Development of Highly Sensitive Infrared Spectrometer with Femtosecond Time Resolution

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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.