Ogura T, Yoshikawa S, Kitagawa T
Biochemistry. 1985 Dec 17;24(26):7746-52. doi: 10.1021/bi00347a037.
Occurrence of photoreduction of bovine cytochrome c oxidase was confirmed with the difference absorption spectra and oxygen consumption measurements for the enzyme irradiated with laser light at 406.7, 441.6, and 590 nm. The resonance Raman spectra were obtained under the same experimental conditions as those adopted for the measurements of oxygen consumption and difference absorption spectra. The photoreduction was more effective upon irradiation at shorter wavelengths and was irreversible under anaerobic conditions. However, upon aeration into the cell, the original oxidized form was restored. It was found that aerobic laser irradiation produces a photo steady state of the catalytic dioxygen reduction and that the Raman scattering from this photo steady state probes cytochrome a2+ and cytochrome a3(3)+ separately upon excitations at 441.6 and 406.7 nm, respectively. The enzyme was apparently protected from the photoreduction in the spinning cell with the spinning speed between 1 and 1500 rpm. These results were explained satisfactorily with the reported rate constant for the electron transfer from cytochrome a to cytochrome a3 (0.58 s-1) and a comparable photoreduction rate of cytochrome a. The anaerobic photoreduction did give Raman lines at 1666 and 214 cm-1, which are characteristic of the ferrous high-spin cytochrome a3(2)+, but they were absent under aerobic photoreduction. The formyl CH = O stretching mode of the a3 heme was observed at 1671 cm-1 for a2+a3(2)+CO but at 1664 cm-1 for a2+a3(2)+CN-, indicating that the CH = O stretching frequency reflects the pi back-donation to the axial ligand similar to the oxidation state marker line (v4).
通过对在406.7、441.6和590nm波长激光照射下的牛细胞色素c氧化酶进行差示吸收光谱和氧消耗测量，证实了该酶光还原现象的发生。在与氧消耗和差示吸收光谱测量相同的实验条件下获得了共振拉曼光谱。较短波长照射时光还原更有效，且在厌氧条件下是不可逆的。然而，向细胞中通空气后，可恢复为原来的氧化形式。发现有氧激光照射产生催化双氧还原的光稳态，并且在441.6和406.7nm激发下，来自该光稳态的拉曼散射分别单独探测细胞色素a2+和细胞色素a3(3)+。当旋转细胞转速在1至1500rpm之间时，该酶显然受到保护而免受光还原。利用报道的从细胞色素a到细胞色素a3的电子转移速率常数（0.58 s-1）和细胞色素a相当的光还原速率，对这些结果进行了令人满意的解释。厌氧光还原确实在1666和214 cm-1处给出了拉曼谱线，这是亚铁高自旋细胞色素a3(2)+的特征谱线，但在有氧光还原下不存在。对于a2+a3(2)+CO，a3血红素的甲酰基CH = O伸缩振动模式在1671 cm-1处观察到，而对于a2+a3(2)+CN-，在1664 cm-1处观察到，这表明CH = O伸缩频率反映了与氧化态标记线（v4）类似的对轴向配体的π反馈。
