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

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, FMNox, to FMN semiquinone, FMNH-, (quantum efficiency around 1%) which further reduced to FMN hydroquinone, FMNredH2 or FMNredH- (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. (c) 2007 Elsevier B.V. All rights reserved.