Femtosecond JR spectroscopy: Methods and applications to protein dynamics

We have developed a fs JR absorption spectrometer that provides high sensitivity in both near and mid-JR regions. High sensitivity is attained using a dual beam approach: A fs broadband IR probe pulse is split into sample and reference pulses which are routed through and around the sample, respectively. The two pulses are directed through a monochromator before impinging on photodetectors. The ratio ofthe sample and reference signals, i.e., the sample transmittance at a particular wavelength, is measured to a precision of ≈ 1 % per laser shot. Signal averaging at the 1 .5 kHz repetition rate of the laser reduces the random noise level to less than 5 ×10-4 rms in absorbance units after 1 sec of signal averaging. Femtosecond near-JR probe pulses are produced by continuum generation and provide spectral coverage out to 1 .tm. Femtosecond mid-IR probe pulses are generated by differencefrequency mixing an amplified portion of the continuum with the fundamental in a 1 mm Li103 crystal and provide spectral coverage from 3 .3 to 5. 5 .μm. The spectral resolution of the spectrometer is determined by the bandpass of the monochromator, which is routinely set to 10 cm and 3 cm' for near and mid-JR wavelengths, respectively. The time resolution is < 300 fs in both near- and mid-IR regions. This spectrometer has been used to probe the dynamics of conformational relaxation of myoglobin (Mb) and the dynamics ofligand motion after photodissociation of MbCO.