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

发展了一种大g各向异性无序系统的二维章动谱新技术。该方法基于章动频率由g值确定的事实，g值属于垂直于外部磁场的方向。该方法使人们能够确定一个非常小的g值（通常小于1）和g主值的相对符号。将该方法应用于马心低自旋蛋氨酸肌红蛋白.章动实验脉冲EPR仪器最重要的部分是微波谐振器。强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气温度测量的环隙谐振腔稳定样品保持器.利用谱模拟技术对无序样品的章动信号进行了分析。门控积分和ESE-FT方法作为检测技术进行了比较。分析表明，非共振效应会引起选通积分谱的畸变和移动。这些伪影可以通过ESE-FT技术去除。3.观察到低自旋马心高铁肌红蛋白在冷冻溶液中的二维章动谱。利用ESE-FT技术，得到了不受非共振影响的章动谱。的光谱进行了比较与理论预测的章动谱的磁场依赖性。证实了章动频率由与外磁场正交的方向的g主值决定。用该方法可以观察到氰根离子络合物的g值很小，g=0.93.理论分析表明，无序系统的章动谱可以确定g主值的相对符号。

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.