title: Geminate rebinding in R-state hemoglobin: kinetic and computational evidence for multiple hydrophobic pockets.
abstract: Biphasic geminate rebinding of CO to myoglobin upon flash photolysis has been associated to ligand distribution in hydrophobic cavities, structurally detected by time-resolved crystallography, xenon occupancy, and molecular simulations. We show that the time course of CO rebinding to human hemoglobin also exhibits a biphasic geminate rebinding when the protein is entrapped in wet nanoporous silica gel. A simple branched kinetic scheme, involving the bound state A, the primary docking site C, and a secondary binding site B was used to calculate the microscopic rates and the time-dependent population of the intermediate species. The activation enthalpies of the associated transitions were determined in the absence and presence of 80% glycerol. Potential hydrophobic docking cavities within the alpha and beta chains of hemoglobin were identified by computational modeling using xenon as a probe. A hydrophobic pocket on the distal side of the heme, corresponding to Xe4 in Mb, and a nearby site that does not have a correspondence in Mb were detected. Neither potential xenon sites on the proximal side nor a migration channel from the distal to proximal site was located. The small enthalpic barriers between states B and C are in very good agreement with the location of the xenon sites on the distal side. Furthermore, the connection between the two xenon sites is relatively open, explaining why the decreased mobility of the protein with viscosity only slightly perturbs the energetics of ligand migration between the two sites.
authors.affiliation: Dipartimento di Fisica, Università degli Studi di Parma, Parco Area delle Scienze 7/A, 43100 Parma, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Sottini
authors.forename: Stefania
authors.initials: S
authors.lastname: Abbruzzetti
authors.forename: Francesca
authors.initials: F
authors.lastname: Spyrakis
authors.forename: Stefano
authors.initials: S
authors.lastname: Bettati
authors.forename: Luca
authors.initials: L
authors.lastname: Ronda
authors.forename: Andrea
authors.initials: A
authors.lastname: Mozzarelli
authors.forename: Cristiano
authors.initials: C
authors.lastname: Viappiani
mesh_terms: D001665:Binding Sites; D002248:Carbon Monoxide / Q000737:chemistry / Q000378:metabolism; D003198:Computer Simulation; D006454:Hemoglobins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008956:Models, Chemical; D008958:Models, Molecular; D010782:Photolysis; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits; D014783:Viscosity
chemical_list: D006454:Hemoglobins; D021122:Protein Subunits; D002248:Carbon Monoxide