The UV-induced DNA lesions, cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4 photoproducts), can be directly photorepaired by CPD photolyases and 6-4 photolyases, respectively. The fully reduced flavin (hydroquinone, HQ) cofactor is required for the catalysis of both types of these photolyases. On the other hand, flavin cofactor in the semireduced state, semiquinone, can be utilized by photolyase homologs, the cryptochromes. However, the evolutionary process of the transition of the functional states of flavin cofactors in photolyases and cryptochromes remains mysterious. In this work, we investigated three representative photolyases (Escherichia coli CPD photolyase, Microcystis aeruginosa DASH, and Phaeodactylum tricornutum 6-4 photolyase). We show that the residue at a single site adjacent to the flavin cofactor (corresponding to Ala377 in E. coli CPD photolyase, hereafter referred to as site 377) can fine-tune the stability of the HQ cofactor. We found that, in the presence of a polar residue (such as Ser or Asn) at site 377, HQ was stabilized against oxidation. Furthermore, this polar residue enhanced the photorepair activity of these photolyases both in vitro and in vivo. In contrast, substitution of hydrophobic residues, such as Ile, at site 377 in these photolyases adversely affected the stability of HQ. We speculate that these differential residue preferences at site 377 in photolyase proteins might reflect an important evolutionary event that altered the stability of HQ on the timeline from expression of photolyases to that of cryptochromes.

紫外线诱导的 DNA 损伤、环丁烷嘧啶二聚体 (CPD) 和嘧啶 (6-4) 嘧啶酮光产物 (6-4 光产物) 可以分别通过 CPD 光解酶和 6-4 光解酶直接进行光修复。完全还原的黄素（氢醌，HQ）辅因子是这两种光解酶的催化所必需的。另一方面，半还原状态的黄素辅因子半醌可被光解酶同系物隐花色素利用。然而，光解酶和隐花色素中黄素辅因子功能状态转变的进化过程仍然是个谜。在这项工作中，我们研究了三种具有代表性的光解酶（大肠杆菌CPD 光解酶、铜绿微囊藻DASH 和三角褐指藻）6-4 光解酶）。我们表明，与黄素辅因子相邻的单个位点（对应于大肠杆菌CPD 光解酶中的 Ala377，以下称为位点 377）的残基可以微调 HQ 辅因子的稳定性。我们发现，在 377 位点存在极性残基（如 Ser 或 Asn）时，HQ 可以稳定地抵抗氧化。此外，这种极性残基增强了这些光解酶在体外和体内的光修复活性. 相反，在这些光解酶的 377 位点替换疏水残基，如 Ile，对 HQ 的稳定性产生不利影响。我们推测光解酶蛋白中 377 位点的这些不同的残基偏好可能反映了一个重要的进化事件，该事件改变了 HQ 在从光解酶表达到隐花色素表达的时间线上的稳定性。