMN) in the wild-type light, oxygen and voltage sensitive domain LOV2 of the photoreceptor phot from the green alga Chlamydomonas reinhardtii is studied. Actually a LOV2-His protein (LOV2 domain bound at N-terminal to 15 His aminoacids via a Gly aminoacid) expressed in an Escherichia coli strain is investigated. For fresh samples stored in the dark an initial fluorescence quantum yield phi(F) = 0.12 +/- 0.01 and an effective fluorescence lifetime of tau(F) = 2.4 +/- 0.1 ns are determined. Blue-light photo-excitation generates an intermediate photoproduct (flavin-C(4a)cysteinyl adduct with absorption peak at 390 nm) resulting in an intensity-dependent fluorescence quenching. In the aqueous solutions at pH 8 approximately 3.8 % of the FMN molecules are not bound to the protein binding pocket, whereas 96.2 % are non-covalently bound. Even at high-intensity light excitation at 428 nm a fraction of about 7 % of the non-covalently bound FMN remains non-converted to an FMN-Cys adduct because of photo-induced back-relaxation of the adduct to non-covalently bound FMN. Two holo-LOV2-His conformations with different adduct recovery time constants are revealed by spectrally and temporally resolved fluorescence and absorption measurements: A fraction of about 48 % forms FMN-Cys adducts with a fast recovery time constant of tau(Ad,f) = 19 +/-2 s in the dark, and the rest forms adducts with a slow recovery time constant of tau(F,s) = 5.5 +/- 1 min. Prolonged blue light irradiation of the flavin-C(4a)-cysteinyl adducts reduces their ability to recover back in the dark to noncovalently bound FMN (photo-induced permanent adduct formation). Numerical simulations of the intensity-dependent absorption depletion reveals a quantum yield of intermediate photo-adduct formation of phi(Ad,b) = 0.9 +/- 0.1. Simulation of the adduct absorption dynamics gives a quantum yield of photo-induced adduct back-relaxation of phi(Ad,b) = 0.15 +/- 0.01 and a quantum yield of photoinduced permanent adduct formation of phi(Ad,p) = (2.6 +/- 0.5) x 10(-4). (C) 2004 Elsevier B.V. All rights reserved.