Development of Highly Sensitive Infrared Spectrometer with Femtosecond Time Resolution

介绍了飞秒时间分辨中红外瞬变吸收光谱仪的设计和性能。该光谱仪由一套商用钛宝石飞秒激光器研制而成，采用差频混频技术。用该光谱仪在800~2800 cm~(-1)cm~(-1)的中红外波段进行了瞬时吸收测量，对重复频率为1 kHz的差值吸收灵敏度为1×10~(-4)~(-4)。在亚皮秒可见光或紫外光泵浦下，获得了250FS的时间分辨率。以一氧化碳在肌红蛋白中的结合和解离过程的研究为例，用飞秒可见光泵浦中红外探测光谱研究了一氧化碳引起的转录调节蛋白CooA的变构作用。当血红素被激发到Soret带时，在大约1979 cm~(-1)&gt~(-1)处检测到瞬时漂白，这是与血红素结合的CO的吸收区域。漂白信号表现出非指数衰减，时间常数分别为56ps和290ps，这是由于CO与血红素的重新结合引起的。约97%的游离CO以双亲方式重组。CooA中的双链重组速度明显快于肌红蛋白和血红蛋白。根据各向异性的测量，计算出束缚CO相对于卟啉平面的角度约为78度。检测到结合CO的漂白剂中红外光谱发生了漂移，其特征时间为160ps。结果表明，光谱漂移是由于不同蛋白质构象，特别是活性构象和中间构象中结合CO的频率不同造成的。因此，追踪了CooA中与生物相关的构象变化。讨论了观测到的构象变化的可能归属。

The design and performance of a femtosecond time-resolved mid-infrared transient absorption spectrometer is illustrated. The spectrometer was developed from a set of commercially available Ti : sapphire femtosecond lasers utilizing difference frequency mixing technique. With this spectrometer, the transient absorption in the mid-infrared region from 800 to 2800 cm^<-1> was detected with a sensitivity of 1×10^<-4> for difference absorbance at a repetition rate of 1 kHz. With a sub-picosecond visible or ultraviolet pump, a time-resolution of 250fs has been obtained. A study on the binding and dissociation processes of carbon monoxide in myoglobin is presented as an example of its applications.CooA, which is a transcriptional regulator heme protein allosterially triggered by CO, is studied by femtosecond visible-pump mid-IR-probe spectroscopy. Transient bleaching upon excitation of the heme into Soret band is detected at around 1979cm^<-1>, which is the absorption region of the CO bound to the heme. The bleach signal shows a nonexponential decay with time constants of 56 ps and 290 ps, caused by the rebinding of the CO to the heme. About 97% of dissociated CO recombines geminately. The geminate recombination rate in CooA is significantly faster than that in myoglobin and hemoglobin. The angle of the bound CO with respect to the porphyrin plane is calculated to be about 78 degrees based on the anisotropy measurements. A shift of the bleach mid-IR spectrum of the bound CO is detected having characteristic time of 160 ps. It is suggested that the spectral shift is caused by a difference in the frequency of the bound CO in different protein conformations, particularly in active conformation and in intermediate one. Thus, the biologically relevant conformation change in CooA was traced. Possible assignment of the observed conformation change is discussed.