Sensitivity enhancement in EPR and electron-nuclear double resonance by pulsed polarization schemes

在这个提议中，我们计划研究脉冲极化方案，以提高EPR，特别是电子核双共振（ENDOR）实验的灵敏度。后一种技术是酶催化机理研究的基础，例如在核糖核苷酸还原酶（RNR）中，阐明形成的自由基中间体的结构，这些中间体只能在非常小的体积和浓度（<< 100微升和<< 100微摩尔）下才能获得。目前，脉冲ENDOR实验的低灵敏度大大加剧了这些研究，其灵敏度比相应的电子自旋回波（ESE）实验小1到2个数量级。为了解决这一问题，将广泛测试源自核磁共振和动态核极化的脉冲极化方案，如交叉极化（CP）技术，以优化电子和原子核之间的相干极化转移。在第二步中，这些方案将被扩展到在测量原子核与电子自旋相互作用的超精细光谱的endortype实验中检测产生的核极化。所谓的CP-ENDOR实验的整体性能（灵敏度和分辨率）将与传统的Davies和Mims ENDOR在实际样品上的应用进行比较，例如在RNR催化循环中自由基中间体的光谱中。

In this proposal we plan to examine pulse polarization schemes to enhance the sensitivity of EPR and specifically electron nuclear double resonance (ENDOR) experiments. This latter technique is fundamental in mechanistic studies of enzyme catalysis, such as in ribonucleotide reductase (RNR), to elucidate the structure of formed radical intermediates obtainable only in very small volumes and concentrations (<< 100 microliter and << 100 micromolar). Currently, these investigations are considerably aggravated by the well-known low sensitivity of pulsed ENDOR experiments, which is about 1 to 2 orders of magnitude smaller than in the respective electron spin echo (ESE) experiment. To address this issue, pulsed polarization schemes derived from nuclear magnetic resonance and dynamic nuclear polarization, such as the cross-polarization (CP) technique, will be extensively tested to optimize coherent polarization transfer between electron and nuclei. In a second step, these schemes will be extended to detect the generated nuclear polarization in an ENDOR-type of experiment that measures the hyperfine spectrum of nuclei interacting with the electron spin. The overall performance (sensitivity and resolution) of a so-called CP-ENDOR experiment will be compared with traditional Davies and Mims ENDOR in applications on real samples, such as in the spectra of the radical intermediates in RNR catalytic cycle.