Electronic structure studies of flavin radicals

生物化学和生物物理学中最基本的问题之一是，如何通过载脂蛋白对辅酶的调节来实现特定催化反应的辅因子特异性。因此，未配对电子自旋密度是探测芳族辅助因子自由基态前沿轨道的重要物理量。通过这种方法，可以评估电子结构的贡献。电子顺磁共振（EPR）和基于电子-核双共振（ENDOR）或超精细亚能级相关（HYSCORE）光谱等方法观察到的超精细耦合常数是由磁性原子核位置或附近的电子自旋密度决定的。在这个项目中，我们力求尽可能完整地绘制出直接构成黄素辅因子的pi-环系统的原子的超精细映射，特别是碳和氮。以前的文献报道大多依靠与7,8-二甲基异alloxazine环结合的质子的超精细耦合来得出顺磁性黄素电子结构的间接结论。利用选择性13C-和15n标记的黄素辅酶同位素库，我们应用脉冲EPR方法直接绘制了各种蛋白质（光解酶，趋光素的LOV2结构域，黄氧还蛋白）中黄素半醌的pi-环系统的超精细结构。在项目扩展中，我们将努力使用混合量子力学/分子力学（QM/MM）计算对超精细映射进行复杂的理论分析。我们期望从结果中预测，哪些特定的蛋白质-辅因子相互作用对未配对电子自旋密度的功能特异性分布至关重要，这些分布反映在超精细耦合的大小和各向异性中。

One of the most fundamental questions in biochemistry and biophysics is, how cofactor specificity is achieved for a particular catalytic reaction via the modulation of the coenzyme by the apoprotein. To this end, the unpaired electron-spin density is an important physical quantity to probe frontier orbitals of aromatic cofactors in their radical state. By such an approach, the contribution of electronic structure may be evaluated. Hyperfine coupling constants as observed by electron paramagnetic resonance (EPR) and thereupon-based methods, such as electron-nuclear double resonance (ENDOR) or hyperfine sublevel correlation (HYSCORE) spectroscopy, are determined by the electron-spin densities at or near the positions of magnetic nuclei. In this project we strive for an as complete as possible hyperfine mapping of the atoms directly composing the pi-ring system of the ubiquitously encountered flavin cofactor, specifically, of the carbons and the nitrogens. Previous reports in the literature mostly relied on presenting hyperfine couplings of protons bound to the 7,8-dimethyl isoalloxazine ring to draw, rather indirect, conclusions on the electronic structure of paramagnetic flavins. Using a library of selectively 13C- and 15N-labeled flavin coenzyme isotopologs, we have applied pulsed EPR methods to directly map the hyperfine structure of the pi-ring system of flavin semiquinones in various proteins (photolyase, LOV2 domain of phototropin, flavodoxin) that stabilize the neutral radical form. In the project extension, we will strive for sophisticated theoretical analyses of the hyperfine mappings using hybrid quantum-mechanical/molecular-mechanical (QM/MM) computations. We expect from the results to predict, which specific protein-cofactor interactions are critical for the function-specific distribution of unpaired electron-spin density that is reflected in the size and anisotropy of hyperfine couplings.