title: Probing the alpha 1 beta 2 interface of human hemoglobin by mutagenesis. Role of the FG-C contact regions.
abstract: The allosteric transition of hemoglobin involves an extensive reorganization of the alpha 1 beta 2 interface, in which two contact regions have been identified. This paper concerns at the effect of two mutations located in the "switch" (alpha C3 Thr --> Trp) and the "flexible joint" (beta C3 Trp --> Thr). We have expressed and characterized one double and two single mutants: Hb alpha T38W/beta W37T, Hb beta W37T, and Hb alpha T38W, whose structure has been determined by crystallography. We present data on: (i) the interface structure in the contact regions, (ii) oxygen and CO binding kinetics and cooperativity, (iii) dissociation rates of deoxy tetramers and association rates of deoxy dimers, and (iv) the effect of NaI on deoxy tetramer dissociation rate constant. All the mutants are tetrameric and T-state in the deoxygenated derivative. Reassociation of deoxygenated dimers is not modified by interface mutations. DeoxyHb alpha T38W/beta W37T dissociate much faster. We propose a binding site for I- at the switch region. The single mutants binds O2 cooperatively; the double one is almost non-cooperative, a feature confirmed by CO binding. The functional data, analyzed with the two-state model, indicate that these mutations reduce the value of the allosteric constant LO.
authors.affiliation: Department of Biochemical Sciences A. Rossi Fanelli, University of Rome La Sapienza, Italy.
authors.forename: B
authors.initials: B
authors.lastname: Vallone
authors.forename: A
authors.initials: A
authors.lastname: Bellelli
authors.forename: A E
authors.initials: AE
authors.lastname: Miele
authors.forename: M
authors.initials: M
authors.lastname: Brunori
authors.forename: G
authors.initials: G
authors.lastname: Fermi
mesh_terms: D000495:Allosteric Site; D001704:Biopolymers; D002248:Carbon Monoxide / Q000378:metabolism; D006454:Hemoglobins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000378:metabolism; D011487:Protein Conformation
chemical_list: D001704:Biopolymers; D006454:Hemoglobins; D002248:Carbon Monoxide; D010100:Oxygen