Role of the middle residue in the triple tryptophan electron transfer chain of DNA photolyase:: Ultrafast spectroscopy of a Trp→Phe mutant
Role of the middle residue in the triple tryptophan electron transfer chain of DNA photolyase:: Ultrafast spectroscopy of a Trp→Phe mutant
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DOI：
10.1021/jp063686b
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发表时间：
2006-08-17
影响因子：
3.3
通讯作者：
Vos, Marten H.
中科院分区：
文献类型：
--
作者：
Lukacs, Andras;Eker, Andre P. M.;Vos, Marten H.
大肠杆菌DNA光解酶将半还原黄嘌呤二核苷酸辅因子FADH（.）光还原为FADH（-）涉及三个色氨酸(W)残基，形成紧密间隔的电子转移链FADH(.)- w382 - w359 - w306。为了研究这一过程，我们构建了一个突变体光解酶，其中W359被苯丙氨酸(F)取代。利用飞秒光谱法监测其光产物，激发态FADH(。*被发现在大约30秒内腐烂，与野生型（WT）光解酶相似。然而，与WT相反，在W359F突变型光解酶中，基态FADH（.）几乎随着其激发态的衰减而完全恢复，尽管存在初级电子给体W382，但在任何时候都没有观察到可测量的黄素减少。因此，W359F光解酶的行为似乎与许多其他黄素蛋白一样，其中黄素激发态被芳香残基的非常短暂的氧化所猝灭。我们的分析表明，这两个电荷复合的初级电荷分离状态FADH(-)W382(。+)和（在WT中）从W359到W382的电子转移(。+)在时间常数< 4 ps时发生，比初始的W382 -> FADH（）快得多。*电子转移步骤。我们的结果提供了第一个实验表明，芳香残基之间的电子转移可以在类似于10(-12)s的时间尺度上发生。
Photoreduction of the semi-reduced flavin adenine dinucleotide cofactor FADH(.) in DNA photolyase from Escherichia coli into FADH(-) involves three tryptophan ( W) residues that form a closely spaced electron-transfer chain FADH(.)-W382-W359-W306. To investigate this process, we have constructed a mutant photolyase in which W359 is replaced by phenylalanine (F). Monitoring its photoproducts by femtosecond spectroscopy, the excited-state FADH(.)* was found to decay in similar to 30 ps, similar as in wild type (WT) photolyase. In contrast to WT, however, in W359F mutant photolyase the ground-state FADH(.) fully recovered virtually concomitantly with the decay of its excited state and, despite the presence of the primary electron donor W382, no measurable flavin reduction was observed at any time. Thus, W359F photolyase appears to behave like many other flavoproteins, where flavin excited states are quenched by very short-lived oxidation of aromatic residues. Our analysis indicates that both charge recombination of the primary charge separation state FADH(-)W382(.+) and (in WT) electron transfer from W359 to W382(.+) occur with time constants < 4 ps, considerably faster than the initial W382 -> FADH(.)* electron-transfer step. Our results provide a first experimental indication that electron transfer between aromatic residues can take place on the time scale of similar to 10(-12) s.