| Abstract
| - Biphasic geminate rebinding of CO to myoglobin upon flash photolysis has been associated toligand distribution in hydrophobic cavities, structurally detected by time-resolved crystallography, xenonoccupancy, and molecular simulations. We show that the time course of CO rebinding to human hemoglobinalso exhibits a biphasic geminate rebinding when the protein is entrapped in wet nanoporous silica gel. Asimple branched kinetic scheme, involving the bound state A, the primary docking site C, and a secondarybinding site B was used to calculate the microscopic rates and the time-dependent population of theintermediate species. The activation enthalpies of the associated transitions were determined in the absenceand presence of 80% glycerol. Potential hydrophobic docking cavities within the α and β chains ofhemoglobin were identified by computational modeling using xenon as a probe. A hydrophobic pocket onthe distal side of the heme, corresponding to Xe4 in Mb, and a nearby site that does not have acorrespondence in Mb were detected. Neither potential xenon sites on the proximal side nor a migrationchannel from the distal to proximal site was located. The small enthalpic barriers between states B and Care in very good agreement with the location of the xenon sites on the distal side. Furthermore, theconnection between the two xenon sites is relatively open, explaining why the decreased mobility of theprotein with viscosity only slightly perturbs the energetics of ligand migration between the two sites.
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