Real-time tracking of CO migration and binding in the α and β subunits of human hemoglobin via 150-ps time-resolved Laue crystallography

We have developed the method of picosecond Laue crystallography and used this capability to probe ligand dynamics in tetrameric R-state hemoglobin (Hb). Time-resolved, 2 angstrom-resolution electron density maps of photolyzed HbCO reveal the time-dependent population of CO in the binding (A) and primary docking (B) sites of both alpha and beta subunits from 100 ps to 10 mu s. The proximity of the B site in the beta subunit is about 0.25 angstrom closer to its A binding site, and its k(BA) rebinding rate (similar to 300 mu s (1)) is six times faster, suggesting distal control of the rebinding dynamics. Geminate rebinding in the beta subunit exhibits both prompt and delayed geminate phases. We developed a microscopic model to quantitatively explain the observed kinetics, with three states for the alpha subunit and four states for the beta subunit. This model provides a consistent framework for interpreting rebinding kinetics reported in prior studies of both HbCO and HbO(2). Published by Elsevier B.V.