Single residue modification of only one dimer within the hemoglobin tetramer reveals autonomous dimer function

Abstract

The mechanism of cooperativity in the human hemoglobin tetramer (a dimer of ␣␤ dimers) has historically been modeled as a simple two-state system in which a low-affinity structural form (T) switches, on ligation, to a high-affinity form (R), yielding a net loss of hydrogen bonds and salt bridges in the dimer-dimer interface. Modifications that weaken these cross-dimer contacts destabilize the quaternary T tetramer, leading to decreased cooperativity and enhanced ligand affinity, as demonstrated in many studies on symmetric double modifications, i.e., a residue site modified in both ␣-or both ␤-subunits. In this work, hybrid tetramers have been prepared with only one modified residue, yielding molecules composed of a wild-type dimer and a modified dimer. It is observed that the cooperative free energy of ligation to the modified dimer is perturbed to the same extent whether in the hybrid tetramer or in the doubly modified tetramer. The cooperative free energy of ligation to the wild-type dimer is unperturbed, even in the hybrid tetramer, and despite the overall destabilization of the T tetramer by the modification. This asymmetric response by the two dimers within the same tetramer shows that loss of dimer-dimer contacts is not communicated across the dimer-dimer interface, but is transmitted through the dimer that bears the modified residue. These observations are interpreted in terms of a previously proposed dimer-based model of cooperativity with an additional quaternary (T͞R) component.M any tertiary and quaternary conformational events important for catalysis and cooperativity in biological macromolecules are energetically unfavorable, and must be driven by favorable free energy of ligand binding. In the case of human hemoglobin (Hb), initial binding of O 2 to the heme Fe drives intrinsically unfavorable conformation change within the globin. The associated energetic ''penalty'' results in lower binding constants for initial ligation. However, penalties for the additional binding steps are progressively reduced, generating Hb's characteristic sigmoidal binding curve. Contributing to this curve are the partially ligated Hb intermediates, composed of two different subunit types (␣ and ␤) with four binding sites (␣ 1 , ␤ 1 , ␣ 2 , ␤ 2 ). Up to four labile ligands (O 2 ) are present on each tetramer in multiple configurations of site occupancy within at least two distinct quaternary structures (T and R). The challenge in understanding the mechanism of Hb cooperativity has been to identify and measure the separate energetic penalties for each O 2 binding step, and then to correlate individual penalties with specific structural events.To approach a resolution of this complex problem, energetic components of the system's ligation intermediates were evaluated with no prior assumptions as to their quaternary structures. Analysis of the thermodynamic linkages between dimertetramer assembly and O 2 binding permitted the ligand binding constant of the noncooperative free dimer to be used as the thermodynamic reference s...

Nitrite Reductase Activity of Sol-Gel-encapsulated Deoxyhemoglobin

“…Functional and spectroscopic studies are consistent with equilibrium populations of these different functionally distinct conformations being modulated by added heterotropic effectors (54,64), with the degree and symmetry of partial ligand binding (65,66,69), and with the nature of the surrounding solvent/environment (44, 70 -72). The present study shows that nitrite reductase activity is also responsive to this tuning of conformational distributions and, thus, provides a biophysical framework for understanding the interplay between hemoglobin enzymatic reactivity and the complex in vivo physiological milieu.…”

“…That x-ray study as well as functional and spectroscopic results (64) obtained on semi-Hbs (dimeric ␣ 1 ␤ 1 derivatives composed of one apo and one holo globin chain) suggest that intra-T-state communication occurring during this loosening process is likely to be facilitated through a bending of the ␣␤ dimers. Thermodynamic studies have directly provided indications of intra dimer communication within the T-state (65,66).…”

Allosteric Hemoglobin Assembly: Diversity and Similarity

“…Despite many elegant models and very extensive study, there is no universal agreement concerning the mechanism of cooperativity by human hemoglobin. One intriguing recent finding is that of Ackers and colleagues suggesting cooperativity within each ␣␤ dimer (17). Although controversial, this idea finds support from recent structural results (18) and suggests an intriguing parallel with cooperative invertebrate hemoglobins, which are assembled from dimeric units that likely possess intrinsic cooperativity.…”

Protein structure change studied by hydrogen-deuterium exchange, functional labeling, and mass spectrometry

“…A great deal of information on regulatory structure change in many proteins is now available, but mainly in a qualitative pictorial sense from ''before and after'' crystallographic or NMR views. How these changes participate in energy transduction and translocation has been little explored (19)(20)(21)(22).The HX work described here is directed toward the goal of specifying the individual allosterically important structure changes, the energetic contribution of each, and how they interact to produce the allosteric function. The methods used here provide complete protein coverage, nearly site resolution, and high-throughput efficiency.…”

“…A great deal of information on regulatory structure change in many proteins is now available, but mainly in a qualitative pictorial sense from ''before and after'' crystallographic or NMR views. How these changes participate in energy transduction and translocation has been little explored (19)(20)(21)(22).…”