The objective of the proposed research is the study of the ligand dynamics of carbonmonoxide(CO) nitric oxide (NO) in hemeproteins and the correlated protein conformational changes using transient mid-infrared absorption, picosecond Raman, and transient visible/near-infrared absorption techniques. The research on the dynamics of the protein cage will probe the amide I infrared optical marker, the iron-histidine Raman line and the 4- and 5- coordination visible/near-infrared absorption spectrum differences in a series of experiments aimed at correlating the barrier fluctuations with the observed ligand dynamics. In the Phe29 mutant myoglobin, the Phe29 residue acts as a very high barrier to ligand diffusion, separating the ligands into two groups: the ones before and after the Phe29 residue. The infrared monitoring of these two species creates a new handle for the study of barrier fluctuations. The optical marker associated with each of these two species will be monitored. This will permit the study of the diffusional process of the outgoing species as a function of pH, temperature and viscosity. Myoglobin under pH3 conditions should present larger barrier fluctuations, while low temperature measurements are likely to freeze those barriers. From these measurements one should be able to infer what are the modulators of NO diffusion. The short lived species will provide information about the near heme environment. During the photodeligation process, both a bleach corresponding to the loss of the bound species and an increase in absorption due to the creation of the new unbound species occur. These changes appear in different spectral regions and they can tell us about the dynamics of recombination, escape rate from the heme pocket, etc. The bleached absorption an isotropy identifies the mean orientation of the bound species (A-states) with respect to the heme, while the unbound species absorption an isotropy identifies orientational details about the B-states. We will create a reliable, user friendly, widely tunable (2--l0 millimicron) highly sensitive, femtosecond transient infrared spectrophotometer. It will incorporate an innovative AC monochromator and a powerful and flexible Dual Ti:Sapphire Regenerative Laser Amplifier. The AC monochromator will allow for full spectrum acquisition at each shot, as well as full spectrum normalization at 4 kHz repetition rate. The proposed experimental schemes are expected to bring a 512-fold improvement upon the existing experimental setups. They will create a femtosecond time- resolution apparatus with FTIR (Fourier Transform Infrared Spectrophotometer) sensitivity.
