Energetics of subunit assembly and ligand binding in human hemoglobin

Abstract

An extensive and self-consistent set of thermodynamic properties has recently been established for the coupled processes of subunit assembly and ligand binding (oxygen and protons) in human hemoglobin. The resulting thermodynamic values permit a consideration of the possible sources of energetic terms accounting for stability of the tetrameric quaternary structures at different stages of ligation, and of the possible sources of cooperative energy. The analysis indicates that: (a) The change in buried surface ara upon oxygenation (i.e., hydrophobic stabilization) does not play a dominant role in stabilizing the unliganded tetramer relative to the liganded tetramer. (b) The pattern of enthalpic and entropic contributions to the free energies of dimer-tetramer. (c) The thermodynamic results are consistent with a dominant role of increased hydrogen bond formation in the deoxy quaternary structure. (d) Within tetramers the variation in free energy for successive oxygenation steps arises from both enthalpic and entropic contributions and the enthalpic contributions are almost entirely attributable to the heats of Bohr proton release. At pH 7.4 the pattern of thermodynamic values suggests that a large contribution to the free energy of cooperativity may arise from the energetics of Bohr proton release. It is suggested that a combination of proton ionization and hydrogen bonding may account for the main energetic features of cooperativity. Possible contributions from fluctuation behavior cannot presently be evaluated.

Ligand Linked Assembly of Scapharca Dimeric Hemoglobin

“…(Oxidation of HbI results in a hemichrome species that shows extensive dissociation, with an association constant of about 3 ϫ 10 3 M Ϫ1 at pH 7.0 (29).) Thermodynamic coupling between ligation and subunit dissociation (14) predicts that given a tighter assemblage in the deoxy state compared with the oxy state, oxygen affinity of HbI should increase upon subunit dissociation. To test this prediction, we carried out oxygen binding experiments on solutions with hemoglobin concentrations between 0.2 and 8 M heme.…”

“…In human hemoglobin, cooperativity results primarily from quaternary constraints in which the tight deoxy assemblage lowers the intrinsic subunit oxygen affinity, and binding of subsequent ligands leads to a stepwise reduction of these constraints (14). Analysis of equilibrium binding data has suggested that the fourth ligand is bound with higher affinity than isolated chains, an effect termed quaternary enhancement (13).…”

Application of linear free energy relations to protein conformational changes: the quaternary structural change of hemoglobin.

“…First, this number cannot strictly be compared with experimental values for the Ro --To transition,-since there are tertiary conformational changes upon removing the 4 ligands from the R4 molecule, which might affect the buried surface area (30,31,37) (as well as the position of the transition state). Second, the value of 25 cal mold A-2 could be in significant error (38).…”

Probing the energetics of proteins through structural perturbation: sites of regulatory energy in human hemoglobin.

“…A series ofstudies over the past 7 years, of which this work forms a part, has been aimed toward resolving the essential energetic aspects of the hemoglobin mechanism and of correlating the thermodynamic and structural information (cf. refs [15][16][17][18][19][20]. Identification of the sites of regulatory energy change within the hemoglobin molecule, as provided by the results ofthis study, lies at the core of this problem.…”