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传输的分子机理，并建立了蛋白质部分的紫外共振拉曼光谱测量系统。选取了两个典型的系统：一个是含有细菌视紫红质的光驱动质子泵系统，另一个是含有细胞色素C氧化酶的电子驱动质子泵系统。对于细菌视紫红质，考察了发色团的希夫碱质子转移到蛋白质部分时所涉及的中间体，并重点讨论了当蛋白质部分不接受质子时会发生什么。对于细胞色素c氧化酶，证实了光还原的发生，并测定了从α-亚铁血红素到α-亚铁血红素的电子转移速率。结果表明，好氧光还原产生的光稳定态主要由&lt；a^(2+)&gt；&lt；a(_3^(3+))&gt；组成，其&lt；a^(2+)&gt；和&lt；a(_3^(3+))&gt；分别可用441.6 nm和406.7 nm激发选择性地探测到。指出了Fe-His伸缩拉曼强度对酶活性的敏感性，并讨论了含氧中间体的瞬时拉曼光谱。利用SPEX-1260单色仪，配以1200 Gr/mm和200 nm闪耀光栅，建立了一套观测200-240 nm紫外光激发下的拉曼光谱系统。激发光由Nd：YAG激光器和拉曼移位器产生。该系统获得了高质量的芳香族氨基酸残基光谱。

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.