Photocycle dynamics of the E149A mutant of cryptochrome 3 from Arabidopsis thaliana.
The E149A mutant of the cryDASH member cryptochrome 3 (cry3) from Arabidopsis thaliana was characterized in vitro by optical absorption and emission spectroscopic studies. The mutant protein non-covalently binds the chromophore flavin adenine dinucleotide (FAD). In contrast to the wild-type protein it does not bind N5,N10-methenyl-5,6,7,8-tetrahydrofolate (MTHF). Thus, the photo-dynamics caused by FAD is accessible without the intervening coupling with MTHF. In dark adapted cry3-E149A, FAD is present in the oxidized form (FAD(ox)), semiquinone form (FADH(.)), and anionic hydroquinone form (FAD(red)H(-)). Blue-light photo-excitation of previously unexposed cry3-E149A transfers FAD(ox) to the anionic semiquinone form (FAD()(-)) with a quantum efficiency of about 2% and a back recovery time of about 10s (photocycle I). Prolonged photo-excitation leads to an irreversible protein re-conformation with structure modification of the U-shaped FAD and enabling proton transfer. Thus, a change in the photocycle dynamics occurs with photo-conversion of FAD(ox) to FADH(.), FADH(.) to FAD(red)H(-), and thermal back equilibration in the dark (photocycle II). The photocycle dynamics of cry3-E149A is compared with the photocycle behaviour of wild-type cry3 and other photo-sensory cryptochromes.
Institut II, Experimentelle und Angewandte Physik, Universität Regensburg, Universitätstrasse 31, D-93053 Regensburg, Germany.
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P
Zirak
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A
Penzkofer
J
J
Moldt
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R
Pokorny
A
A
Batschauer
L-O
LO
Essen
D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000737:chemistry*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000378:metabolism; D056931:Cryptochromes / Q000737:chemistry* / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013763:Tetrahydrofolates / Q000737:chemistry / Q000378:metabolism
D029681:Arabidopsis Proteins; D056931:Cryptochromes; D011994:Recombinant Proteins; D013763:Tetrahydrofolates; D005182:Flavin-Adenine Dinucleotide; C018092:5,10-methenyltetrahydrofolate; C515145:Cry3 protein, Arabidopsis