Yan Luyao, Cao Xiaodan, Wang Lijuan, Chen Jie, Sancar Aziz, Zhong Dongping
Center for Ultrafast Science and Technology, School of Chemistry and Chemical Engineering, School of Physics and Astronomy, Zhang Jiang Institute for Advanced Study, Shanghai Jiao Tong University, Shanghai 200240, China.
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599.
Proc Natl Acad Sci U S A. 2024 Dec 10;121(50):e2417633121. doi: 10.1073/pnas.2417633121. Epub 2024 Dec 2.
Photolyase and cryptochrome belong to a group of structurally similar flavoproteins but with two distinct functions of DNA repair as a photoenzyme and signal transduction as a photoreceptor, respectively, under blue-light illumination. Here, we studied a recently discovered bifunctional cryptochrome (CraCRY) with focus on its repair of UV-induced pyrimidine-pyrimidone (6-4) photoproduct (6-4PP). We used femtosecond spectroscopy and site-directed mutagenesis to map out the critical elementary steps by following the dynamics of initial reactants, various intermediates, and final products. We observed initial direct ultrafast electron tunneling from the hydroquinone flavin cofactor to 6-4PP in 300 ps through an intervening adenine as a mediator, minimizing the electron bifurcation of a two-step hopping pathway bridged also by the adenine as an intermediate. The subsequent proton transfer (PT) from the neighboring histidine to anionic 6-4PP in 2 ns is critical and competes with the futile back electron transfer in 151 ps. Mutations of either of two histidines in the active site nearly abolish repair, indicating their essential role on repair reactivity and structural integrity. These results elucidate the electron-coupled PT mechanism and the repair photocycle of this bifunctional cryptochrome at the molecular level, further supporting that the hydroquinone flavin is the active state in vivo for dual functions or one of the two functions is excessive.
光解酶和隐花色素属于一组结构相似的黄素蛋白，但在蓝光照射下分别具有作为光酶进行DNA修复和作为光感受器进行信号转导这两种不同功能。在此，我们研究了最近发现的双功能隐花色素（CraCRY），重点关注其对紫外线诱导的嘧啶 - 嘧啶酮（6 - 4）光产物（6 - 4PP）的修复作用。我们使用飞秒光谱和定点诱变技术，通过追踪初始反应物、各种中间体和最终产物的动力学过程来确定关键的基本步骤。我们观察到，在300皮秒内，对苯二酚黄素辅因子通过作为介质的腺嘌呤将初始直接超快电子隧穿至6 - 4PP，从而使同样由腺嘌呤作为中间体桥接的两步跳跃途径的电子分叉最小化。随后在2纳秒内，相邻组氨酸向阴离子型6 - 4PP的质子转移（PT）至关重要，且与151皮秒内的无效反向电子转移相互竞争。活性位点上两个组氨酸中任一个发生突变几乎都会使修复作用消失，这表明它们对修复反应性和结构完整性起着至关重要的作用。这些结果在分子水平上阐明了这种双功能隐花色素的电子耦合质子转移机制和修复光循环，进一步支持了对苯二酚黄素是体内具有双重功能的活性状态，或者其中一种功能是多余的。
