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Article Dans Une Revue Journal of Photochemistry and Photobiology B: Biology Année : 2009

Spectroscopic characterization of a (6-4) photolyase from the green alga Ostreococcus tauri

Résumé

The cofactor content of OtCPF1, a (6-4) photolyase isolated from the green marine alga Ostreococcus tauri, was characterized by steady-state absorption and fluorescence spectroscopy. The heterologously expressed, GST-fused, purified protein (MW: 89 kDa) is non-covalently bound to flavin adenine dinucleotide (FAD), with a flavin to apoprotein molecular ratio of 64%. No light-harvesting chromophore was found in this protein. In freshly purified OtCPF1, FAD is present in three different redox states: the fully oxidized form (FADox, 82%), the neutral semiquinone (FADH°, 14%) and the fully reduced anion (FADH-, 4%). Keeping the sample in the dark, at 5°C, yields oxidation of FADH° and FADH-, partial release of FAD to the solution and slow degradation of the protein. Upon steady-state blue-light irradiation of OtCPF1 at 450 nm, photoreduction processes leading to an accumulation of stable FADH° and FADH- species are observed. We demonstrate that this accumulation is due to the presence of an external electron donor agent in the purification buffer. Composition changes observed under steady-state photoexcitation are interpreted in terms of photoinduced reductions of FADox and FADH° states and competitive back reactions. Specific irradiation by red light at 620 nm shows both photoreduction of FADH° to FADH- and irreversible oxidation of FADH° to FADox. The photoinduced oxidation reaction is believed to be indirectly caused by the external donor agent present in the buffer. Photoexcitation is also shown to stabilize the binding of FAD to the protein. We suggest this effect to be due to slight changes in the protein conformation, possibly strengthening the hydrogen-bonding network surrounding FAD.

Dates et versions

hal-00389814 , version 1 (29-05-2009)

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Citer

Anwar Usman, Johanna Brazard, Monique M. Martin, Pascal Plaza, Marc Heijde, et al.. Spectroscopic characterization of a (6-4) photolyase from the green alga Ostreococcus tauri. Journal of Photochemistry and Photobiology B: Biology, 2009, 96, pp.38. ⟨10.1016/j.jphotobiol.2009.04.003⟩. ⟨hal-00389814⟩
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