Photo-reduction of flavin mononucleotide to semiquinone form in LOV domain mutants of blue-light receptor phot from Chlamydomonas reinhardtii

摘要

The photo-excitation dynamics of the mutants LOV1-C57S and LOV2-C250S of the LOV-domains of the phototropin photoreceptor phot from the green alga Chlamydomonas reinhardtii is investigated by absorption and fluorescence studies.The LOV domains fused to a maltose binding protein (MBP) are expressed in Escherichia coli.The mutants were studied under aerobic conditions in aqueous solution at pH 8.Blue-light exposure reduced the fully oxidized flavin mononucleotide,FMN_(ox),to FMN semiquinone,FMNH~(centre dot),(quantum efficiency around 1%) which further reduced to FMN hydroquinone,FMN_(red)H2 or FMN_(red)H- (quantum efficiency ca.3 x 10~(-5)).In the dark both reduced forms recovered back to the oxidized form on a minute timescale.Besides photoreduction,blue-light photo-excitation of the mutants resulted in photoproduct formation (efficiency in the 2 x 10~(-4)-10~(-3) range).Photo-reaction schemes for the mutants are discussed.