Development of a New Two-dimensional Pulsed EPR Method and an Application to Heme-proteins

发展了一种新的大g各向异性无序系统的二维章动光谱技术。该方法基于章动频率由属于垂直于外部磁场的方向的g值确定的事实。该方法使人们能够确定非常小的g值(通常小于1)和g-主值的相对符号。该方法被应用于从马心中提取的低自旋MET肌红蛋白。用于章动实验的脉冲EPR仪器中最重要的部件是微波谐振器。一个强大而均匀的微波场是必不可少的。将环隙谐振器与矩形腔谐振器进行了比较。设计并制作了用于氦温度测量的环隙谐振器的稳定样品夹持器。用谱模拟技术分析了无序样品的章动信号。比较了门控积分和ESE-FT两种检测方法。分析表明，由于非共振效应，在门控积分中存在一定的频谱失真和漂移。这些伪影可以通过ESE-FT技术去除。观察了低自旋马心MET-肌红蛋白在冰冻溶液中的二维章动光谱。利用ESE-FT技术，得到了不受非共振影响的章动谱。将这些谱与章动谱随磁场变化的理论预测进行了比较。证实章动频率是由与外加磁场垂直的方向的g主值决定的。用这种方法可以观察到很小的氰离子络合物的g值，g=0.93。理论分析表明，g-主值的相对符号可以由无序系统的章动谱来确定。

A new technique of two-dimensional nutation spectroscopy for a disordered system with large g-anisotropy was developed. This method is based on the fact that a nutational frequency is determined by the g value which belongs to the direction perpendicular to the external magnetic field. The method enables one to determine a very small g-value (typically less than 1) and relative sign of g-principal values. The method is applied to low-spin met myoglobin from horse heart.1. The most important part of the pulsed EPR instrument for the nutational experiment is the microwave resonator. A strong and homogeneous microwave field is essential. A loop-gap resonator was compared with a rectangular cavity resonator. A stable sample holder of a loop-gap resonator for helium temperature measurements was designed and constructed.2. A nutation signal from a disordered sample was analyzed by a spectral simulation technique. A gated integration and ESE-FT methods were compared as a detection technique. The analysis showed that there was some distortion and shift of a spectrum in a gated integration caused by off-resonance effects. These artifacts could be removed by the ESE-FT technique.3. Two-dimensional nutation spectra were observed for low-spin horse heart met-myoglobin in frozen solution. By means of the ESE-FT technique, the nutation spectra those were free from off-resonance effects were obtained. The spectra were compared with theoretical predictions for magnetic field dependence of a nutational spectrum. It is confirmed that a nutation frequency was determined by the g-principal value of the direction orthogonal to the external magnetic field. The very small g-value of cyanide ion complex, g=0.93, could be observed by this method.4. It was predicted by the theoretical analysis that relative sign of g-principal values could be determined by a nutation spectrum from a disordered system.