UMR 8640 CNRS-ENS-UPMC, Département de Chimie, Ecole Normale Supérieure, 24 rue Lhomond, 75005 Paris, France.
J Am Chem Soc. 2010 Apr 7;132(13):4935-45. doi: 10.1021/ja1002372.
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 (WH(351)) in 340 ps, hence, with the overall Brettel-Vos mechanism.
两种新型黄素蛋白（OtCPF1 和 OtCPF2）的光激活动力学研究，这些蛋白属于绿藻海胆虫的隐色体光解酶家族（CPF）。我们通过宽带紫外可见飞秒吸收光谱研究了它们的光激活动力学。在激发蛋白发色团辅因子，氧化形式的黄素腺嘌呤二核苷酸（FAD(ox)）时，我们在两种情况下都观察到 FAD(ox)的超快光还原：OtCPF1 为 390 fs，OtCPF2 为 590 fs。尽管这种超快电子转移已经在其他黄素蛋白和 CPF 成员中得到报道，但目前的结果是首次通过机制的全光谱特征来证明。光产物光谱的分析允许鉴定色氨酸为最初的电子供体。发现该残基被氧化为其质子化自由基阳离子形式（WH(+）），而 FAD(ox)被还原为 FAD(-）。随后在皮秒和亚纳秒范围内观察到动力学，主要由光产物瞬态信号的双指数部分衰减描述（OtCPF1 为 9 和 81 ps，OtCPF2 为 13 和 340 ps），同时光谱变化减少，而长寿命光产物在纳秒时间尺度上仍然存在。我们根据 Brettel 和 Vos 小组提出的模型来解释这些观察结果，该模型描述了大肠杆菌 CPD 光解酶（EcCPD）中 FADH(*)的光还原是沿着三个色氨酸残基的链进行的顺序电子转移，尽管在这种情况下，限速步骤是 30 ps 内的原始光还原。在本研究中，激发 FAD(ox)允许揭示以下步骤，并通过光谱将它们分配给沿色氨酸链的空穴跳跃过程，同时在每个步骤伴随部分电荷复合。此外，通过同源建模进行的结构分析使我们能够提出 FAD 和保守色氨酸三联体的相对取向的暂定结构。最后，对 OtCPF2 进行的初步瞬态各向异性测量的结果最终与 340 ps 时远端色氨酸残基（WH(351)）的氧化很好地兼容，因此与整体 Brettel-Vos 机制兼容。
