title: Dynamics of allostery in hemoglobin: roles of the penultimate tyrosine H bonds.
abstract: The tyrosine residues adjacent to the C termini of the hemoglobin (Hb) subunits, alphaY140 and betaY145, are expected to play important structural roles, because the C termini are the loci of T-state quaternary salt-bridges, and because the tyrosine side-chains bridge the H and F helices via H bonds to the alphaV93 and betaV98 carbonyl groups. These roles have been investigated via measurements of oxygen binding, (1)H NMR spectra, resonance Raman (RR) spectra, and time-resolved resonance Raman (TR(3)) spectra on site mutants in which the Hcdots, three dots, centeredF H bonds are eliminated by replacing the tyrosine residues with phenylalanine. The TR(3) spectra confirm the hypothesis, based on TR(3) studies of wild-type Hb, that the Hcdots, three dots, centeredF H bonds break and then re-form during the sub-microsecond phase of the R-T quaternary transition. The TR(3) spectra support the inference from other mutational studies that the alphabeta dimers act as single dynamic units in this early phase, motions of the E and F helices being coupled tightly across the dimer interface. Formation of T quaternary contacts occurs at about the same rate in the mutants as in HbA. However, these contacts are weakened substantially by the Y/F substitutions. Equilibrium perturbations are apparent also, especially for the alpha-subunits, in which relaxation of the Fe-His bond, strengthening of the Acdots, three dots, centeredE interhelical H bond, and weakening of the "switch" quaternary contact in deoxyHb are all apparent. Structural effects are less marked for the beta-chain Y/F replacement, but the Bohr effect is reduced by 25%, indicating that the salt-bridge and H bond interactions of the adjacent C terminus are loosened. The alpha-chain replacement reduces the Bohr effect much more, consistent with the global perturbations detected by the structure probes.
authors.affiliation: Department of Chemistry, Princeton University, NJ 08544, USA.
authors.forename: Janina
authors.initials: J
authors.lastname: Kneipp
authors.forename: Gurusamy
authors.initials: G
authors.lastname: Balakrishnan
authors.forename: Ruopian
authors.initials: R
authors.lastname: Chen
authors.forename: Tong-Jian
authors.initials: TJ
authors.lastname: Shen
authors.forename: Sarata C
authors.initials: SC
authors.lastname: Sahu
authors.forename: Nancy T
authors.initials: NT
authors.lastname: Ho
authors.forename: Janel L
authors.initials: JL
authors.lastname: Giovannelli
authors.forename: Virgil
authors.initials: V
authors.lastname: Simplaceanu
authors.forename: Chien
authors.initials: C
authors.lastname: Ho
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Spiro
mesh_terms: D000494:Allosteric Regulation; D002248:Carbon Monoxide / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry* / Q000235:genetics; D006860:Hydrogen Bonding; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010100:Oxygen / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013059:Spectrum Analysis, Raman; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D006454:Hemoglobins; D011994:Recombinant Proteins; D014443:Tyrosine; D002248:Carbon Monoxide; D010100:Oxygen