Fourier-Transform Infrared Study of the Photoactivation Process of Xenopus (6-4) Photolyase

Photolyases (PHRs) are blue light-activated DNA repair enzymes that maintain genetic integrity by reverting UV-induced photoproducts into normal bases. The flavin adenine dinucleotide (FAD) chromophore of PHRs has four different redox states: oxidized (FAD(ox)), anion radical (FAD(center dot-)), neutral radical (FADH(center dot)), and fully reduced (FADH(-)). We combined difference Fourier-transform infrared (FTIR) spectroscopy with UV-visible spectroscopy to study the detailed photoactivation process of Xenopus (6-4) PHR. Two photons produce the enzymatically active, fully reduced PHR from oxidized FAD: FAD(ox) is converted to semiquinone via light induced one electron and one proton transfers and then to FADH(-) by light induced one electron transfer. We successfully trapped FAD(center dot-) at 200 K, where electron transfer occurs but proton transfer does not UV-visible spectroscopy following 450 nm illumination of FAD(ox) at 277 K defined the FADH(center dot)/FADH(-) mixture and allowed calculation of difference FTIR spectra among the four redox states. The absence of a characteristic C=O stretching vibration indicated that the proton donor is not a protonated carboxylic acid. Structural changes in Trp and Tyr are suggested by UV-visible and FTIR analysis of FAD(center dot-) at 200 K. Spectral analysis of amide I vibrations revealed structural perturbation of the protein's beta-sheet during initial electron transfer (FAD(center dot-) formation), a transient increase in alpha-helicity during proton transfer (FADH(center dot) formation), and reversion to the initial amide I signal following subsequent electron transfer (FADH(-) formation). Consequently, in (6-4) PHR, unlike cryptochrome-DASH, formation of enzymatically active FADH(-) did not perturb alpha-helicity. Protein structural changes in the photoactivation of (6-4) PHR are discussed on the basis of these FTIR observations.