Quantum Yield Measurements of Short-Lived Photoactivation Intermediates in DNA Photolyase: Toward a Detailed Understanding of the Triple Tryptophan Electron Transfer Chain
详细信息 查看全文
- 作者：Martin Byrdin ; Andras Lukacs ; Viruthachalam Thiagarajan ; Andr P. M. Eker ; Klaus Brettel ; Marten H. Vos
- 刊名：Journal of Physical Chemistry A
- 出版年：2010
- 出版时间：March 11, 2010
- 年：2010
- 卷：114
- 期：9
- 页码：3207-3214
- 全文大小：274K
- 年卷期：v.114,no.9(March 11, 2010)
- ISSN：1520-5215
文摘
The light-dependent DNA repair enzyme photolyase contains a unique evolutionary conserved triple tryptophan electron transfer chain (W382−W359−W306 in photolyase from E. coli) that bridges the 15 Å distance between the buried flavin adenine dinucleotide (FAD) cofactor and the surface of the protein. Upon excitation of the semireduced flavin (FADH°), electron transfer through the chain leads to formation of fully reduced flavin (FADH−; required for DNA repair) and oxidation of the most remote tryptophan residue W306, followed by its deprotonation. The thus-formed tryptophanyl radical W306°+ is reduced either by an extrinsic reductant or by reverse electron transfer from FADH−. Altogether the kinetics of these charge transfer reactions span 10 orders of magnitude, from a few picoseconds to tens of milliseconds. We investigated electron transfer processes in the picosecond−nanosecond time window bridging the time domains covered by ultrafast pump−probe and “classical” continuous probe techniques. Using a recent dedicated setup, we directly show that virtually no absorption change between 300 ps and 10 ns occurs in wild-type photolyase, implying that no charge recombination takes place in this time window. In contrast, W306F mutant photolyase showed a partial absorption recovery with a time constant of 0.85 ns. In wild-type photolyase, the quantum yield of FADH− W306°+ was found at 19 ± 4%, in reference to the established quantum yield of the long-lived excited state of [Ru(bpy)3]2+. With this yield, the optical spectrum of the excited state of FADH° can be constructed from ultrafast spectroscopic data; this spectrum is dominated by excited state absorption extending from below 450 to 850 nm. The new experimental results, taken together with previous data, allow us to propose a detailed kinetic and energetic scheme of the electron transfer chain.