Abstract
Cryptochromes are flavin-based photoreceptors occurring throughout the biological kingdom, which regulate growth and development in plants and are involved in the entrainment of circadian rhythms of both plants and animals. A number of recent theoretical works suggest that cryptochromes might also be the receptors responsible for the sensing of the magnetic field of the earth (e.g. in insects, migratory birds, or migratory fish). Cryptochromes undergo forward light-induced reactions involving electron transfer to excited state flavin to generate radical intermediates, which correlate with biological activity. Here, we give evidence of a mechanism for the reverse reaction, namely dark reoxidation of protein-bound flavin in Arabidopsis thaliana cryptochrome (AtCRY1) by molecular oxygen that involves formation of a spin-correlated FADH(•)-superoxide radical pair. Formation of analogous radical pairs in animal cryptochromes might enable them to function as magnetoreceptors.
Highlights
Photolyases and the mechanism of their action have been subject to extensive studies in the last five decades [3, 9], the photochemistry and the signaling pathways of cryptochromes are just beginning to become known
This is in marked contrast to photolyases, where photoreduction results in formation of the fully reduced (FADHϪ) form, which is active in DNA repair [11]
We show that the reaction is dependent on molecular oxygen concentration, that it occurs on a relatively slow time scale, and that the reaction from FADH1⁄7 to FADox occurs by a distinct mechanism depending on whether the FADH1⁄7 radical is formed by oxidation of FADHϪ or by reduction of FADox
Summary
Protein Expression and Purification—AtCRY1 protein was prepared by a well established procedure described elsewhere [20] with the only exception that 50 mM phosphate buffer was used instead of 50 mM Tris-HCl buffer (same pH, 7.5) for the purification procedures. Fresh AtCRY1 samples (in 0.5 M NaCl, 50 mM phosphate buffer, pH 7.5) were rid of oxygen by nitrogen purging for 2 h (on ice), and the flavin chromophore was subsequently photoreduced by blue LED (30 min, on ice, 465 nm, light intensity of ϳ1,500 mol mϪ2 sϪ1). After this treatment, virtually no oxidized flavin remained, and the samples contained a mix of reduced and 10 –30% semireduced flavin (complete reduction to 100% FADHϪ could not be achieved even at this blue light irradiance). 10 l of buffer (0.5 M NaCl, 50 mM phosphate buffer, pH 7.5) were added (to see the effect of the addition of a small volume of liquid possibly having a slightly different O2 concentration) followed by 10 l of catalase (c ϭ 1 mg/ml) in the same buffer
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