Department of Frontier Materials, Nagoya Institute of Technology, Nagoya 466-8555, Japan.
Biochemistry. 2012 Jul 24;51(29):5774-83. doi: 10.1021/bi300530x. Epub 2012 Jul 13.
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(•-)), 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: 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(•-) 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(•)/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(•-) at 200 K. Spectral analysis of amide I vibrations revealed structural perturbation of the protein's β-sheet during initial electron transfer (FAD(•-) formation), a 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 these FTIR observations.
光解酶（PHRs）是一种蓝光激活的 DNA 修复酶，通过将 UV 诱导的光产物逆转回正常碱基来维持遗传完整性。PHRs 的黄素腺嘌呤二核苷酸（FAD）发色团有四个不同的氧化还原态：氧化态（FAD(ox)）、阴离子自由基（FAD(•-)）、中性自由基（FADH(•)）和完全还原态（FADH(-)）。我们结合差示傅里叶变换红外（FTIR）光谱和紫外可见光谱研究了非洲爪蟾（6-4）PHR 的详细光激活过程。两个光子产生酶活性的完全还原的 PHR 从氧化 FAD：FAD(ox)通过光诱导的单电子和单质子转移转化为半醌，然后通过光诱导的单电子转移转化为 FADH(-)。我们成功地在 200 K 时捕获了 FAD(•-)，其中发生电子转移但质子转移不发生。在 277 K 下用 450 nm 光照射 FAD(ox)后进行的紫外可见光谱分析确定了 FADH(•)/FADH(-)混合物，并允许计算四个氧化还原态之间的差 FTIR 光谱。特征 C=O 伸缩振动的缺失表明质子供体不是质子化的羧酸。在 200 K 时对 FAD(•-)进行的紫外可见和 FTIR 分析表明色氨酸和酪氨酸的结构发生了变化。酰胺 I 振动的光谱分析揭示了初始电子转移（FAD(•-)形成）过程中蛋白质β-片层的结构扰动、质子转移（FADH(•)形成）过程中α-螺旋性的短暂增加以及随后电子转移（FADH(-)形成）后恢复到初始酰胺 I 信号。因此，与隐色体-DASH 不同，（6-4）PHR 中酶活性 FADH(-)的形成并未扰乱α-螺旋性。根据这些 FTIR 观察结果，讨论了（6-4）PHR 光激活过程中蛋白质结构的变化。
