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

摘要

The photoactivation dynamics of two new flavoproteins (OtCPF1 and OtCPF2) of the cryptochromenphotolyase family (CPF), belonging to the green alga Ostreococcus tauri, was studied by broadband UV-visnfemtosecond absorption spectroscopy. Upon excitation of the protein chromophoric cofactor, flavin adeninendinucleotide in its oxidized form (FADox), we observed in both cases the ultrafast photoreduction of FADox:nin 390 fs for OtCPF1 and 590 fs for OtCPF2. Although such ultrafast electron transfer has already beennreported for other flavoproteins and CPF members, the present result is the first demonstration with fullnspectral characterization of the mechanism. Analysis of the photoproduct spectra allowed identifyingntryptophan as the primary electron donor. This residue is found to be oxidized to its protonated radicalncation form (WH•+), while FADox is reduced to FAD•-. Subsequent kinetics were observed in the picosecondnand subnanosecond regime, mostly described by a biexponential partial decay of the photoproduct transientnsignal (9 and 81 ps for OtCPF1, and 13 and 340 ps for OtCPF2), with reduced spectral changes, while anlong-lived photoproduct remains in the nanosecond time scale. We interpret these observations within thenmodel proposed by the groups of Brettel and Vos, which describes the photoreduction of FADH• within E.ncoli CPD photolyase (EcCPD) as a sequential electron transfer along a chain of three tryptophan residues,nalthough in that case the rate limiting step was the primary photoreduction in 30 ps. In the present study,nexcitation of FADox permitted to reveal the following steps and spectroscopically assign them to the holehoppingnprocess along the tryptophan chain, accompanied by partial charge recombination at each step.nIn addition, structural analysis performed by homology modeling allowed us to propose a tentative structurenof the relative orientations of FAD and the conserved tryptophan triad. The results of preliminary transientnanisotropy measurements performed on OtCPF2 finally showed good compatibility with the oxidation ofnthe distal tryptophan residue (WH351) in 340 ps, hence, with the overall Brettel-Vos mechanism.