Martin Ryan, Lacombat Fabien, Espagne Agathe, Dozova Nadia, Plaza Pascal, Yamamoto Junpei, Müller Pavel, Brettel Klaus, de la Lande Aurélien
PASTEUR, Département de chimie, École normale supérieure, UPMC Univ. Paris 06, CNRS, PSL Research University, 75005 Paris, France.
Phys Chem Chem Phys. 2017 Sep 20;19(36):24493-24504. doi: 10.1039/c7cp04555g.
Photolyases are flavoenzymes repairing UV-induced lesions in DNA, which may be activated by a photoreduction of their FAD cofactor. In most photolyases, this photoreduction proceeds by electron transfer along a chain of three tryptophan (Trp) residues, connecting the flavin to the protein surface. Much less studied, animal (6-4) photolyases (repairing pyrimidine-pyrimidone (6-4) photoproducts) are particularly interesting as they were recently shown to have a longer electron transfer chain, counting four Trp residues. Using femtosecond polarized transient absorption spectroscopy, we performed a detailed analysis of the photoactivation reaction in the (6-4) photolyase of Xenopus laevis with oxidized FAD. We showed that the excited flavin is very quickly reduced (∼0.5 ps) by a nearby tryptophan residue, yielding FAD˙ and WH˙ radicals. Subsequent kinetic steps in the picosecond regime were assigned to the migration of the positive charge along the Trp tetrad, in competition with charge recombination. We propose that the positive charge is actually delocalized over various Trp residues during most of the dynamics and that charge recombination essentially occurs through the proximal tryptophanyl radical. Oxidation of the fourth tryptophan is thought to be reached about as fast as that of the third one (∼40 ps), based on a comparison with a mutant protein lacking the distal Trp, implying ultrafast electron transfer between these two residues. This unusual mechanism sheds light on the rich diversity of electron transfer pathways found in various photolyases, and evolution-related cryptochromes alike.
光解酶是一种黄素酶，可修复DNA中紫外线诱导的损伤，其黄素腺嘌呤二核苷酸（FAD）辅因子的光还原作用可激活该酶。在大多数光解酶中，这种光还原作用是通过电子沿着三个色氨酸（Trp）残基组成的链转移来进行的，该链将黄素与蛋白质表面相连。研究较少的动物（6-4）光解酶（修复嘧啶 - 嘧啶酮（6-4）光产物）特别有趣，因为最近发现它们具有更长的电子转移链，包含四个Trp残基。我们使用飞秒偏振瞬态吸收光谱，对非洲爪蟾（Xenopus laevis）（6-4）光解酶中氧化型FAD的光激活反应进行了详细分析。我们发现，激发态的黄素会被附近的色氨酸残基非常迅速地还原（约0.5皮秒），产生FAD˙和WH˙自由基。皮秒时间尺度上的后续动力学步骤被归因于正电荷沿着Trp四联体的迁移，同时伴有电荷复合竞争。我们提出，在大多数动力学过程中，正电荷实际上是在各个Trp残基上离域的，并且电荷复合基本上是通过近端色氨酸自由基发生的。基于与缺乏远端Trp的突变蛋白的比较，认为第四个色氨酸的氧化速度与第三个色氨酸的氧化速度大致相同（约40皮秒），这意味着这两个残基之间存在超快电子转移。这种不同寻常的机制揭示了各种光解酶以及与进化相关的隐花色素中发现的丰富多样的电子转移途径。
