The Flash−Quench Technique in Protein−DNA Electron Transfer: Reduction of the Guanine Radical by Ferrocytochrome c
Electron transfer from a protein to oxidatively damaged DNA, specifically from ferrocytochrome c to the guanine radical, was examined using the flash−quench technique. Ru(phen)_2dppz^(2+) (dppz = dipyridophenazine) was employed as the photosensitive intercalator, and ferricytochrome c (Fe^(3+) cyt c), as the oxidative quencher. Using transient absorption and time-resolved luminescence spectroscopies, we examined the electron-transfer reactions following photoexcitation of the ruthenium complex in the presence of poly(dA-dT) or poly(dG-dC). The luminescence-quenching titrations of excited Ru(phen)_2dppz^(2+) by Fe^(3+) cyt c are nearly identical for the two DNA polymers. However, the spectral characteristics of the long-lived transient produced by the quenching depend strongly upon the DNA. For poly(dA-dT), the transient has a spectrum consistent with formation of a [Ru(phen)_2dppz^(3+), Fe^(2+) cyt c] intermediate, indicating that the system regenerates itself via electron transfer from the protein to the Ru(III) metallointercalator for this polymer. For poly(dG-dC), however, the transient has the characteristics expected for an intermediate of Fe^(2+) cyt c and the neutral guanine radical. The characteristics of the transient formed with the GC polymer are consistent with rapid oxidation of guanine by the Ru(III) complex, followed by slow electron transfer from Fe^(2+) cyt c to the guanine radical. These experiments show that electron holes on DNA can be repaired by protein and demonstrate how the flash−quench technique can be used generally in studying electron transfer from proteins to guanine radicals in duplex DNA.
© 2000 American Chemical Society. Received January 20, 2000. Publication Date (Web): July 28, 2000. We are grateful for grants from the NIH (GM 49216 to J.K.B.), the NSF (MCB981-7338 to E.D.A.S.), and Mount St. Mary's College Professional Development Fund (to E.D.A.S.) for financial support of this work. We also thank Prof. E. Margoliash, Dr. J. Rack, and Dr. I. Dmochowski for helpful discussions and Dr. O. Schiemann, Dr. H.-A. Wagenknecht, and C. Treadway for carefully reading the original manuscript.