Transient Infrared Spectroscopy of Carboxyhemoglobin Photodeligation

Ligand binding to and unbinding from proteins is a much studied problem important in biophysics. By and large, fast time-resolved studies until now have been electronic spectroscopy and resonance Raman spectroscopy which focus on the behavior of the protein chromophore. Here we report the use of transient infrared spectroscopy to look directly at the CO ligand with subpicosecond resolution after 585 nm photoexcitation of carboxyhemoglobin (HbCO) in D sub 2 O at 300K. The excitation pulses have 0.3 ps duration and are synchronized with 0.3 ps infrared probe pulses which are generated by difference frequency mixing of 585 nm radiation and a white light continuum in LiIO sub 3. Angle tuning the nonlinear crystal enables us to vary its output from 2.5micron to 5.5micron. At a given angle, the infrared pulses have ~300 cm sup (-1) bandwidth. Here they span the range between 1900 and 2200 cm sup (-1) corresponding to the CO stretching region of the spectrum. Vibrational resolution is recovered by placing a monochromator and InSb detector after the sample. A variable pathlength delay allows us to map out the ligand dynamics.