The nature of ligand motion within proteins has been investigated by m
easuring femtosecond time-resolved infrared (IR) spectra of CO photodi
ssociated from the haem of myoglobin. Upon dissociation, the CO rotate
s approximately 90 degrees and becomes trapped within a ligand docking
site located near the binding site. Two trajectories, distinguished s
pectroscopically and kinetically with time constants of 0.20+/-0.05 ps
and 0.52+/-0.10 ps, lead to CO located within the docking site with o
pposite orientations. The protein reorganizes about the 'docked' CO wi
th a time constant of 1.6+/-0.3 ps and quickly establishes an energeti
c barrier that inhibits the reverse rebinding process.