Performance of a time-resolved IR facility for assessment of protonation T states and polarity changes in carboxyl groups in a large membrane protein, mammalian cytochrome c oxidase, under turnover conditions in a sub-millisecond time resolution
Performance of a time-resolved IR facility for assessment of protonation T states and polarity changes in carboxyl groups in a large membrane protein, mammalian cytochrome c oxidase, under turnover conditions in a sub-millisecond time resolution
复制标题
时间分辨红外设备的性能，用于在亚毫秒时间分辨率的周转条件下评估大型膜蛋白、哺乳动物细胞色素 C 氧化酶中羧基的质子化 T 状态和极性变化
DOI：
10.1016/j.bbabio.2018.05.015
复制
发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Satoru Nakashima
中科院分区：
文献类型：
--
作者：
Chen Li;Tatsuhito Nishiguchi;Kyoko Shinzawa-Itoh;Shinya Yoshikawa;Takashi Ogura;Satoru Nakashima
时间分辨红外光谱分析的质子化和极性变化的羧基参与质子泵酶的营业额条件下是必不可少的，阐明其质子泵机制。我们开发了一种新的时间分辨红外设备，通过引入流动系统，将高浓度的粘性蛋白质溶液转移到一个薄的（50 μm）流动池中，该流动池配备了一个高灵敏度的红外光谱仪，该光谱仪采用光谱宽度为350 cm− 1的飞秒中红外脉冲激光器作为红外白色光源，配备了多通道MCT检测器。该设备配备了O2供应系统，能够在亚毫秒时间尺度上对O2还原进行红外测量，并结合由CO闪光光解在COOH（1725-1770 cm−1）区域引发的牛细胞色素氧化酶（CcO）的质子泵送，精度约为10 μO.D.。在背景OD下为1.该设备确定了在~1744 cm− 1处的谱带强度变化，可归因于反应开始后1 ms内羧基的质子化以及单电子转移到O2还原中心。结果表明，该设施检测到质子化的一个单一的羧基包括在大型蛋白质，如CcO（210 kDa）。目前的设备还通过检测1750 cm− 1和1760 cm−1附近的谱带位移，灵敏地识别COOH基团的极性变化，而没有显著的强度变化。这些研究结果表明，该设备的性能足够高，为理解蛋白质羧基的质子转移机制提供关键信息。
Time-resolved IR analyses for the protonation and polarity changes of carboxyl groups involved in proton pump enzymes under turnover conditions are indispensable for elucidation of their proton-pump mechanisms. We have developed a new time-resolved infrared facility by introducing a flow system for transferring highly concentrated and thus viscous protein solution to a thin (50 μm) flow cell equipped in a highly sensitive IR spectrometer constructed with the femtosecond mid-IR pulse laser with spectral width of 350 cm−1as an IR white light source equipped with multi-channel MCT detector. This facility equipped with O2supply system enables the sub-millisecond time scale infrared measurements of the O2reduction coupled with proton pumping by bovine cytochromecoxidase (CcO) initiated by CO-flash photolysis in the COOH (1725–1770 cm−1) region with the accuracy of about 10 μO.D. under the background O.D. of 1. The facility identifies a band intensity change at ~1744 cm−1assignable to protonation of a carboxyl group coupled with a single electron transfer to the O2reduction center within 1 ms after initiation of the reaction. The results suggest that the facility detects protonation of a single carboxyl group included in large proteins like as CcO (210 kDa). The present facility sensitively identifies also polarity changes in COOH group by detecting shifts of the bands near 1750 cm−1and 1760 cm−1, without significant intensity changes. These findings show the performance of this facility sufficiently high for providing crucial information for understanding the proton transferring mechanisms of protein carboxyl groups.