Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Quantum Beam Science Research Directorate, National Institutes for Quantum and Radiological Science and Technology, Tokai-mura, Ibaraki 319-1106, Japan.
Sci Rep. 2017 Feb 22;7:43162. doi: 10.1038/srep43162.
Flavin compounds such as flavin adenine dinucleotide (FAD), flavin mononucleotide and riboflavin make up the active centers in flavoproteins that facilitate various oxidoreductive processes. The fine structural features of the hydrogens and valence electrons of the flavin molecules in the protein environment are critical to the functions of the flavoproteins. However, information on these features cannot be obtained from conventional protein X-ray analyses at ordinary resolution. Here we report the charge density analysis of a flavoenzyme, NADH-cytochrome b reductase (b5R), at an ultra-high resolution of 0.78 Å. Valence electrons on the FAD cofactor as well as the peptide portion, which are clearly visualized even after the conventional refinement, are analyzed by the multipolar atomic model refinement. The topological analysis for the determined electron density reveals the valence electronic structure of the isoalloxazine ring of FAD and hydrogen-bonding interactions with the protein environment. The tetrahedral electronic distribution around the N5 atom of FAD in b5R is stabilized by hydrogen bonding with CH of Tyr65 and amide-H of Thr66. The hydrogen bonding network leads to His49 composing the cytochrome b-binding site via non-classical hydrogen bonds between N5 of FAD and CH of Tyr65 and O of Tyr65 and CH of His49.
黄素化合物，如黄素腺嘌呤二核苷酸（FAD）、黄素单核苷酸和核黄素，构成了黄素蛋白中的活性中心，促进了各种氧化还原过程。蛋白质环境中黄素分子的氢和价电子的精细结构特征对黄素蛋白的功能至关重要。然而，这些特征的信息无法从常规的蛋白质 X 射线分析在普通分辨率下获得。在这里，我们报告了黄素酶 NADH-细胞色素 b 还原酶（b5R）在超高质量分辨率 0.78 Å下的电荷密度分析。即使在常规精修后，FAD 辅因子和肽部分上的价电子也可以通过多极原子模型精修进行分析。确定的电子密度的拓扑分析揭示了 FAD 的异咯嗪环的价电子结构以及与蛋白质环境的氢键相互作用。b5R 中 FAD 的 N5 原子周围的四面体电子分布通过 Tyr65 的 CH 和 Thr66 的酰胺-H 与 FAD 的氢键稳定。氢键网络导致 His49 通过 FAD 的 N5 与 Tyr65 的 CH 和 Tyr65 的 O 与 His49 的 CH 之间的非经典氢键与细胞色素 b 结合位点组成。
