Direct Observation of Ligand Rebinding Pathways in Hemoglobin Using Femtosecond Mid-IR Spectroscopy

The dynamics of NO rebinding in hemoglobin (Hb) was directly observed using femtosecond mid-IR spectroscopy after photodeligation of NO from HbNO in D<sub>2</sub>O at 283 K. Time-resolved spectra of bound NO appeared to have a single feature peaked at 1616 cm<sup>–1</sup> but were much better described by two Gaussians with equal intensities but different rebinding kinetics, where the feature at 1617 cm<sup>–1</sup> rebinds faster than the one at 1614 cm<sup>–1</sup>. It is possible that the two bands each correspond to one of two subunit constituents of the tetrameric Hb. Transient absorption spectra of photodeligated NO revealed three evolving bands near 1858 cm<sup>–1</sup> and their red-shifted replicas. The red-shifted replicas arise from photodeligated NO in the vibrationally excited <i>v</i> = 1 state. More than 10% of the NO was dissociated into the vibrationally excited <i>v</i> = 1 state when photolyzed by a 580 nm pulse. The three absorption bands for the deligated NO could be attributed to three NO sites in or near the heme pocket. The kinetics of the three transient bands for the deligated NO, as well as the recovery of the bound NO population, was most consistent with a kinetics scheme that incorporates time-dependent rebinding from one site that rapidly equilibrates with the other two sites. The time dependence results from a time-dependent rebinding barrier due to conformational relaxation of protein after deligation. By assigning each absorption band to a site in the heme pocket of Hb, a pathway for rebinding of NO to Hb was proposed.