title: Significance of beta116 His (G18) at alpha1beta1 contact sites for alphabeta assembly and autoxidation of hemoglobin.
abstract: The role of heterotetramer interaction sites in assembly and autoxidation of hemoglobin is not clear. The importance of beta(116His) (G-18) and gamma(116Ile) at one of the alpha1beta1 or alpha1gamma1 interaction sites for homo-dimer formation and assembly in vitro of beta and gamma chains, respectively, with alpha chains to form human Hb A and Hb F was assessed using recombinant beta(116His)(-->)(Asp), beta(116His)(-->)(Ile), and beta(112Cys)(-->)(Thr,116His)(-->)(Ile) chains. Even though beta chains (e.g., 116 His) are in monomer/tetramer equilibrium, beta(116Asp) chains showed only monomer formation. In contrast, beta(116Ile) and beta(112Thr,116Ile) chains showed homodimer and homotetramer formation like gamma-globin chains which contain 116 Ile. Assembly rates in vitro of beta(116Ile) or beta(112Thr,116Ile) chains with alpha chains were 340-fold slower, while beta(116Asp) chains promoted assembly compared to normal beta-globin chains. These results indicate that amino acid hydrophobicity at the G-18 position in non-alpha chains plays a key role in homotetramer, dimer, and monomer formation, which in turn plays a critical role in assembly with alpha chains to form Hb A and Hb F. These results also suggest that stable dimer formation of gamma-globin chains must not occur in vivo, since this would inhibit association with alpha chains to form Hb F. The role of beta(116His) (G-18) in heterotetramer-induced stabilization of the bond with oxygen in hemoglobin was also assessed by evaluating autoxidation rates using recombinant Hb tetramers containing these variant globin chains. Autoxidation rates of alpha(2)beta(2)(116Asp) and alpha(2)beta(2)(116Ile) tetramers showed biphasic kinetics with the faster rate due to alpha chain oxidation and the slower to the beta chain variants whose rates were 1.5-fold faster than that of normal beta-globin chains. In addition, NMR spectra of the heme area of these two hemoglobin variant tetramers showed similar resonance peaks, which are different from those of Hb A. Oxygen-binding properties of alpha(2)beta(2)(116His)(-->)(Asp) and alpha(2)beta(2)(116His)(-->)(Ile), however, showed slight alteration compared to Hb A. These results suggest that the beta116 amino acid (G18) plays a critical role in not only stabilizing alpha1beta1 interactions but also in inhibiting hemoglobin oxidation. However, stabilization of the bonds between oxygen and heme may not be dependent on stabilization of alpha1beta1 interactions. Tertiary structural changes may lead to changes in the heme region in beta chains after assembly with alpha chains, which could influence stability of dioxygen binding of beta chains.
authors.affiliation: The Children's Hospital of Philadelphia, Division of Hematology and NMR Core Facility, Philadelphia, Pennsylvania 19104, USA. adachi@email.chop.edu
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Adachi
authors.forename: Yi
authors.initials: Y
authors.lastname: Yang
authors.forename: Vinaysagar
authors.initials: V
authors.lastname: Lakka
authors.forename: Suzanne
authors.initials: S
authors.lastname: Wehrli
authors.forename: Konda S
authors.initials: KS
authors.lastname: Reddy
authors.forename: Saul
authors.initials: S
authors.lastname: Surrey
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D002850:Chromatography, Gel; D002942:Circular Dichroism; D005319:Fetal Hemoglobin / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D006441:Hemoglobin A / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006639:Histidine / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D006454:Hemoglobins; D011994:Recombinant Proteins; D006418:Heme; D006639:Histidine; D006441:Hemoglobin A; D005319:Fetal Hemoglobin; D010100:Oxygen