Evidence of electron transfer from peptides to DNA: Oxidation of DNA-bound tryptophan using the flash-quench technique

A flash-quench method has been employed to probe electron-transfer reactions from peptides to DNA. The photoexcited intercalators [Ru(phen)(2)(dppz)](2+) (phen = 1,10-phenanthroline; dppz = dipyridophenazine) and [Ru(phen)(bpy')(dppz)](2+) (bpy' = 4-(4'-methyl-2,2'-bipyridyl)valerate) are quenched by a nonintercalating and weakly bound electron-transfer quencher to generate the corresponding DNA-bound Ru(III) complexes in situ. Both Ru(III) complexes are powerful ground-state oxidants, capable of oxidizing guanine in DNA or DNA-bound tryptophan of the intercalating peptide, Lys-Trp-Lys. In mixed-sequence oligonucleotide duplexes containing [Ru(phen)(bpy')(dppz)](2+) tethered at one end, damage to distant guanines is observed by gel electrophoresis, consistent with the mobility of the electron through the DNA duplex. This damage at guanines is observed in both the presence and absence of Lys-Trp-Lys, but the presence of the peptide affects the distribution. in flash-quench experiments using mixed-sequence oligonucleotides or poly(dG.dC) in the presence of Lys-Trp-Lys, transient absorption spectroscopy reveals a signal at lambda = 510 nm assigned to the tryptophan radical; it decays on the time scale of 60-250 mu s. The final peptide product of this electron-transfer reaction has been described by UV/vis spectroscopy and mass spectrometry. No DNA-peptide adducts were detected. Significantly, the tryptophan radical is not observed in reactions with Ru(III) bound to poly(dA.dT), an observation that suggests the intermediacy of the guanine radical cation in generating the tryptophan radical. These results indicate that charge migration from tryptophan to [Ru(phen)(bpy')(dppz)](3+) occurs to produce the tryptophan radical and that this process is DNA mediated. This work establishes methodology to probe tryptophan intercalation in DNA by protein or peptides. Moreover, this methodology demonstrates an electron-transfer event between peptides and DNA and suggests the consideration of such events within the cell.