The Flash-Quench Technique in Protein-DNA Electron Transfer: Reduction of the Guanine Radical by Ferrocytochrome c

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• 作者：Eric D. A. Stemp and Jacqueline K. Barton
• 刊名：Inorganic Chemistry
• 出版年：2000
• 出版时间：August 21, 2000
• 年：2000
• 卷：39
• 期：17
• 页码：3868 - 3874
• 全文大小：189K
• 年卷期：v.39,no.17(August 21, 2000)
• ISSN：1520-510X

文摘

Electron transfer from a protein to oxidatively damaged DNA, specifically from ferrocytochrome c to the guanineradical, was examined using the flash-quench technique. Ru(phen)₂dppz²⁺ (dppz = dipyridophenazine) wasemployed as the photosensitive intercalator, and ferricytochrome c (Fe³⁺ cyt c), as the oxidative quencher. Usingtransient absorption and time-resolved luminescence spectroscopies, we examined the electron-transfer reactionsfollowing photoexcitation of the ruthenium complex in the presence of poly(dA-dT) or poly(dG-dC). Theluminescence-quenching titrations of excited Ru(phen)₂dppz²⁺ by Fe³⁺ cyt c are nearly identical for the two DNApolymers. However, the spectral characteristics of the long-lived transient produced by the quenching dependstrongly upon the DNA. For poly(dA-dT), the transient has a spectrum consistent with formation of a [Ru(phen)₂dppz³⁺, Fe²⁺ cyt c] intermediate, indicating that the system regenerates itself via electron transfer fromthe protein to the Ru(III) metallointercalator for this polymer. For poly(dG-dC), however, the transient has thecharacteristics expected for an intermediate of Fe²⁺ cyt c and the neutral guanine radical. The characteristics ofthe transient formed with the GC polymer are consistent with rapid oxidation of guanine by the Ru(III) complex,followed by slow electron transfer from Fe²⁺ cyt c to the guanine radical. These experiments show that electronholes on DNA can be repaired by protein and demonstrate how the flash-quench technique can be used generallyin studying electron transfer from proteins to guanine radicals in duplex DNA.