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

Abstract

Photolyases (PHRs) are blue-light activated DNA repair enzymes that maintain genetic integrity by reverting UV-induced photoproducts into normal bases. The FAD chromophore of PHRs has four different redox states: oxidized (FADox), anion radical (FAD•−), neutral radical (FADH•) 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: FADox 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•− at 200 K, where electron transfer occurs, but proton transfer does not. UV-visible spectroscopy following 450-nm illumination of FADox at 277 K defined the FADH•/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 from UV-visible and FTIR analysis of FAD•− at 200 K. Spectral analysis of amide-I vibrations revealed structural perturbation of the protein’s β-sheet during initial electron transfer (FAD•− formation), transient increase in α-helicity during proton transfer (FADH• 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 α-helicity. Protein structural changes in the photoactivation of (6-4) PHR are discussed on the basis of the present FTIR observations.

Essential Role of an Unusually Long-lived Tyrosyl Radical in the Response to Red Light of the Animal-like Cryptochrome aCRY

“…However, FTIR spectroscopy revealed structural changes in the protein moiety only in the transition from FADH ⅐ to the anionic fully reduced state (FADH Ϫ ) of aCRY providing further support for FADH ⅐ as the dark state of the chromophore (16). Strikingly, these changes in turn structures were not detected in the closely related Xenopus laevis (6 -4) photolyase (17). The lifetime of FADH ⅐ state in vitro was strongly sensitive to alterations of the pH but not of the oxygen level in contrast to other cryptochromes (18 -20).…”

“…Red Light-induced FTIR Difference Spectroscopy on aCRY⌬CCT-The previously recorded FTIR difference spectrum of the wild type (16) showed changes in turn structures in the light-induced conversion from FADH ⅐ to FADH Ϫ that are not present in (6 -4) photolyase (17). To further investigate a possible involvement of the CCT in the red light response of aCRY, difference spectra of aCRY⌬CCT were taken after red light exposure.…”

Structural role of two histidines in the (6-4) photolyase reaction

“…However, the amount of purified protein was low (0.26 mg of purified protein from 1 L of culture), which is disadvantageous for future studies using isotope-labeling and mutations. Therefore, we used a Histag system in this study as described previously [ 27 ]. Briefly, the wild-type and mutant genes of Xenopus (6-4) PHR containing a His-tag at the N-terminus were expressed in E. coli C41(DE3) strain, and purified by a TALON Metal Affinity Resin column (TAKARA) and a HiTrap Heparin HP column (GE Healthcare).…”

“…In FTIR measurements, we used redissolved samples, as established in previous studies [ 21 , 25 – 27 ]. First, we added 2 μL of the sample solution containing 1 mM (6-4) PHR in 50 mM Tris-HCl buffer (pH 8.0) and 200 mM NaCl on an IR window (BaF 2 ).…”

“…This has been proven for photosynthetic reaction centers [ 15 , 16 ], animal and microbial rhodopsins [ 17 – 19 ], and various flavin-binding proteins [ 20 , 21 ]. To gain structural and chemical insights into the activation and repair processes of PHR, we recently established a method to measure light-induced FTIR spectra of CPD PHR [ 21 – 24 ] and (6-4) PHR [ 21 , 25 – 27 ]. In the latter case, the repair spectra clearly contain characteristic phosphate vibrations of the T(6-4)T dimer on the negative side (before repair) and normal DNA on the positive side (after repair) [ 25 ].…”