Resonance Raman Studies on Reaction Mechanism of Photolyases: Structural Characteristics of the Active Site and Photo-repair Mechanism of UV-damaged DNA

Ultraviolet light radiation in the wavelength range between 260 and 320 nm causesdamage to DNA by forming the dimerization of adjacent pyrimidines in the same DNAstrand. This covalentry linked dimer influences the replication and transcription, and leadsto cell death or skin cancer. Most (70-80%) of UV-induced DNA lesions is cyclobutanepyrimidine dimer (CPD) and, a lesser extent (20-30%) is (6-4) photoproduct. These twomajor types of UV-damaged DNA are repaired under illumination with near-UV/visiblelight by CPD photolyase and (6-4) photolyase, respectively. The both photolyases containflavin adenine dinucleotide (FAD) as an essential cofactor for DNA repairing. The structureand catalytic mechanism of CPD photolyase have been extensively studied. However, itsdetailed structure of the active site containing CPD is uncertain until now. The structure of (6-4) photolyase is less well studied. The genes for this enzyme exhibit a sequencesimilarity to CPD photolyase, especially in the FAD binding sites. Such high similarityindicates a similar structure and reaction mechanism in two photolyases. Unexpectedly,(6-4) photolyase presents a much lower quantum yield compared to that of CPD photolyase,indicating the differences in structure and reaction mechanism between (6-4) and CPDphotolyase. Such precise differences between CPD photolyase and (6-4) photolyaseregarding substrate binding and DNA repair needs to be clarified. To investigate the unclear structure and environment of the active site in (6-4)photolyase, we measured resonance Raman spectra of (6-4) photolyase having neutralsemiquinoid and oxidized forms of FAD, which were selectively intensity enhanced byexcitations at 568.2 and 488.0 nm, respectively. DFT calculations were carried out for thefirst time on the neutral semiquinone. The marker band of a neutral semiquinone at 1606cm-1 in H2O. whose frequency is the lowest among various flavoenzymes, apparently splitsinto two comparable bands at 1594 and 1608 cm-1 in D2O, and similarly that at 1522 cm-1 in H2O does into three bands at 1458, 1508, and 1536 cm-1 in D2O. This D2O effect was recognized only after being oxidized once and photoreduced to form a semiquinone again,but not by simple H/D exchange of solvent. Some Raman bands of the oxidized form wereobserved at significantly low frequencies (1621, 1576 cm-1)and with band splittings(1508/1493, 1346/1320 cm-1). These Raman spectral characteristics indicate strong H-bondinginteractions (at N5-H, N1), a fairly hydrophobic environment, and an electron-lacking feature inbenezene ring of the FAD cofactor, which seems to specifically control the reactivity of (6-4) photolyase. To clarify the structure of active site upon substrate binding and the mechanism of DNA repair, weexamined the resonance Raman spectra of complexes between damaged DNA and the neutralsemiquinoid and oxidized forms of (6-4) and CPD photolyases. The marker band for a neutralsemiquinoid flavin and band I of the oxidized flavin, which are derived from the vibrations of thebenzene ring of flavin adenine dinucleotide (FAD), were shifted to lower freauencies upon binding of damaged DNA by CPD photolyase but not by (6-4) photolyase, indicating that CPD interacts with thebenzene ring of FAD directly but that (6-4) photoproduct does not. Bands II and VII of the oxidizedflavin and the 1398/1391 cm-1 bands of the neutral semiquinoid flavin, which may reflect the bending ofthe U-shaped FAD, were altered upon substrate binding suggesting that CPD and (6-4) photoproductinteract with the adenine ring of FAD, When substrate is bound, there is an upshiftesd 1528 cm-1 band ofthe neutral semiquinoid flavin in CPD photolyase, indicating a weekend hydrogen bond at N5-H ofFAD, and in (6-4) photolyase, band X is downshifted, indicating a strengthened hydrogen bond at N3-Hof FAD. These Raman spectra led us to conclude that the two photolyases have different electron transfer mechanisms as well as different hydrogen bonding environments, which account for the higherredox potential of CPD photolyase. This work revealed that the FAD in (6-4) photolyase is characterized by an electronlocalized structure, and binds to the protein in a fairly hydrophobic and strong hydrogen bonding environment, Specially, a stronger H-bonding at N5-H of FAD was identified for (6-4) photolyase, which may result in the low quantum yield for DNA-repair of thisenzyme. Besides, UV-damaged DNA contacts the benzene ring of FAD only in CPDphotolyase and the adenine ring of FAD in both photolyases. These structures indicate that the election transfer during DNA -repair between isoalloxazine and UV-damaged DNA in CPD photolyase is direct, whereas that in (6-4) photolyase is not direct and bridged byadenine