Photoreduction of the Folate Cofactor in Members of the Photolyase Family

Julia Moldt,Richard Pokorny,Christian Orth,Uwe Linne,Yann Geisselbrecht,Mohamed A Marahiel,Lars-Oliver Essen,Alfred Batschauer

doi:10.1074/jbc.m109.018697

Julia Moldt, Richard Pokorny + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m109.018697

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Abstract

Cryptochromes and DNA photolyases are related flavoproteins with flavin adenine dinucleotide as the common cofactor. Whereas photolyases repair DNA lesions caused by UV radiation, cryptochromes generally lack repair activity but act as UV-A/blue light photoreceptors. Two distinct electron transfer (ET) pathways have been identified in DNA photolyases. One pathway uses within its catalytic cycle, light-driven electron transfer from FADH(-)* to the DNA lesion and electron back-transfer to semireduced FADH(o) after photoproduct cleavage. This cyclic ET pathway seems to be unique for the photolyase subfamily. The second ET pathway mediates photoreduction of semireduced or fully oxidized FAD via a triad of aromatic residues that is conserved in photolyases and cryptochromes. The 5,10-methenyltetrahydrofolate (5,10-methenylTHF) antenna cofactor in members of the photolyase family is bleached upon light excitation. This process has been described as photodecomposition of 5,10-methenylTHF. We show that photobleaching of 5,10-methenylTHF in Arabidopsis cry3, a member of the cryptochrome DASH family, with repair activity for cyclobutane pyrimidine dimer lesions in single-stranded DNA and in Escherichia coli photolyase results from reduction of 5,10-methenylTHF to 5,10-methyleneTHF that requires the intact tryptophan triad. Thus, a third ET pathway exists in members of the photolyase family that remained undiscovered so far.

Highlights

DNA photolyases and cryptochromes2 form a large family of related flavoproteins with DNA repair activity and photoreceptor function, respectively
In contrast to wild type, the absorption peak of fully oxidized FAD increases in the W356F mutant upon UV-A treatment, and the final decrease in 380-nm absorption is very small (Fig. 2, E and F). This increase in the amount of FADox in the W356F mutant under UV-A irradiation can be explained by electron donation to 5,10-methenylTHF by residual FADHϪ but a lack of photoreduction of the flavin caused by the interrupted tryptophan triad
The minor change seen in 380-nm absorbance (Fig. 2E) between the original spectrum and the spectrum after UV-A irradiation signifies less change in the 5,10-methenylTHF content than occurred

Summary

Photoreduction of Folate

Arabidopsis thaliana contain polyglutamated 5,10-methenyltetrahydrofolate (5,10-methenylTHF) as the second chromophore [1, 12, 17, 20, 21] (see Fig. 1B for folate structures). Excitation of the 5,10-methenylTHF antenna chromophore at its absorption peak at 380 nm causes a likewise photoreduction of the catalytic FAD [1, 27, 28, 30, 31]. Irreversible bleaching of the 380-nm peak is observed under high irradiance UV-A or camera flash illumination [28, 30]. This irreversible bleaching goes along with release of the folate cofactor from the protein moiety [30] and was named photodecomposition of 5,10-methenylTHF [28]. Bleaching at 380 nm does not reflect destruction but is a specific chemical conversion of the second chromophore

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DISCUSSION