DETERMINATION OF RATES AND YIELDS OF INTERCHROMOPHORE (FOLATE-]FLAVIN) ENERGY-TRANSFER AND INTERMOLECULAR (FLAVIN-]DNA) ELECTRON-TRANSFER IN ESCHERICHIA-COLI PHOTOLYASE BY TIME-RESOLVED FLUORESCENCE AND ABSORPTION-SPECTROSCOPY
DETERMINATION OF RATES AND YIELDS OF INTERCHROMOPHORE (FOLATE-]FLAVIN) ENERGY-TRANSFER AND INTERMOLECULAR (FLAVIN-]DNA) ELECTRON-TRANSFER IN ESCHERICHIA-COLI PHOTOLYASE BY TIME-RESOLVED FLUORESCENCE AND ABSORPTION-SPECTROSCOPY
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DOI：
10.1021/bi00111a011
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发表时间：
1991-11-26
期刊：
影响因子：
2.9
通讯作者：
SANCAR, A
中科院分区：
文献类型：
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作者：
KIM, ST;HEELIS, PF;SANCAR, A
大肠杆菌DNA光裂合酶是一种光修复环丁烷嘧啶二聚体的酶，它含有两个发色团辅因子：1，5-二氢黄素腺嘌呤二核苷酸（FADH 2）和5，10-亚甲基四氢叶酸（MTHF）。先前的工作已经表明，MTHF是将能量转移到FADH 2 Cofactor的主要感光体; FADH 2单重激发态然后通过电子转移修复光二聚体。在这项研究中，我们已经确定了时间分辨荧光和吸收光谱的这些物理过程的速率常数。从时间分辨荧光，我们发现从MTHF到FADH 2和FADH度的能量转移分别以4.6 X 10（9）和3.0 X 10（10）s-1的速率发生，从FADH 2到嘧啶二聚体的电子转移以5.5 X 10（9）s-1的速率发生。使用长距离能量转移的福斯特理论并假设K2 = 2/3，在MTHF-FADH 2对的情况下，发色团间距离估计为22埃，在MTHF-FADH度对的情况下，发色团间距离估计为21埃。皮秒吸收光谱鉴定了MTHF单态，其衰变以产生FADH 2的第一激发单态。MTHF和FADH 2单重态的寿命和发色团间能量转移速率，以及通过时间分辨荧光测量的从FADH 2到DNA的电子转移速率，与通过皮秒激光闪光光解获得的值非常一致。类似地，荧光或吸收寿命的叶酸耗尽的酶与和没有光二聚体的研究表明，FADH 2，在其单重激发态，转移一个电子的二聚体与89%的效率。FADH 2与光二聚体之间的距离计算为约1.5 μ m。14埃。
Escherichia coli DNA photolyase, which photorepairs cyclobutane pyrimidine dimers, contains two chromophore cofactors, 1,5-dihydroflavin adenine dinucleotide (FADH2) and 5,10-methenyltetrahydrofolate (MTHF). Previous work has shown that MTHF is the primary photoreceptor which transfers energy to the FADH2 CofaCtor; the FADH2 singlet excited state then repairs the photodimer by electron transfer. In this study, we have determined the rate constants for these photophysical processes by time-resolved fluorescence and absorption spectroscopy. From time-resolved fluorescence, we find that energy transfer from MTHF to FADH2 and FADH-degrees occurs at rates of 4.6 X 10(9) and 3.0 X 10(10) s-1, respectively, and electron transfer from FADH2 to a pyrimidine dimer occurs at a rate of 5.5 X 10(9) s-1. Using Forster theory for long-range energy transfer and assuming K2 = 2/3, the interchromophore distances were estimated to be 22 angstrom in the case of the MTHF-FADH2 pair and 21 angstrom for the MTHF-FADH-degrees pair. Picosecond absorption spectroscopy identified an MTHF single state which decays to yield the first excited singlet state of FADH2. The lifetimes of MTHF and FADH2 singlets and the rates of interchromophore energy transfer, as well as the rate of electron transfer from FADH2 to DNA measured by time-resolved fluorescence, were in excellent agreement with the values obtained by picosecond laser flash photolysis. Similarly, fluorescence or absorption lifetime studies of the folate-depleted enzyme with and without photodimer suggest that FADH2, in its singlet excited state, transfers an electron to the dimer with 89% efficiency. The distance between FADH2 and the photodimer was calculated to be ca. 14 angstrom.