Structural Study on Electron Tunneling and Proton Relay in Proteins by Using Ultraviolet Resonance Raman Spectroscopy

利用共振拉曼光谱研究了质子通过生物膜的分子机制，并建立了一套蛋白质分子紫外共振拉曼光谱测量系统。两个典型的系统，一个是光驱动的质子泵系统的细菌视紫红质和另一个是电子驱动的质子泵系统的细胞色素c氧化酶。对于细菌视紫红质中间体涉及的时间区域时，发色团的席夫碱质子转移到蛋白质部分进行了检查和讨论的重点是会发生什么时，蛋白质部分不接受质子。对于细胞色素c氧化酶的光还原的发生进行了确认和电子转移速率从血红素的血红素<a_3>进行了测定。结果表明，好氧光还原反应产生了一个主要由&lt;a^（2+）&gt; &lt;a（_3^（3+））&gt;组成的光稳态，其&lt;a^（2+）&gt;和&lt;a（_3^（3+））&gt;血红素可分别被441.6和406.7 nm的激发光选择性地探测。指出了Fe-His伸缩拉曼强度对酶活性的敏感性，并对含氧中间体的瞬态拉曼光谱进行了讨论。利用dac 1260单单色仪和1200 gr/mm、200 nm闪耀光栅，建立了200-240 nm紫外光激发的拉曼光谱观测系统。激发光由Nd：YAG激光器和拉曼移相器产生。该系统带来了芳香族氨基酸残基的高质量的光谱。

Molecular mechanism of the vectorial proton transportation through biological membrane was investigated by using resonance Raman spectroscopy, and a system for measuring UV resonance Raman spectra of protein moieties was constructed. Two typical systems were picked up; one is a light driven proton pumping system having bacteriorhodopsin and the other is an electron-driven proton pumping system containing cytochrome c oxidase. For bacteriorhodopsin intermediates involved in a time region when the Schiff base proton of the chromophore was transferred to the protein moiety were examined and discussion was focused on what would happen when the protein moiety did not accept the proton. For cytochrome c oxidase the occurrence of photoreduction was confirmed and the electron transfer rate from the a heme to the <a_3> heme was determined. It was found that the aerobic photoreduction generates a photosteady state composed mainly of <a^(2+)> <a(_3^(3+))> , and that its <a^(2+)> and <a(_3^(3+))> hemes can be selectively probed with the 441.6 and 406.7 nm excitations, respectively. Sensitivity of the Fe-His stretching Raman intensity to the enzymic activity was pointed out, and transient Raman spectra of oxygenated intermediates were discussed. A system to observe Raman spectra excited by UV light around 200-240 nm was set up with a SPEX 1260 single monochromator equipped with a 1200 gr/mm and 200 nm blaze grating. Excitation light was derived from Nd:YAG laser and Raman shifter. The system brought about spectra of good quality for aromatic amino acid residues.