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Spectro−Temporal Characterization of the Photoactivation Mechanism of Two New Oxidized Cryptochrome/Photolyase Photoreceptors

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Johanna Brazard^†, Anwar Usman^†, Fabien Lacombat^†, Christian Ley^†, Monique M. Martin^†, Pascal Plaza*^†, Laetitia Mony^‡, Marc Heijde^§, Gérald Zabulon^§ and Chris Bowler^§

UMR 8640 CNRS-ENS-UPMC, Département de Chimie, Ecole Normale Supérieure, 24 rue Lhomond, 75005 Paris, France, UMR 8601 CNRS, Laboratoire de Chimie et Biochimie Pharmacologies et Toxicologiques, Université Paris Descartes, 12 rue de l’Ecole de médecine, 75006 Paris, France, and UMR 8186 CNRS-ENS, Département de Biologie, Ecole Normale Supérieure, 46 rue d’Ulm, 75005 Paris, France

J. Am. Chem. Soc., 2010, 132 (13), pp 4935–4945

DOI: 10.1021/ja1002372

Publication Date (Web): March 12, 2010

Pascal.Plaza@ens.fr, †

UMR 8640 CNRS-ENS-UPMC, Département de Chimie, Ecole Normale Supérieure.

, ‡

UMR 8601 CNRS, Laboratoire de Chimie et Biochimie Pharmacologies et Toxicologiques, Université Paris Descartes.

, §

UMR 8186 CNRS-ENS, Département de Biologie, Ecole Normale Supérieure.

Section:

General Biochemistry

Abstract

The photoactivation dynamics of two new flavoproteins (OtCPF1 and OtCPF2) of the cryptochrome photolyase family (CPF), belonging to the green alga Ostreococcus tauri, was studied by broadband UV−vis femtosecond absorption spectroscopy. Upon excitation of the protein chromophoric cofactor, flavin adenine dinucleotide in its oxidized form (FAD_ox), we observed in both cases the ultrafast photoreduction of FAD_ox: in 390 fs for OtCPF1 and 590 fs for OtCPF2. Although such ultrafast electron transfer has already been reported for other flavoproteins and CPF members, the present result is the first demonstration with full spectral characterization of the mechanism. Analysis of the photoproduct spectra allowed identifying tryptophan as the primary electron donor. This residue is found to be oxidized to its protonated radical cation form (WH^•+), while FAD_ox is reduced to FAD^•−. Subsequent kinetics were observed in the picosecond and subnanosecond regime, mostly described by a biexponential partial decay of the photoproduct transient signal (9 and 81 ps for OtCPF1, and 13 and 340 ps for OtCPF2), with reduced spectral changes, while a long-lived photoproduct remains in the nanosecond time scale. We interpret these observations within the model proposed by the groups of Brettel and Vos, which describes the photoreduction of FADH^• within E. coli CPD photolyase (EcCPD) as a sequential electron transfer along a chain of three tryptophan residues, although in that case the rate limiting step was the primary photoreduction in 30 ps. In the present study, excitation of FAD_ox permitted to reveal the following steps and spectroscopically assign them to the hole-hopping process along the tryptophan chain, accompanied by partial charge recombination at each step. In addition, structural analysis performed by homology modeling allowed us to propose a tentative structure of the relative orientations of FAD and the conserved tryptophan triad. The results of preliminary transient anisotropy measurements performed on OtCPF2 finally showed good compatibility with the oxidation of the distal tryptophan residue (WH₃₅₁) in 340 ps, hence, with the overall Brettel−Vos mechanism.

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