Restricted rotational motion of CO in a protein internal cavity: Evidence for nonseparating correlation functions from IR pump-probe spectroscopy

The strongly restricted orientational motion of CO molecules trapped in the Xe4 internal cavity of myoglobin mutant L29W-S108L is investigated by polarization-dependent mid-infrared pump-probe spectroscopy at cryogenic temperatures. Following an ultrafast initial decay, the signal anisotropy reaches an asymptotic value that is significantly larger than the prediction from the well-known relation r=2/5 < P-2[mu(0)center dot mu(tau)]>, based on previously established potential parameters. This discrepancy is explained by showing that the full four-point correlation function describing third-order spectroscopy does not factorize in systems where its fast decay is dominated by restricted reorientation of the transition dipole moments.