Kim S T, Heelis P F, Okamura T, Hirata Y, Mataga N, Sancar A
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill 27599.
Biochemistry. 1991 Nov 26;30(47):11262-70. doi: 10.1021/bi00111a011.
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 Förster theory for long-range energy transfer and assuming K2 = 2/3, the interchromophore distances were estimated to be 22 A in the case of the MTHF-FADH2 pair and 21 A 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 A.
大肠杆菌DNA光解酶可对环丁烷嘧啶二聚体进行光修复，它含有两种发色团辅因子，即1,5 - 二氢黄素腺嘌呤二核苷酸（FADH₂）和5,10 - 亚甲基四氢叶酸（MTHF）。先前的研究表明，MTHF是主要的光感受器，可将能量传递给FADH₂辅因子；然后FADH₂单重激发态通过电子转移修复光二聚体。在本研究中，我们通过时间分辨荧光和吸收光谱法测定了这些光物理过程的速率常数。通过时间分辨荧光，我们发现从MTHF到FADH₂以及FADH°的能量转移速率分别为4.6×10⁹和3.0×10¹⁰ s⁻¹，并且从FADH₂到嘧啶二聚体的电子转移速率为5.5×10⁹ s⁻¹。使用Förster远程能量转移理论并假设K₂ = 2/3，估计MTHF - FADH₂对的发色团间距离为22 Å，MTHF - FADH°对的发色团间距离为21 Å。皮秒吸收光谱法鉴定出一种MTHF单重态，其衰减产生FADH₂的第一激发单重态。MTHF和FADH₂单重态的寿命、发色团间能量转移速率以及通过时间分辨荧光测量的从FADH₂到DNA的电子转移速率，与皮秒激光闪光光解获得的值非常一致。同样，对有和没有光二聚体的叶酸耗尽酶进行的荧光或吸收寿命研究表明，处于单重激发态的FADH₂以89%的效率将电子转移给二聚体。计算得出FADH₂与光二聚体之间的距离约为14 Å。
