Characterization of Excited State Etheno-Fad: A Probe of the Role of Adenine in DNA Photolyase

Abstract

Using femtosecond transient absorption spectroscopy, we have characterized flavin 1,N6 - ethenoadenine dinucleotide (e-FAD), an analog of flavin adenine dinucleotide (FAD), to understand the role of adenine in the photoinduced electron transfer (PET) reaction between reduced FAD (∗FADH-) and thymine dimers during its repair in DNA photolyase. The adenine in FAD may serve as a virtual or real intermediate in the PET reaction. e-FAD was used to modify the driving force between the adenine and its partners, providing data on parameters and processes that dictate the kinetics and pathways of electron transport. The neutral oxidized and the fully reduced anionic states of e-FAD, FAD, and flavin mononucleotide (FMN) in free solution exhibited multi-exponential decay kinetics that reflect their excited state quenching pathways and possible conformational heterogeneity. Generally, the introduction of the e-Ade group leads to much faster excited state decay in the reduced state. To investigate this further, the evolution of the excited state was probed in the visible, near-UV, and UVB spectral regions to determine whether PET was responsible for the short-lived excited state. The significance of these results to the role of the FAD adenine in DNA photolyase are discussed.