T at 365 nm (UVP; eight W), the flavin cofactor is stabilized at
T at 365 nm (UVP; 8 W), the flavin cofactor is stabilized at the FADstate beneath anaerobic circumstances. The neutral semiquinone (FADH EcPL was prepared by mutation of W382F in EcPL plus the anionic DP Biological Activity hydroquinone (FADH EcPL was stabilized below anaerobic circumstances immediately after purge with argon and subsequent photoreduction. Femtosecond Absorption Spectroscopy. All the femtosecond-resolved measurements had been carried out employing the transient-absorption approach. The experimental layout has been detailed previously (24). Enzyme preparations with oxidized (FAD) and anionic semiquinone (FAD flavin were excited at 480 nm. For enzyme with neutral semiquinone (FADH, the pump wavelength was set at 640 nm. For the anionic hydroquinone (FADH type of the enzyme, we used 400 nm as the excitation wavelength. The probe wavelengths had been tuned to cover a wide range of wavelengths from 800 to 260 nm. The instrument time resolution is about 250 fs and all the experiments were carried out in the magic angle (54.7. Samples had been kept stirring in the course of irradiation to avoid heating and photobleaching. Experiments together with the neutral FAD and FADHstates have been carried out under aerobic circumstances, whereas those together with the anionic FADand FADHstates have been executed under anaerobic circumstances. All experiments have been performed in quartz cuvettes with a 5-mm optical length except that the FADHexperiments probed at 270 and 269 nm have been carried out in quartz cuvettes with a 1-mm optical length. ACKNOWLEDGMENTS. This function is supported in component by National Institutes of Overall health Grants GM074813 and GM31082, the Camille Dreyfus TeacherScholar (to D.Z.), the American Heart Association fellowship (to Z.L.), plus the Ohio State University Pelotonia fellowship (to C.T. and J.L.).18. Byrdin M, Eker APM, Vos MH, Brettel K (2003) Dissection on the triple tryptophan electron transfer chain in Escherichia coli DNA photolyase: Trp382 is the primary donor in photoactivation. Proc Natl Acad Sci USA 100(15):8676681. 19. Kao Y-T, et al. (2008) Ultrafast dynamics of flavins in five redox states. J Am Chem Soc 130(39):131323139. 20. Seidel CAM, Schulz A, Sauer MHM (1996) Nucleobase-specific quenching of fluorescent dyes. 1. Nucleobase one-electron redox potentials and their correlation with static and dynamic quenching efficiencies. J Phys Chem one hundred(13):5541553. 21. Gindt YM, Schelvis JPM, Thoren KL, Huang TH (2005) Substrate binding modulates the reduction prospective of DNA photolyase. J Am Chem Soc 127(30):104720473. 22. Vicic DA, et al. (2000) Oxidative repair of a thymine dimer in DNA from a distance by a covalently linked organic intercalator. J Am Chem Soc 122(36):8603611. 23. Byrdin M, et al. (2010) Quantum yield measurements of short-lived photoactivation intermediates in DNA photolyase: Toward a detailed understanding of your triple tryptophan electron transfer chain. J Phys Chem A 114(9):3207214. 24. Saxena C, ACAT2 Synonyms Sancar A, Zhong D (2004) Femtosecond dynamics of DNA photolyase: Power transfer of antenna initiation and electron transfer of cofactor reduction. J Phys Chem B 108(46):180268033. 25. Park HW, Kim ST, Sancar A, Deisenhofer J (1995) Crystal structure of DNA photolyase from Escherichia coli. Science 268(5219):1866872. 26. Zoltowski BD, et al. (2011) Structure of full-length Drosophila cryptochrome. Nature 480(7377):39699. 27. Balland V, Byrdin M, Eker APM, Ahmad M, Brettel K (2009) What tends to make the difference among a cryptochrome and DNA photolyase A spectroelectrochemical comparison on the flavin redox trans.